| BackgroundIn practical applications, forensic entomology is mainly used for the determination of the postmortem interval (PMI). There are two main ways to infer the PMI:Firstly, to study the growth and development laws of the sarcosaphagous insects on the corpses with the PMI. Secondly, to study the insects that are on the corpses and their corresponding succession to provide important clues for inferring the PMI. In different regions, the insect species and their living habits are not the same. China has a vast territory, and the entomological evidence from one region can not be used for the forensic practice in other regions directly. Therefore, creating local databases with related ecological data for the identification of sarcosaphagous insects is strongly recommended. Additionally, it is important to expand the investigations of various regions based on the previous research experience, to investigate the sarcosaphagous insect community succession, to find the dominant species of local regions and to study their growth and development laws. At that point we can establish local database and estimation system suitable for sarcosaphagous insects collected from different sites of Changsha. The rapid and accurate identification of sarcosaphagous insect species, especially the sarcosaphagous calliphorids, is a crucial step for estimation of the PMI. Calliphorids have important forensic significance, because they are usually the first colonizers, and then spawn and breed the next generations on the corpses. The obvious morphological characteristics to differentiate some closely related blowfly species or blowfly larvaes are still lacking. As a strong complement to the morphological identification method, molecular identification technique is a fast, easy, and effective method. Due to the diverse geographical environment in China, Calliphorids have a wide range of species, so more genetic resources still need to be collected for identification studies. It is necessary to collect calliphorid samples nationwide, and find some new molecular markers for calliphorid identification. Meanwhile, it is important to perfect the developmental duration tables and the growth curves of the sarcosaphagous flies as well as the succession data of the sarcosaphagous insects. However, such data are still scarce in China.Objectives1. To collect the calliphorid samples in multiple regions of China, and to investigate the species and distribution of some common calliphorids. To study the insect communities and their succession laws on the pig’s corpses in the region of Changsha in summer,.2. To identify the species of130calliphorids collected from28regions in China by278bp COI gene fragment and272bp COI fragment. To detect the ability of two gene fragments to identify some types of blowfly species. To set up and perfect the molecular marker system for Calliphoridae in China.3. To raise three dominant fly species and study their growth and development laws. To lay the foundation for local practice of forensic entomology.Methods1. Animal corpses are used to trap calliphorids in28regions of China in different latitudes and longitudes. Putting pig corpses at selected outdoor and indoor sites for succession experiment in Changsha in summer for three years.2. The primers for amplification of short COI fragment are designed by Primer5program, mtDNA of all samples are extracted by improved DNA extraction method for grinding tissues or Proteinase K+phenol/chloroform/isoamyl alcohol method, and mtDNA is amplified by the Promega PCR Kit. Conventional agarose gel electrophoresis is used for the detection of DNA and PCR products. Amplification products are purified by gel extraction kit and the purified products are analyzed by dideoxy-chain termination method of Sanger. Sequencing results are aligned by Chromas and clustering analysis is performed by ClustalW software. Finally, the sequences are analyzed by Mega4.0and phylogenetic tree is constructed. Gene data of sarcosaphagous calliphorids collected from different geographic regions and climate conditions are uploaded to GenBank database for accumulating Chinese calliphorid genetic data.3. L. sericata, C. Megacephala and B. peregrine are raised under the room-temperature in Changsha in summer. The body length and weight of the larvae, the length, width and weight of the pupae are measured, and the growth curves over the time are established.Results1. Calliphorids were trapped from28regions across17provinces in the country within three years, samples were obtained and preliminary morphological identification was executed.130samples of13calliphorid species were selected.2. Succession experiments were carried out at selected outdoor and indoor sites in Changsha in summer for three years.29sarcosaphagous species were found on the pig corpses belonging to three orders and15families respectively.3.278bp COI fragment and272bp COI fragment were used for the identification of all kinds of calliphoridae species collected all over the country. These results show that the fragments are suitable for identification of the sarcosaphagous calliphoridae species, especially some closely related calliphorid species.4. When the278bp COI fragments were used for identification, the intraspecific geographical differences were discovered in species L. sericata (Meigen,1826) and L. Caesar (Linnaeus,1758). And when272bp COI fragments were used for identification, the intraspecific geographical differences were discovered in species Chrysomya megacephala (Fabricius,1794) and Protophormia terraenovae (Robineau-Desvoidy,1830).5. Three sarcosaphagous fly species were successfully raised, i.e. L. sericata, C. megacephala and Boerttcherisca peregrine (Robineau-Desvordy,1830), and the growth charts of the larval body length and weight, fly pupae length, width and weight changes over the time were drawn. The results indicate that the larval body length and time relations coincide with cubic equation L=aT+bT2+cT+d and fly pupa weight is in line with the simple equation W=aT+b. The correlation was a significant difference (R2>0.9>,P<0.05).(L represents the length of the maggots, W represents the weight of the pupal, T represents the time, a, b, c and d represent constants).Conclusion1. A number of calliphorid samples are obtained in various districts around the country. The data concerning species and distribution of calliphorid flies are enriched.2. The sucession experiments have been carried out in Changsha region for three years, which will have practical forensic significance for the PMI estimation.3. Short fragments of COI (278bp and272bp) have the advantage of being fast, simple, economic and effective in identification of species. They are suitable for grassroot forensic workers.4. A large number of COI fragments have been uploaded into the GenBank database. The molecular maker system of calliphorid flies has been preliminarily set up in China.5. Some forensically important sarcosaphagous fly species have been raised, and a preliminary study has been done into the growth and development laws about them. Some basic and scientific data have been obtained. They will serve as theoretical basis for the forensic work. They will also provide methods for the future research of the growth and development laws of the sarcosaphagous flies. |
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