The transposon vector containing enhanced green fluorescent protein (EGFP) was injected into early housefly (Musca domestica L.) eggs by microinjection method to realize stable gene expression in vivo for verification, and to study housefly gene function. A borosilicate glass micro injection needle suitable for microinjection of housefly eggs was made, the softening treatment conditions of housefly egg shells were explored, and a microinjection technology platform suitable for housefly was constructed with a high-precision microsyringe Nanoject Ⅲ as the main body. The recombinant plasmid PiggyBac-[3×P3]-EGFP containing the eye-specific 3×P3 promoter and EGFP and the stable genetic expression helper plasmid pHA3pig helper were microinjected into the treated housefly eggs. After emergence, the eye luminescence was observed, and the expression and transcription level of EGFP were detected. The results showed that the normal hatching rate of housefly eggs was 55% when rinsed in bleaching water for 35 s. The hardness of the egg shell treated for 35 s was suitable for injection and the injection needle was not easy to break. About 3% of the emerged housefly eyes had green fluorescence. Through further molecular detection, EGFP specific fragments with a size of 750 bp were amplified from DNA and RNA of housefly. Through the technical platform, the stable expression of reporter genes in housefly can be conveniently and effectively realized, and a bioreactor with housefly as the main body can be established, which provides certain reference value for subsequent research on housefly gene function.
Animals ; Animals, Genetically Modified ; Gene Expression ; Genes, Reporter ; Green Fluorescent Proteins/genetics* ; Houseflies/genetics* ; Microinjections

OBJECTIVETo 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.

METHODSThe 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).

RESULTSIn 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.

CONCLUSIONThe 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.

Animals ; Chitin ; metabolism ; Gastrointestinal Tract ; metabolism ; Houseflies ; metabolism ; Insect Proteins ; metabolism ; Larva ; metabolism ; Proteomics

We purified a novel mannose binding lectin form Musca domestica pupae by affinity chromatography on Con A-Sepharose 4B and DEAE weak anion-exchange chromatography. By SDS-PAGE, MBL-1 yielded a single band with the molecular weight of 24 kDa. It was a glycoprotein detected by periodic acid-schiffs staining reaction, with 97.36% protein and 2.1% oligosaccharide. Meanwhile, the results of beta-elimination reaction, infrared spectroscopy, atomic force microscopy and protein sequencing instrument show that MBL-1 was an ellipsoidal-shaped monomer with 60-100 nm in diameter. N-glycoside bond linked oligosaccharide chain and the N-terminal blocked peptide chain. Further study suggested that MBL-1 promote the proliferation of macrophage in a concentration-dependent manner. The scanning electron microscope analysis shows that MBL-1 promoted the activation of macrophages. These results show that MBL-1 purified from Musca domestica pupae possesses immune regulation effect, serving a reference basis to develop natural immune-modulator.
Animals ; Glycoproteins ; analysis ; Houseflies ; chemistry ; Immunomodulation ; immunology ; physiology ; Macrophages ; immunology ; Mannose-Binding Lectin ; chemistry ; physiology ; Oligosaccharides ; analysis ; Pupa ; chemistry

In medicolegal investigations, correct identification of the necrophagous fly species collected around and on the corpse is an essential step for estimating the postmortem interval (PMI). Therefore, forensic pathologists and entomologists investigating deaths due to violent crimes need a rapid, easy-to-use protocol to identify fly species found on corpses. A rapid and robust DNA-based tool that can distinguish between various immature and mature species from the Calliphoridae, Muscidae, and Sarcophagidae families would be ideal for such investigations. To date, the DNA barcode initiative is the best approach for identifying species-specific nucleotide sequences. We have developed 3 sequence-characterized amplified region (SCAR)-based identification systems derived from the Abdominal-B homeobox sequences of 17 fly species belonging to the Muscidae and Sarcophagidae. The flies used in this study were collected in Korea. These assay systems can classify 17 forensically important fly species into the dipteran family group and reliably distinguish them from inter- and intraspecific fly species through a 2-step multiplex PCR. This novel approach may also be used as an alternative to conventional DNA-based identification methods.
Base Sequence ; Cadaver ; Crime ; Diptera ; DNA ; Genes, Homeobox ; Humans ; Korea ; Multiplex Polymerase Chain Reaction ; Muscidae ; Sarcophagidae

To screen the candidate genes associated with Musca domestica antibacterial peptides using DNA microarray technique, the hybrid probes were designed from the conservative domains of the encoded area of the insect antibacterial peptide genes in GenBank with biology software Designer 2.0, and were synthesized by a chemical process, with the assistance of the automated Gen III Microarray Spotter, those oligo probes were printed on a special ready-made glass, and a cDNA microarray was constructed. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by Escherichia coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analyzed using the software of MIDAS, fifteen valid hybridization signals were detected through two times of hybridization and scanning (the positive samples as a control were excluded). DNA microarray technique can be successfully applied screen the candidate genes associated with Musca domestica antibacterial peptides, and further provide significant evidence to discover its antibacterial peptide new genes.
Animals ; Antimicrobial Cationic Peptides ; genetics ; Base Sequence ; Gene Expression Profiling ; Genes, Insect ; Houseflies ; genetics ; growth & development ; Larva ; genetics ; growth & development ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; methods ; Oligonucleotide Probes ; chemistry

OBJECTIVETo identify necrophagous fly species from different regions in China using inter-simple sequence repeat (ISSR) melocular markers and analyze their genetic difference and relationship.

METHODSFive carrion fly species were collected from 12 cities and regions in China, including M.domestica, Lucilia sericata, Chrysomyia megacephala, Helicophagella melanura, and Boetthcherisca peregrina. Twenty-two ISSR primers were designed and synthesized, from which 8 were selected to identify the necrophagous fly species. Cluster analysis was conducted based on distance matrices using unweighted pair group method.

RESULTSTotally 121 amplification samples were obtained using the 8 primers, and 679 clear and stable bands were visualized including 516 bands with polymorphisms. M.domestica, Lucilia sericata, Chrysomyia megacephala, Helicophagella melanura, and Boethcherisca peregrina from different regions in China produced their specific PCR band spectra. M. domestica from 10 different regions in China showed different inheritance patterns of the markers. Species-specific ISSR fragment was found among the necrophagous flys pecies. Cluster analysis among the most abundant carrion fly species demonstrated that M.domestica from 10 different regions could be divided into 4 groups at different levels. Most of the Chrysomyia megacephala and Lucilia sericata could be clustered in one tree.

CONCLUSIONThis study represents the first identification of the common necrophagous fly species in China. ISSR-PCR-based identification of the species reveals the genetic diversity and genotypic difference among M.domestica from 10 cities and regions in China.

Animals ; China ; Genetic Markers ; Genetic Variation ; Minisatellite Repeats ; genetics ; Muscidae ; classification ; genetics ; Phylogeny ; Polymerase Chain Reaction ; methods ; Random Amplified Polymorphic DNA Technique ; methods ; Repetitive Sequences, Nucleic Acid ; Species Specificity

A forensic entomological study conducted in an oil palm plantation in Tanjung Sepat, Selangor, Malaysia on 3 August 2007 revealed that a housefly, Musca domestica Linnaeus oviposited its eggs on a freshly dead pig. This finding indicated that housefly might play an important role in forensic investigation in determining post-mortem interval (PMI), although it was not yet found in human corpses or any animal carrion. This preliminary paper presented a first record of Musca domestica eggs found on animal carcass in the country.
Houseflies ; Swine ; Malaysia ; L ; occurrence

DNA barcoding was recently introduced to molecular identification of forensically important fly species. So, we have analysed the barcode region (687 nucleotides) of mitochondrial cytochrome oxidase subunit I (COI) gene for four species of Muscidae flies collected from Korea. The sequences were aligned and analysed to construct a phylogenetic tree using DNA Star 5.01(DNAStar Inc) and MEGA 3.1 program(Kumar, Tamura, Nei 2004). Intraspecific variation was not noted between M.stabulans individual to each other. Intraspecific variation ranges of other species were 0.1%, 0.1~0.3% and 0.1~0.6% for O.leucostoma, M.angustifrons and M.domestica, respectively. Interspecific percent distance was minimal(9.7~10.0%) between M.stabulans and M.angustifrons. Other species showed above 10% distance from each other. The result showed that four species of Muscidae fly species (Muscina angustifrons, Muscina stabulans, Ophyra leucostoma and Musca domestica) were identifiable from each other with analysis of barcode region of COI gene. Therefore, we conclude that species identification of forensically important Muscidae flies used in this study is possible with percent distance of sequences of COI barcode region, but more species and individuals should be examined to be confident about the conclusion.
Cytochromes* ; Diptera ; DNA* ; Electron Transport Complex IV* ; Korea* ; Muscidae*

OBJECTIVETo study the inducible antibacterial activity of the hemolymph from housefly larva and analyze the antibacterial molecules.

METHODSThe hemolymph was collected from the third instar housefly larvae 48 h after pricking treatment. Nine standard bacterial strains were used for determination of the antibaterial activity of the collected hemolymph and its combination with ampicillin. The anti-yeast activity of the hemolymph and its mixture with fluconazol was also assayed. The antibacterial molecules in the hemolymph was analyzed by SDS-PAGE.

RESULTSThe growth of E.coli, Staphylococcus aureus, Staphylococcus albus, subserotypes of Shigeila flexneri, Bacillus proteus, Bacillus subtilis, Bacillus typhi, Bacillus paratyphosus, and Micrococcus lysodeikticus could be inhibited by the hemolymph collected from housefly larva, and the effect differed significantly between the groups (Plt;0.001). The hemolymph produced the strongest antibacterial activity against Micrococcus lysodeikticus, and the combination of the hemolymph with ampicillin most conspicuously inhibited the growth of Staphylococcus albus. The hemolyph and fluconazol exhibited obvious synergistic effect against yeast. SDS-PAGE identified some specific antibacterial molecules in the hemolymph.

CONCLUSIONThe induced hemolymph from housefly larva possesses strong antibacterial and antifungal activities especially against Micrococcus lysodeikticus. The hemolymph in combination with ampicillin produces the strongest effect against Staphylococcus albus, and fluconazol can significantly enhance the anti-yeast activity of the hemolymph through a synergistic mechanism.

Ampicillin ; pharmacology ; Animals ; Anti-Bacterial Agents ; pharmacology ; Antifungal Agents ; pharmacology ; Bacteria ; drug effects ; Electrophoresis, Polyacrylamide Gel ; Fluconazole ; pharmacology ; Hemolymph ; immunology ; Houseflies ; immunology ; Larva ; immunology ; Microbial Sensitivity Tests ; Yeasts ; drug effects

Estimation of postmortem interval (PMI) in a putrefied corpse has been a long theme in the forensic medicine. Insects, especially necrophagous fly species are now utilized as indicators of PMI because the first visitors to a dead body are usually known to be blow fly species (Family Calliphoridae). House flies (Family Muscidae) are later visitors but they are very significant in forensic entomology because of their worldwide distribution. Entomologic evidences recovered from the scene are often immature individuals such as eggs, maggots and pupae. Because growth rates and ecological characteristics are different among fly species, accurate species identification is essential. As species identification in immature stages is very difficult or even impossible to an expert entomologist, many researchers are trying to identify fly species by molecular techniques. Authors analyzed 400bp of mitochondrial COI gene sequences of six Muscidae fly species (Fannia prisca, Muscina angustifrons, Muscina stabulans, Musca domestica, Hydrotaea dentipes and Ophyra leucostoma). In spite of limited number of flies analyzed in this study, all six fly species have different haplotype of COI gene and shows minimal intraspecific variation. This result shows that six fly species analyzed in this study can be discriminated each other by COI gene sequence analysis. But, more individuals from various geographic region should be analyzed to apply this result to a forensic entomology practice.
Cadaver ; Cytochromes* ; Diptera* ; Eggs ; Electron Transport Complex IV* ; Entomology ; Forensic Medicine ; Haplotypes ; Houseflies ; Insects ; Larva ; Muscidae ; Ovum ; Pupa ; Sequence Analysis