OBJECTIVES: To identify the common sarcosaprophagous flies in the Yangtze River Delta based on mitochondrial cytochrome c oxidase subunit Ⅰ(COⅠ) gene sequence and verify the reliability of this method. METHODS: Seven common genetically stable sarcosaprophagous flies in three families and six genera were collected from large domestic pig carcasses placed in the field and cultured in the laboratory for many generations. The whole genome DNA was extracted and the COⅠ gene fragment was amplified. The forward and reverse sequencing was followed by splicing. The base composition of the amplified fragment and the rate of interspecific evolutionary divergence were analyzed by software such as Mega 7.0.26. The phylogenetic tree of COⅠ gene sequence of common necrophagous flies in the Yangtze River Delta was established by neighbor joining (NJ) method and unweighted pair-group method with arithmetic means (UPGMA) method. RESULTS: The average base composition of different flies was A(30.14%), T(38.23%), C(15.98%), G(15.65%). The rate of interspecific evolutionary divergence ranged from 2.2% to 15.3%, the lowest rate was between Chrysomya megacephala and Chrysomya pinguis, the highest rate was between Muscina stabulans and Boettcherisca peregrina. CONCLUSIONS COⅠ gene can be used to identify the common necrophagous flies in the Yangtze River Delta.
Animals ; Cadaver ; Diptera/genetics* ; Phylogeny ; Reproducibility of Results ; Rivers

Objective To identify the species of common necrophagous flies in Fujian Province by gene fragment sequences of mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） and 16S ribosomal deoxyribonucleic acid （16S rDNA）, and to explore the identification efficacy of these two molecular markers. Methods In total 22 common necrophagous flies were collected from the death scenes in 9 different regions in Fujian Province and DNA was extracted from the flies after morphological identification. The gene fragments of COⅠ and 16S rDNA were amplified and sequenced. All the sequences were uploaded to GeneBank and BLAST and MEGA 10.0 software were used to perform sequence alignment, homology analysis and intraspecific and interspecific genetic distance analysis. The phylogenetic trees of DNA fragment sequences of COⅠ and 16S rDNA of common necrophagous flies in Fujian Province were established by unweighted pair-group method with arithmetic means （UPGMA）, respectively. Results The flies were classified into 6 species, 5 genera and 3 families by morphological identification. The results of gene sequence analysis showed that the average number of interspecific and intraspecific genetic distance of 16S rDNA ranged from 1.8% to 8.9% and 0.0% to 2.4%, respectively. The average number of interspecific and intraspecific genetic distance of COⅠ ranged from 7.2% to 13.6% and 0.0% to 6.3%, respectively. Conclusion The gene sequences of COⅠ and 16S rDNA can accurately identify the species of different necrophagous flies, and 16S rDNA showed higher value in species identification of common calliphoridae necrophagous flies in Fujian Province.
Animals ; DNA, Ribosomal/genetics* ; Diptera/genetics* ; Humans ; Phylogeny ; RNA, Ribosomal, 16S/genetics* ; Sequence Analysis, DNA ; Species Specificity

Objective To assess the feasibility of using 28S ribosomal RNA （28S rRNA） and mitochondrial cytochrome c oxidase subunit Ⅰ （COⅠ） gene sequences of nine necrophagous Calliphorid flies for the identification of common necrophagous Calliphorid flies, and to provide technical support for postmortem interval （PMI） estimation. Methods Twenty-three Calliphorid flies were collected and identified morphologically, and DNA were extracted from legs. The gene fragments of 28S rRNA and COⅠ were amplified and sequenced, then the sequence alignment was performed with BLAST. The composition of obtained sequences was analyzed and evolutionary divergence rate between species and intraspecies were established. The phylogeny tree was constructed with neighbor-joining method. Results The 23 necrophagous Calliphorid flies were identified to 9 species of 5 genera. The 715 bp from 28S rRNA and 637 bp from COⅠ gene were obtained and the online BLAST result showed more than 99% of similarity. The phylogeny tree showed that the necrophagous flies could cluster well into 9 groups, which was consistent with morphological identification results. The intraspecific difference in 28S rRNA was 0 and the interspecific difference was 0.001-0.033. The intraspecific difference in COⅠ was 0-0.008 and the interspecific difference was 0.006-0.101. Conclusion Combined use of 28S rRNA and COⅠ gene sequence fragments can effectively identify the nine Calliphorid flies in this study. However, for closely related blowfly species, more genetic markers should be explored and used in combination in future.
Animals ; DNA, Mitochondrial/genetics* ; Diptera/genetics* ; Phylogeny ; RNA, Ribosomal, 28S/genetics* ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVETo demonstrate a noninvasive large mammalian genetic sampling method using blood meal obtained from a tabanid fly.

METHODSBlood meal was recovered from the abdomen of an engorged tabanid fly (Haematopota sp.) which was captured immediately after biting a Sumatran rhino in captivity. The blood was applied on to a Whatman FTA(®) blood card. Subsequent laboratory work was conducted to extract, amplify and sequence the DNA from the sample. Validation was done by sampling the hair follicles and blood samples from the rhinoceros and subjecting it to the same laboratory process.

RESULTSBLAST search and constructed phylogenetic trees confirmed the blood meal samples were indeed from the rhino.

CONCLUSIONSThis method could be used in the field application to noninvasively collect genetic samples. Collection of tabanids and other haematophagous arthropods (e.g. mosquitoes and ticks) and other blood-sucking parasites (e.g. leeches and worms) could also provide information on vector-borne diseases.

Animals ; Diet ; veterinary ; Diptera ; genetics ; physiology ; Endangered Species ; Female ; Food Chain ; Indonesia ; Insect Bites and Stings ; blood ; veterinary ; Molecular Sequence Data ; Perissodactyla ; Phylogeny ; Polymerase Chain Reaction ; veterinary ; Sequence Analysis, DNA ; veterinary

OBJECTIVE: To explore the application of a 289bp fragment of the 16S rDNA gene to identify various species of sarcosaphagous Calliphorid flies. METHODS: Twenty-six Calliphorid flies were collected from 14 Chinese provinces. All specimens were properly assigned into three genera and six species. The DNA of the pectoralis was extracted using CTAB method. Then PCR amplification was done for the 289 bp fragment of the 16S rDNA gene. The PCR products were then purified and sequenced, and the obtained sequences were uploaded to GenBank. The phylogenetic tree was built by the neighbor-joining method and intraspecific and interspecific divergences were calculated by sequence analysis. RESULTS: The above 26 sarcosaphagous flies could be well clustered according to different genera and species. The evolutional intraspecific values were all zero, the evolutional interspecific variations varied from 0.3% to 6.5%. CONCLUSION The 289 bp fragment of the 16S rDNA of sarcosaphagous flies can be effectively used to identify most of the flies at species level. This method appears to be fast and low dissipative, which might be used to estimate postmortem interval by sarcosaphagous flies.
Animals ; DNA Primers ; DNA, Mitochondrial/genetics* ; DNA, Ribosomal/genetics* ; Diptera/genetics* ; Entomology ; Forensic Medicine/methods* ; Phylogeny ; Polymerase Chain Reaction/methods* ; RNA, Ribosomal, 16S/genetics* ; Rabbits ; Sequence Analysis, DNA ; Species Specificity

Species identification of sarcosaphagous insects is one of the important steps in forensic research based on the knowledge of entomology. Recent studies reveal that the application of molecular biology, especially the mtDNA sequences analysis, works well in the species identification of sarcosaphagous insects. The molecular biology characteristics, structures, polymorphism of mtDNA of sarcosaphagous insects, and the recent studies in species identification of sarcosaphagous insects are reviewed in this article.
Amino Acid Sequence ; Animals ; Base Sequence ; DNA, Mitochondrial/genetics* ; Diptera/genetics* ; Electron Transport Complex IV/genetics* ; Entomology ; Forensic Medicine/methods* ; Genes, Mitochondrial/genetics* ; Insecta/genetics* ; Polymerase Chain Reaction ; Polymorphism, Genetic ; RNA, Ribosomal/genetics* ; Sequence Analysis, DNA ; Species Specificity

OBJECTIVE: To compare effects of three different methods for mtDNA extraction from common sarcosaphagous insects including cetyl trimethyl ammonium bromide (CTAB) method, sodium dodecyl sulfate-potassium acetate (SDS-KAc) method and sodium dodecyl sulfate-proteinase K (SDS-PK) method. METHODS: Seventy-two insects from four species [Chrysomya megacephala (Fabricius, 1784), Eusilpha bicolor (Fairmaire, 1896), Paraeutrichopus pecoudi (Mateu, 1954), Vespa velutina (Lepeletier, 1836)] were collected from the corpses of the rabbits in Changsha district. The total DNA of above samples was extracted by CTAB, SDS-Kac and SDS-PK methods. The purity and concentration of DNA were examined by protein-nucleic acid spectrophotometry, and mtDNA were amplified by specific primers and PCR products were detected by agarose gel electrophoresis. Then PCR products were sequenced and subsequently up-loaded to GenBank. RESULTS: mtDNA was successfully extracted with three methods from most of the samples. The SDS-PK method was better in DNA purity compared to other methods and the CTAB method was superior in extracting DNA from old samples, while SDS-KAc method showed no significant difference for extraction effects of different samples. CONCLUSION The most appropriate method should be chosen depending on different situations. SDS-PK method is expected to obtain high-quality DNA, while CTAB method is preferred in extracting obsolete samples. SDS-KAc method is low cost and can be used in various kinds of preliminary experiments.
Animals ; Coleoptera/genetics* ; DNA Primers ; DNA, Mitochondrial/isolation & purification* ; Diptera/genetics* ; Electrophoresis, Agar Gel ; Entomology ; Forensic Medicine/methods* ; Gene Amplification ; Insecta/genetics* ; Polymerase Chain Reaction/methods* ; Quaternary Ammonium Compounds/chemistry* ; Rabbits ; Reproducibility of Results ; Sequence Analysis, DNA ; Sodium Dodecyl Sulfate/chemistry*

OBJECTIVE: Using CO II sequences to identify common species of carrion-breeding flies and larvae. METHODS: flies and larvae were collected on the corpses of rats in Zhengzhou district, DNA was extracted, CO II sequences were amplified and sequenced. Clustalx and MEGA 4.0 software were used to analyze the gene sequences and to construct the phylogenetic trees. RESULTS: There was no significant gene difference between adults and larvae. COII gene sequences could be used to identify Boettcherisca peregrina, Aldrichina grahami and Lucilia illustris but they could not distinguish Lucilia cuprina from the Lucilia sericata because of their close evolutionary distance and single nucleotide polymorphisms in aldrichina grahami and Lucilia illustris populations were found. CONCLUSION CO II sequence of mtDNA in Zhengzhou district can be used effectively to identify some common species of carrion-breeding fly. The method is simple and accurate.
Animals ; Base Sequence ; China ; DNA Primers ; DNA, Mitochondrial/genetics* ; Diptera/genetics* ; Electron Transport Complex IV/genetics* ; Entomology ; Forensic Medicine/methods* ; Genes, Insect ; Larva/genetics* ; Phylogeny ; Polymerase Chain Reaction/methods* ; Rats ; Sequence Analysis, DNA ; Species Specificity

Sarcosaphagous insects are very important to investigate some criminal cases. They are significant useful in estimating post-mortem interval (PMI) and corpse transfer post-mortem. Lucilia are very common sarcosaphagous insects. They like sunshine and are usually the earliest to touch the cadaver. These characteristics and others such as the stages of their larvae development can offer good evidences for criminal case investigation. This paper summarizes details of their application for estimating postmortem interval in recent years and reviews the methods to identify species and to determine the age of adult Lucilia with molecular biology and entomological morphology.
Animals ; DNA, Mitochondrial/genetics* ; Diptera/physiology* ; Entomology/methods* ; Feeding Behavior ; Forensic Medicine/methods* ; Larva/physiology* ; Polymerase Chain Reaction/methods* ; Postmortem Changes ; Seasons ; Sequence Analysis, DNA ; Species Specificity ; Weather

OBJECTIVE: To explore mitochondrial DNA (mtDNA) extraction effects of different parts from sarcosaphagous insects using improved cetyltriethylammnonium bromide (CTAB) method. METHODS: Thirteen Lucilia sericata (Meigen) and 13 Nicrophorus fossor (Erichson) were collected from the corpses of rabbits placed on the outdoor lawn in Huhehot district. Four parts (head, chest muscle, legs and wings) of insect were collected, and the mtDNA of all samples were extracted using CTAB method. The purity and concentration were tested using protein and nucleic acid spectrophotometry. The integrity of the extracted mtDNA and PCR products were checked by agarose gel electrophoresis. The PCR products were sequenced and the obtained sequences were imputed into GenBank for comparison. RESULTS: mtDNA were successfully extracted from 10 head samples, 6 legs samples, 4 wing samples and 13 chest muscle samples of the Lucilia sericata (Meigen). Also, mtDNA were successfully extracted from 5 head samples, 8 legs samples, 3 wing samples and 13 chest muscle samples of the Nicrophorus fossor (Erichson). CONCLUSION mtDNA can be obtained from chest muscle and other parts of sarcosaphagous insects using the improved CTAB method.
Animals ; Coleoptera/genetics* ; DNA, Mitochondrial/isolation & purification* ; Diptera/genetics* ; Electron Transport Complex IV/genetics* ; Electrophoresis, Agar Gel ; Entomology ; Forensic Medicine/methods* ; Polymerase Chain Reaction/methods* ; Rabbits ; Sequence Analysis, DNA ; Species Specificity