Fundamental Research On The Application Of Molecular Identification And Biochemical Characters Of Necrophagous Flies In The Determination Of Postmortem Interval

This dissertation is to build a molecular method to identify flies species of forensic importance and a quick way to determine flies age using biochemical character of necrophagous flies. The results were shown as follows:1. common species identification by a DNA-based approachIdentifying an insect specimen is an important first step in a forensic-entomological analysis. However, diagnostic morphological criteria are lacking for many species particularly in their immature stage. We demonstrate a method for using mitochondrial COII gene sequences analysis to identify six common specimens of the flies likely to be collected from a human corpse in Hangzhou, China, based on two year surveys of the seasonal occurrence of carrion-breeding flies. The sequences of Lucilia sericata form the Genbank were used in comparing with different geographical populations. This technique also provides clear results when applied to the identification of the larvae.2. Identification early larvae of common carrion-breeding flies by two-dimensional gelelectrophoresis finger mapWith the application of 2-DE technology in biology science, the importance of constructing a proteome map is to be considered. Four protein map of early larvae of common carrion-breeding flies were obtained using 2-DE in the range of pH 4-7. After silver stained detection of sots, we find that there are marked difference among these maps. Isoelectric points and molecular weight analysis indicated that 2DE map be benificial to identificantion early larvae of common carrion-breeding flies which are confused in morphology.3. Detection of food source by PCR analysis of the gut contents of Aldrichina grahami(Aldrich) (Diptera: Calliphoridae) During Post-feeding Period.Maggot last meal may be useful in identifying host species. The DNA profile of host species was detectable in the "last meal". In this paper, mitochondrial DNA analysis of gut contents to identify the host of Aldrichina grahami (Aldrich) (Diptera: Calliphoridae) feeding on during post-feeding period. A modified logistic equations was fitted as the form: y=exp(a+b*x)/(l+exp(a+b*x)) to estimate the probability of detectable under five constanttemperatures (16, 20, 24, 28 and 32°C). Our results shows that detected time raged from a maximum of 24 h at 32°C to 42 h at 16°C and a minimum of 12h at 32°C to 30h at 16°C. Furthermore, the food detectable time was also calculated to give the maximal time after larva hatching from egg. These results indicate that, in criminal cases where the maggots away from the corpse, "last meal" should not be overlooked as potentially critical evidence.4. Biochemical character of four species of necrophagous flies during the development oflarvae and pupae.Under 24°C,the four species of flies arrived their maximum body weight for about 3 days after hatched from eggs , after that, the body weight decreased as Daily age increased. . A modified logistic equations was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the body weight and larval age. The weight of flies pupa decreased not obviously, and the linear equations were fitted the relationship between the body weight and the pupa age.Under 24°C,the four species of flies arrived their maximum protein content for about 3 days after hatched from eggs, after that, they changed slightly and the protein content decreased as Daily age increased. A modified logistic equations was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the protein content and larval age. The protein content of flies pupa decreased not obviously, and the linear equations were fitted the relationship between the protein content and the pupa age.Under 24°C,the four species of flies arrived their maximum carbohydrate content for about 4 days after hatched from eggs and after that they changed slightly. A modified logistic equations was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the carbohydrate content and larval age. The carbohydrate content of flies pupa decreased gradually, and the linear equations were fitted the relationship between the carbohydrate content and the pupa age.Under 24°C,the four species of flies arrived their maximum fucose content on different days ■after hatched from eggs, then the fucose content decreased as Daily age increased. A modified logistic equations was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the fucose content and larval age. The fucose content of flies pupa decreasedgradually, and the linear equations were fitted the relationship between the fucose content and the pupa age.Under 24°C,the four species of flies arrived their maximum lipid content for about 4 days after hatched from eggs. A modified logistic equations was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the lipid content and larval age. The lipid content of flies pupa decreased gradually, and the linear equations were fitted the relationship between the lipid content and the pupa age.5. Effect of temperatures on the biochemical characterUnder five constant temperatures, larval body weight increased with increasing age. While they arrived at their maximum body weight, it was decrease slightly. But the time protracted as decreasing temperatures. A modified logistic equation was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the body weight and larval age under five constant temperatures. The body weight decreased obviously during the early pupae period, after that they decreased slightly during the pupae development stage except for the quick decrease in the end of the pupae stage. The linear equations were fitted the relationship between the body weight and the pupa age.Under five constant temperatures, larval protein content increased with increasing age. While they arrived at their maximum value, it was decrease slightly. But the time protracted as decreasing temperatures. A modified logistic equation was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the protein contentand larval age under five constant temperatures. The protein content decreased slightly during the whole pupae stage. The linear equations were fitted the relationship between the protein content and the pupa age.Under five constant temperatures, larval carbohydrate content increased with increasing age. While they arrived at their maximum value, it was decrease slightly. Though the maximum values increased with the increasing temperatures, the time protracted as decreasing temperatures. A modified logistic equation was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the carbohydrate content and larval age under five constant temperatures. The carbohydrate content decreased slightly during during the early pupae period, after that theydecreased slightly during the pupae development stage except for the quick decrease in the end of the pupae stage. The linear equations were fitted the relationship between the carbohydrate content and the pupa age.Under five constant temperatures, larval lipid content increased with increasing age. While they arrived at their maximum value, it was decrease slightly. But the time protracted as decreasing temperatures. A modified logistic equation was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the lipid content and larval age under five constant temperatures. The lipid content decreased slightly during the early pupae period, after that they decreased slightly during the pupae development stage except for the quick decrease in the end of the pupae stage. The linear equations were fitted the relationship between the lipid content and the pupa age.6. Development profile of hemolymph soluble proteins during the larval and pupal stages intwo species of necrophagous fliesThe development profile of soluble hemolymph proteins was analyzed by the SDS-Polyacrylamide gel electrophoresis during the larval and pupal stages of two species of necrophagous flies which was kept at 24 °C. Several distinct component bands were resolved. The quantity of each protein band chaged with development in the larval and pupal stages acted a certain patter. The quantities of band a^ c, A> F and P were differentiated during the larval third instar stage and the quantities of band a^c^A,F and G were differentiated during the pupae stage. Hemolymph proteins from early larvae after hathed from eggs showed distinct age-specific electrophoretic profiles.7. Purification of storage protein of Parasarcophaga crassipalpis and antibody preparationStorage protein of Parasarcophaga crassipalpis while Hymolymph were conducted with (NH4)2SO4 precipitation, Hydrophobic Interaction Chromatography, Gel Filtration Chromatography, and ion exchange chromatography. After that, anti-storage protein rabbit serum was obtained after the purified storage protein injected. We also have purified the serum by purification kit.8. Storage protein content in two species of flesh flies during their development of larvae andpupae by ELISA and temperatures effected onThe result of ELISA detection shows: under five constant temperatures , the storage larval protein content increased obviously with increasing age and after arriving the maximum value of the storage protein content decreased with increasing age slightly. But the time they arrived the maximum value protracted as decreasing temperatures. A modified logistic equation was fitted as the form: y=exp(a+b*x)/(l+exp(c+d*x)) to estimate the relationship between the storage protein content and larval age under five constant temperatures.The lipid content increasing to their pupae maximum value in the early pupae period, after that they decreased slightly during the pupae development stage except for the quick decrease in the end of the pupae stage. The linear equations were fitted the relationship between the storage protein content and the pupa age.Flies of Parasarcophaga crassipalpis were reared under natural temperatures environment. According to the storage protein content of the larvae and pupae, we estimated the Daily age. It was indicated that it was more suitable for estimate the Daily age of larvae than pupae. 9. ConclusionForensic entomology as a new cross-subject depend on advance of new technology to a great extent. At present, it was considered that how to use the character, including the morphological and biochemical characters, is the most important work. And more accurate method such as species identification with DNA chip and age determination with protein chip, will be applied in this area in the future.