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

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

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.

Peptidoglycan recognition protein SC (PGRP-SC) shapes gut microbiota richness, diversity and composition by modulating immunity in the house fly Musca domestica.

The gastrointestinal tract of all animals, including insects, is colonized by a remarkable array of microorganisms which are referred to collectively as the gut microbiota. The hosts establish mutually beneficial interactions with the gut microbiota. However, the mechanisms shaping these interactions remain to be better understood. Here, we investigated the roles of Musca domestica peptidoglycan recognition protein SC (MdPGRP-SC), a secreted pattern recognition receptor, in shaping the gut microbial community structure by using biochemical and high-throughput sequencing approaches. The recombinant MdPGRP-SC (rMdPGRP-SC) could strongly bind various pathogen-associated molecular patterns (PAMPs) including peptidoglycan, lipopolysaccharide and D-galactose, and exhibited mild affinity to ß-1, 3-glucan and D-mannose. Meanwhile, rMdPGRP-SC could also bind different kinds of microorganisms, including gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and yeast (Pichia pastoris). rMdPGRP-SC also exhibited weak antibacterial activity against Bacillus subtilis. Knockdown of MdPGRP-SC by RNAi reduced the persistence of ingested E. coli and a load of indigenous microbiota in the larval gut significantly. In addition, depleted MdPGRP-SC also altered the gut microbiota composition and led to increased ratios of Gram-negative bacteria. We hypothesize that MdPGRP-SC is involved in maintaining gut homeostasis by modulating the immune intensity of the gut through multiple mechanisms, including degrading or neutralizing various PAMPs and selectively suppressing the growth of some bacteria. Considering the functional conservation of the peptidoglycan recognition protein (PGRP) family in insects, the catalytic PGRPs might be promising candidate targets not only for pest and vector control but also for the treatment of bacterial infection in insect farming.

Synthesis, larvicidal efficiency and molecular docking studies of novel annulated pyrano[2,3-c]pyrazoles against Culex pipiens L. and Musca domestica L. larvae.

A number of novel annulated pyrazolopyranopyrimidines were prepared via reaction of iminoether of the corresponding 6-amino-5-cyano-pyrano[2,3-c]pyrazole derivative 1 with different nitrogen nucleophiles. The structure of the synthesized compounds was deduced based on IR, MS, 1H NMR and 13C NMR spectroscopic data. The larvicidal potency of the synthesized compounds against the lab and field strains of Culex pipiens and Musca domestica larvae was evaluated and the structure-activity relationship (SAR) was discussed. The assay revealed that the tested pyranopyrazole derivatives exhibited good larvicidal bio-efficacy whereas iminoether 4 exhibited the highest efficiency, for lab more than field strains of both species. Also, M. domestica larvae were more sensitive to tested compounds than C. pipiens. The field strain showed low resistance ratios to all compounds with only about 2 folds. The inhibitory effects of synthesized molecules on nAChRs were evaluated by molecular docking. Moreover, the cytotoxicity of the newly synthesized compounds against normal human fibroblasts (WI-38) was investigated. The cytotoxic assay showed that derivatives 4 and 5 were not harmful to normal fibroblasts.

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

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

Gene-Level, but Not Chromosome-Wide, Divergence between a Very Young House Fly Proto-Y Chromosome and Its Homologous Proto-X Chromosome.

X and Y chromosomes are usually derived from a pair of homologous autosomes, which then diverge from each other over time. Although Y-specific features have been characterized in sex chromosomes of various ages, the earliest stages of Y chromosome evolution remain elusive. In particular, we do not know whether early stages of Y chromosome evolution consist of changes to individual genes or happen via chromosome-scale divergence from the X. To address this question, we quantified divergence between young proto-X and proto-Y chromosomes in the house fly, Musca domestica. We compared proto-sex chromosome sequence and gene expression between genotypic (XY) and sex-reversed (XX) males. We find evidence for sequence divergence between genes on the proto-X and proto-Y, including five genes with mitochondrial functions. There is also an excess of genes with divergent expression between the proto-X and proto-Y, but the number of genes is small. This suggests that individual proto-Y genes, but not the entire proto-Y chromosome, have diverged from the proto-X. We identified one gene, encoding an axonemal dynein assembly factor (which functions in sperm motility), that has higher expression in XY males than XX males because of a disproportionate contribution of the proto-Y allele to gene expression. The upregulation of the proto-Y allele may be favored in males because of this gene's function in spermatogenesis. The evolutionary divergence between proto-X and proto-Y copies of this gene, as well as the mitochondrial genes, is consistent with selection in males affecting the evolution of individual genes during early Y chromosome evolution.

Molecular Characterization of 14-3-3 Zeta Gene in Musca domestica (Diptera: Muscidae) and Its Roles in Response to Bacterial Infection.

The 14-3-3 gene plays important role in many biological processes, including cell survival, apoptosis, and signal transduction. However, function of the 14-3-3 homologous gene in Musca domestica remains unclear. Here, we identified and characterized the 14-3-3ζ of M. domestica. We found that Md14-3-3ζ gene was highly homologous with other close insects. The qRT-PCR analysis revealed that the Md14-3-3ζ was highly expressed in adults, and was expressed predominantly in hemocytes and fat body. Meanwhile, the expression of Md14-3-3ζ was up-regulated after injecting Escherichia coli and Staphylococcus aureus. Moreover, the recombinant protein rMd14-3-3ζ strongly inhibits the growth of E. coli and S. aureus. Notably, the rMd14-3-3ζ inhibits E. coli and S. aureus by permeating the cell membrane. Taken together, our findings suggested that Md14-3-3ζ is involved in the immune response against bacteria through damaging the cell membrane.

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.

House fly larval grazing alters dairy cattle manure microbial communities.

BACKGROUND: House fly larvae (Musca domestica L.) require a live microbial community to successfully develop. Cattle manure is rich in organic matter and microorganisms, comprising a suitable substrate for larvae who feed on both the decomposing manure and the prokaryotic and eukaryotic microbes therein. Microbial communities change as manure ages, and when fly larvae are present changes attributable to larval grazing also occur. Here, we used high throughput sequencing of 16S and 18S rRNA genes to characterize microbial communities in dairy cattle manure and evaluated the changes in those communities over time by comparing the communities in fresh manure to aged manure with or without house fly larvae. RESULTS: Bacteria, archaea and protist community compositions significantly differed across manure types (e.g. fresh, aged, larval-grazed). Irrespective of manure type, microbial communities were dominated by the following phyla: Euryarchaeota (Archaea); Proteobacteria, Firmicutes and Bacteroidetes (Bacteria); Ciliophora, Metamonanda, Ochrophyta, Apicomplexa, Discoba, Lobosa and Cercozoa (Protists). Larval grazing significantly reduced the abundances of Bacteroidetes, Ciliophora, Cercozoa and increased the abundances of Apicomplexa and Discoba. Manure aging alone significantly altered the abundance bacteria (Acinetobacter, Clostridium, Petrimonas, Succinovibro), protists (Buxtonella, Enteromonas) and archaea (Methanosphaera and Methanomassiliicoccus). Larval grazing also altered the abundance of several bacterial genera (Pseudomonas, Bacteroides, Flavobacterium, Taibaiella, Sphingopyxis, Sphingobacterium), protists (Oxytricha, Cercomonas, Colpodella, Parabodo) and archaea (Methanobrevibacter and Methanocorpusculum). Overall, larval grazing significantly reduced bacterial and archaeal diversities but increased protist diversity. Moreover, total carbon (TC) and nitrogen (TN) decreased in larval grazed manure, and both TC and TN were highly correlated with several of bacterial, archaeal and protist communities. CONCLUSIONS: House fly larval grazing altered the abundance and diversity of bacterial, archaeal and protist communities differently than manure aging alone. Fly larvae likely alter community composition by directly feeding on and eliminating microbes and by competing with predatory microbes for available nutrients and microbial prey. Our results lend insight into the role house fly larvae play in shaping manure microbial communities and help identify microbes that house fly larvae utilize as food sources in manure. Information extrapolated from this study can be used to develop manure management strategies to interfere with house fly development and reduce house fly populations.

Midgut fluxes and digestive enzyme recycling in Musca domestica: A molecular approach.

Insects are reported to have water midgut countercurrents fluxes powering enzyme recovery before excretion, usually known as enzyme recycling. Up to now there is a single, and very incomplete, attempt to relate transporters and channels with countercurrent fluxes. In this work, M. domestica midgut water fluxes were inferred from the concentration of ingested and non absorbable dye along the midgut, which anterior midgut was divided in two sections (A1, A2), the middle in one (M) and the posterior midgut in four (P1, P2, P3, and P4), which led to the finding of additional sites of secretion and absorption. Water is secreted in A1 and A2 and absorbed at the middle midgut (M), whereas in posterior midgut, water is absorbed at P2 and secreted in the other sections, mainly at P4. Thus, a countercurrent flux is formed from P4 to P2. To disclose the involvement of the known water transporters Na+:K+:2Cl- (NKCC) and K+:Cl- (KCC), as well as the water channels aquaporins in water fluxes, their expression was evaluated by RNA-seq analyses from triplicate samples of seven sections along the midgut. MdNKCC1 was expressed in A1, MdNKCC2 was expressed in M1 and P2 and MdKCC in middle and in the most posterior region, thus apparently involved in secretion, absorption and both, respectively. MdNKCC2, MdKCC and aquaporins MdDRIP1 and 2 were confirmed as being apical by proteomics of purified microvillar membranes. The role of NKCC and KCC on midgut water fluxes was tested observing the effect of the inhibitor furosemide. The change of trypsin distribution along the posterior midgut and the increase of trypsin excretion in the presence of furosemide lend support to the proposal that countercurrent fluxes power enzyme recycling and that the fluxes are caused by NKCC and KCC transporters helped by aquaporins.

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly, Musca domestica (Diptera: Muscidae).

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.

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.

Molecular mechanisms associated with acidification and alkalization along the larval midgut of Musca domestica.

To disclose the molecular mechanisms involved in luminal midgut buffering of M. domestica, we used RNA-seq analyses from triplicate samples of seven sections along the midgut to evaluate the expression levels of genes coding for selected manually curated protein sequences. Channels, pumps and transporters were confirmed as being apical by proteomics of purified microvillar membranes. Midgut pH determinations with a microsensor and a pH indicator were carried out in larvae in different diets with or without added compounds to evaluate the role of proteins in buffering. The data suggested that acidification occurs at middle midgut by the action of H+ V-ATPase with protons produced by carbonic anhydrase, followed by chloride ions transported by a K+Cl- symporter. K+ ions are recovered by an apical K+ channel and K+ homeostasis maintained by a basolateral Na+/K+-ATPase. Acidification is also affected by a Na+/H+ exchanger and a multidrug resistance protein. Posterior midgut alkalization results from the action of a NH3 transporter and H+-coupled peptide transporter, mainly in a diet rich in free peptides. A working model was proposed for the midgut luminal acidification and alkalization, as well as for mucosal protection against acid by a neutralized mucus layer.

The roles of mucus-forming mucins, peritrophins and peritrophins with mucin domains in the insect midgut.

Most insects have a gut lined with a peritrophic membrane (PM) consisting of chitin and proteins, mainly peritrophins that have chitin-binding domains. The PM is proposed to originate from mucus-forming mucins (Mf-mucins), which acquired a chitin-binding domain that interlocked with chitin, replacing mucus in function. We evaluated the expression of Mf-mucins and peritrophins by RNA-sequencing (RNA-seq) throughout the midgut of four distantly related insects. Mf-mucins were identified as proteins with high o-glycosylation and a series of uninterrupted Pro/Thr/Ser residues. The results demonstrate that the mucus layer is widespread in insects, and suggest that insect Mf-mucins are derived from those found in other animals by the loss of the cysteine knot and von Willebrand domains. The data also support a role of Mf-mucins in protecting the middle midgut of Musca domestica against acidic buffers. Mf-mucins may also produce a jelly-like material associated with the PM that immobilizes digestive enzymes in Spodoptera frugiperda. Peritrophins with a domain similar to Mf-mucins may be close to the ancestor of peritrophins. Expression data of peritrophins and chitin synthase genes throughout the midgut of M. domestica, S. frugiperda and Tenebrio molitor indicated that peritrophins were incorporated along the PM, according to their preferential sites of formation. Finally, the data support the view that mucus has functions distinct from the PM.

Identification and characterization of two novel C-type lectins from the larvae of housefly, Musca domestica L.

Lectins and antimicrobial peptides (AMPs) are widely distributed in various insects and play crucial roles in primary host defense against pathogenic microorganisms. Two AMPs (cecropin and attacin) have been identified and characterized in the larvae of housefly. In this study, two novel C-type lectins (CTLs) were obtained from Musca domestica, while their agglutinating and antiviral properties were evaluated. Real-time PCR analysis showed that the mRNA levels of four immune genes (MdCTL1, MdCTL2, Cecropin, and Attacin) from M. domestica were significantly upregulated after injection with killed Gram-negative Escherichia coli. Moreover, purified MdCTL1-2 proteins can agglutinate E. coli and Staphylococcus aureus in the presence of calcium ions, suggesting their immune function is Ca2+ dependent. Sequence analysis indicated that typical WND and QPD motifs were found in the Ca2+ -binding site 2 of carbohydrate recognition domain from MdCTL1-2, which was consistent with their agglutinating activities. Subsequently, antiviral experiments indicated that MdCTL1-2 proteins could significantly reduce the infection rate of Spodoptera frugiperda 9 cells by the baculovirus Autographa californica multicapsid nucleopolyhedrovirus, indicating they might play important roles in insect innate immunity against microbial pathogens. In addition, MdCTL1-2 proteins could effectively inhibit the replication of influenza H1 N1 virus, which was similar to the effect of ribavirin. These results suggested that two novel CTLs could be considered a promising drug candidate for the treatment of influenza. Moreover, it is believed that the discovery of the CTLs with antiviral effects in M. domestica will improve our understanding of the molecular mechanism of insect immune response against viruses.

Characterization and Expression Pattern Analysis of the T-Complex Protein-1 Zeta Subunit in Musca domestica L (Diptera).

Chaperonins, belonging to the T-complex protein-1 (TCP-1) family, assist in the correct folding of nascent and misfolded proteins. It is well-known that in mammals, the zeta subunit of the TCP-1 complex (TCP-1ζ) plays a vital role in the folding and assembly of cytoskeleta proteins. This study reported for the first time the cloning, characterization and expression pattern analysis of the TCP-1ζ from Musca domestica, which was named as MdTCP-1ζ. The MdTCP-1ζ cDNA is 1,803 bp long with a 1,596 bp open reading frame that encodes a protein with 531 bp amino acids. The analysis of the transcriptional profile of MdTCP-1ζ using qRT-PCR revealed relatively high expression in the salivary glands and trachea at the tissues while among the developmental stages. The highest expression was observed only in the eggs suggesting that the MdTCP-1ζ may play a role in embryonic development. The expression of MdTCP-1ζ was also significantly induced after exposure to short-term heat shock and infection by Escherichia coli, Staphylococcus aureus, or Candida albicans. This suggested that MdTCP-1ζ may take part in the immune responses of housefly and perhaps contribute to the protection against cellular injury.

A serine protease inhibitor from Musca domestica larva exhibits inhibitory activity against elastase and chymotrypsin.

OBJECTIVE: Insect-derived serine protease inhibitors (serpins) exhibit multiple inhibitory activities, but so far, no functional roles for serpins of Musca domestica have been identified. Here, the functional features of M. domestica serine protease inhibitor (MDSPI16) were characterized. RESULTS: Hundred forty seven differentially expressed genes including the MDSPI16 gene were screened by constructing the subtractive cDNA library. The 1154-bp full-length MDSPI16 gene was cloned, and the recombinant MDSPI16 serpin protein was expressed as a 42.6 kDa protein in an Escherichia coli expression system. The recombinant MDSPI16 protein was purified using Ni-NTA affinity chromatography, and the inhibitory activity of MDSPI16 was assessed. MDSPI16 did not inhibit trypsin, papain, or proteinase K but strongly inhibited elastase (Ki = 2.8 nM) and chymotrypsin (Ki = 28 nM). The inhibitory activity of MDSPI16 remained stable over from 37 to 100 °C and from pH 2 to 12. CONCLUSIONS: The MDSPI16 exhibited inhibitory activity against elastase and chymotrypsin and the inhibitory activity remained stable.

Insect nicotinic receptor interactions in vivo with neonicotinoid, organophosphorus, and methylcarbamate insecticides and a synergist.

The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR [(3)H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on [(3)H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site.

Proteomic Analysis of the Peritrophic Matrix from the Midgut of Third Instar Larvae, Musca domestica.

OBJECTIVE: To better comprehend the molecular structure and physiological function of the housefly larval peritrophic matrix (PM), a mass spectrometry approach was used to investigate the PM protein composition. METHODS: The PM was dissected from the midgut of the third instar larvae, and protein extracted from the PM was evaluated using SDS-PAGE. A 1D-PAGE lane containing all protein bands was cut from top to bottom, the proteins in-gel trypsinised and analysed via shotgun liquid chromatography- tandem mass spectrometry (LC-MS/MS). RESULTS: In total, 374 proteins, with molecular weights varying from 8.225 kD to 996.065 kD and isoelectric points ranging from 3.83 to 11.24 were successfully identified, most identified proteins were mainly related to immunity, digestion, nutrient metabolism and PM structure. Furthermore, many of these proteins were functionally associated with pattern binding, polysaccharide binding, structural constituent of peritrophic membrane and chitin binding, according to Gene Ontology annotation. CONCLUSION: The PM protein composition, which provides a basis for further functional investigations of the identified proteins, will be useful for understanding the housefly larval gut immune system and may help to identify potential targets and exploit new bioinsecticides.

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.