The Investigation Of Constitution And Succession Of Flies Community And The Study Of Molecular Markers Of Their MtDNA In The Identification Of Forensically Important Species

Objective In order to determine the constitution and succession of sarcosaphagous flies community appeared on some animal carcasses in Hohhot district in the summer and autumn; To solve the problems of identification of Sarcosaphagous flies and their larvae and pupae and eggs, for which all of eggs and pupae and most of larvae could not be identified only using their morphological features, analysis was based on each a 278 bp and a 635 bp and a 513bp region of the gene for cytochrome oxidase subunit â…  and â…¡ (CO â…  and CO â…¡) and 16SrDNA encoding region of mtDNA. At the same time, to solve the problems of deduce the region that the geographical species of Sarcosaphagous flies come from by analysis the relations of correlate and regression between hereditary and latitude interval was based on the gene of mtDNA of sarcosaphagous flies. It was help to be sure their geographical species and further to identify the Sarcosaphagous flies and determine the post mortem interval (PMI) and scene of crime (SOC) for forensic doctors.Methods Sixteen rabbits and one dog were killed and some bowels of chickens and fishes were placed outdoors at different time from July to October in two years in Hohhot district. The constitution and succession of sarcosaphagous flies community that appeared on the above cadavers were discovered and recorded; Specimens were collected on the corpses of rabbits on the grassland in the Huhhot and Chengdu district. The mtDNA of flies was extracted using the Improvement in grinding tissue during extracting DNA from small insects technique. Reactions were conducted on a Perkin-Elmer 9600 thermal cycler. PCR products were investigated by horizontal geloseelectrophoresis and vertical non-denaturing polyacrylamide electrophoresis and followed by silver stain and purified using the Nucleic Acid Purification Kit (Shenergy Biocolor Co. Shanghai). Sequences of both strands were obtained by direct sequence of the double-stranded PCR product using one of the PCR primers and the ABI PRISM Big Dye Terminator Cycle Sequencing Kit. Sequence reactions were electrophoresed on ABI Model 377 DNA Sequencers. Using the multiple-alignment program DNAMAN, version 4.0 analyses, carried out the sequence alignments. A neighbour-joining tree using the Tamura and Nei model of nucleotide substitution was constructed using the MEGA2.1 package. At the same time, each a 278 bp and a 635 bp region of the gene for CO â…  and CO â…¡ encoding region of mtDNA of sarcosaphagous flies( include of 23 Drosophila simulans for CO â…  collected from other district abroad and 10 Lucilia sericata and Lucilia cuprina for CO â…¡ from Huhhot and Chengdu district and abroad) was indexed on the GenBank of Medline by Blast path. A neighbour-joining tree using the Tamura and Nei model of nucleotide substitution was constructed using the MEGA2.1 package. The number of inherit intervals of inner-species of different Sarcosaphagous flies above all were analyzes by Kimura's two-parameter model and used for construction the relations between hereditary and latitude interval by SPSS soft and further to determine the relations of correlate and regression of them.Results It showed that there are 10 main species belonging to 3 families and 8 genera were from Diptera, including Musca domestica, Ophyra capensis, Hydrotaea armipes, Muscina stabulans from Muscidae; Lucilia sericata, Chrysomya megacephala, Lucilia cuprina, Aldrichina grahami fromCalliphoridae; Boettcherisca peregrina, Parascarcophaga crassipalpi from Sacrophagidae. Besides, it showed that the sarcosaphagous flies were the first community of sarcosaphagous insects and the obvious regulations of the constitution and succession of sarcosaphagous flies community appeared on above animal carcasses at different postmortem interval; each a 278 bp and a 635 bp region of the gene for CO I and CO II encoding region of mtDNA of sarcosaphagous flies collected from Huhhot and Chengdu district was showed less than 1% sequence divergence within species and about 8% divergence between species. In contrast, with the low level of sequence divergence of sister species, the data obtained from the method of CO â…  and CO â…¡ could not distinguish among taxa from the same species group, i.e. The species within the Lucilia sericata and Lucilia cuprina. But the 513bp region of the gene of 16srDNA was analyzed and distinguished them successfully. In the study of identification of the regions geographical species coming from, it showed the relations between inherit and latitude interval for 10 Lucilia sericata for CO â…¡ from Huhhot and Chengdu district and abroad. Rather than for 23 Drosophila simulans for CO â…  collected from other district abroad and 10 Lucilia cuprina for CO â…¡ from Huhhot and Chengdu district and abroad. Conclusion It may be useful for determining time, manner and place of death for forensic doctors in Hohhot district; each a 278 bp and a 655 bp region of the gene for CO â…  and CO â…¡ encoding region of mtDNA of sarcosaphagous flies can be effectively used for identification of most of them to species level. Owing to the speed, easy and accuracy of current nucleotide sequencing technology, it is likely to enable the reliable identification of sarcosaphagous flies. The 513bp region of the gene ef 16SrDNA of sarcosaphagous flies can be effectively used for identification of most of them in species level. It is likely to the reliable identification of sarcosaphagous flies as a complementary method of analysis the 278bp and a 635bp region of the gene for CO â…  and CO â…¡ encoding region of mtDNA. In the study of identification of the regions geographical species coming from, it was not showed the relations between inherit and latitude interval for all above sarcosaphagous flies and need further to hard work at it.