Identification Of Common Sarcosaphagous Sarcophagid Flies Based On Molecular Markers And The Establishment Of Local Genbanks

IntroductionThe determination of the postmortem interval (PMI) has always been the major emphasis and difficulty in criminal cases detection. The value of forensic entomology for PMI in practical forensic work has been well demonstrated in many case studies, research articles and books by entomologists and forensic investigators. Many sarcophagid species have the potential to estimate the PMI. Accurate and fast identification of sarcophagid species is an initial and crucial step of using entomological evidence in forensic cases. The utility of the forensically important Sarcophagidae (Diptera) for crime scene investigation has been severely restricted, as morphological identification is difficult, especially the identification of females and larvae. To implement the use of sarcophagids for PMI estimation, a method for easy yet accurate species-level identification at any life stage is required. In recent years, the development of molecular biology has been the hope of sarcophagid species identification. Molecular researches of the forensically significant flies have become a routine complement to traditional morphological method. Entomological evidence must be evaluated on a regional scale and the creation of local databases with referred ecological data for sarcophagids identification is strongly recommended. However, molecular data available with respect to the sarcophagid flies are very limited in China. To the best of our knowledge, few studies on genetic identification of Sarcophagidae in China have been published yet. To research the utility of DNA markers for identification of forensically important sarcophagid species in China, and simultaneously to accumulate genetic data for the global study on Sarcophagidae is the key problem to be solved.ObjectivePerforms sarcophagids samples collection and investigate the species and distribution of the common sarcophagids in China. To improve and optimize the DNA extraction method of Sarcophagidae, investigate the evaluation potency of different gene segments. Combined analysis of molecular identification will be studied. Screen out appropriate molecular markers and set up Sarcophagidae Genbank in China. Try to apply forensically important sarcophagid flies and DNA-based identification into forensic practice, and estimate the PMI in real cases.MethodsSamples of sarcophagid flies were trapped by rabbits, dogs and pigs in representative regions of China according to different latitude. Collected samples were identified and numbered by traditional morphological characters by entomologist, then preserved. To obtain enough DNA using improved DNA extraction method from a few tissues of fly. Explore published primers worldwide, and use primer5program to design primers for short fragments according to mtDNA locus like COâ… ,COâ…¡,16SrDNA, ND1,Cytb. Amplify by PCR Kit made by Promega Corporation. Test DNA and PCR products by means of conventional agarose gel electrophoresis. Amplification products were purified by Gel Extraction Kit and analyzed by dideoxy-chain termination method of Sanger. All resultant sequences were aligned using Chromas v1.43, ClustalW, and made cluster analysis by Mega v4.0, then choose DNA markers according to the analysis result. Upload gene data of Sarcosaphagous sarcophagids collected from different regions with various climates to GenBank to accumulate genetic data in China. Maggots collected in case crime scene will be identified by DNA marker. Then, try to estimate PMI based on published thermobiological and growth data. To improve the process and approaches of Sarcosaphagous sarcophagids presented as evidence in court.ResultsSarcophagid samples, including Boerttcherisca peregrine, Helicophagella melanura, Parasarcophaga albiceps, Parasarcophaga dux, and Phallosphaera gravely, were collected from29regions of18provinces between2009and2011. After morphological identification,272specimens were finally selected and numbered. Improved SDS-PK method can extract DNA from thorax, head, leg and wing of Boerttcherisca peregrine. When we use272-bp,278-bp COâ…  segments,289bp,555-bp16Sr RNA segments and196-bp,637-bp COâ…¡ segments to distinguish common Sarcosaphagous sarcophagids, the interspecies divergent usually over5%, some interspecies even over10%, but sometimes interspecies divergent between different species from same genus is below5%. The intraspecies divergent is usually below3%, many species is0. Errors occurred when ND1and Cytb sequences are used in species identification. For example, the interspecies divergent sometimes is less than the intraspecies divergent in the phylogenetic tree. It is beneficial to make use of combined analysis with a more reasonable interspecies and intraspecies divergent and a higher accuracy. The gene sequences, whose molecular identification is in accordance with morphological identification, are uploaded to GenBank in batches. The molecular method applied to the sarcophagid species identification is feasible. Sarcosaphagous sarcophagids serve forensic expertise in PMI evaluation with higher accuracy but relatively less stability due to multifactor.Conclusions1. Sarcophagid samples were collected by animal thermobiological studies in many provinces. The species and distribution of Sarcophagid flies were improved.2. SDS-PK method can extract high quality DNA with few tissues (such as one leg). The short fragments (200-600bp) of16Sr RNA, COâ…  and COâ…¡ can be used as supplementary means of morphological method in identification of sarcophagid species, as the technology used mtDNA is easier to perform and saving more time for forensic scientists within their routine work.3. Great amount of genes with regional representativeness have been uploaded into GenBank, which marks preliminary GenBank of sarcophagids in China have been set up and opens a window for foreign researchers to know about the molecular characters of Chinese Sarcosaphagous sarcophagids.4. It is possible to identify sarcophagid species quickly and exactly in real cases. Moreover, the problem of identity cognizance and PMI estimation can be solved in practice which pushes forensic entomology theory to a higher level.