Species Identification Of Necrophagous Blowflies Using Molecular Marker And Forensic Medicine Application

PART I: Application of mtDNA cytochrome oxidase subunit I gene inspecies identification between common sarcosaphagous fliesObjective To identify species of sarcosaphagous flies by analysisbased on498bp and841bp region of the gene for cytochrome oxidasesubunit I (COI) encoding region of mtDNA and provide assistance for theforensic science application.Methods Sarcosaphagous fly samples in different regions belongingto2families,4genera and6species were collected for morphologicalindentify. MtDNA was extracted and the PCR amplification reaction wasoptimized by two pairs of primers in COI gene. The PCR products wereobtained and purified through agar gel electrophoresis and sequenced onABI Model377DNA sequencers. Sequences of498bp and841bp in COIgene were acquired by multiple-alignment DNAMAN (version6.0) andthe sequence alignments were disposed. BLAST search and homologyalignment was carried out in the GenBank database. Phylogenetic treeswere constructed by the MEGA5.1package. Results28of30sarcosaphagous fly samples collected from5different regions were identified in correct alignment rate of93.33%bysequence I (489bp) of COI gene. The divergence of sequence I within andbetween species was0.1%～1.6%and2.2%～11.2%respectively.Different species can be identified successfully except2samples of luciliacuprina (Wiedemann) and lucilia sericata (Meigen).35of36sarcosaphagous fly samples collected from3diffrent regions wereidentified in correct alignment rate of97.22%in sequence II (841bp) ofCOI gene. The divergence of sequence II within and between species was0.2%～1.5%and2.7%～10.6%respectively. All the different species canbe identified successfully by the phylogenetic trees.Conclusion Methods of sequence homology alignment andphylogenetic tree of COI gene was an important supplementary means inthe species identification of sarcosaphagous flies, which realized the rapid,accurate and easy identification in forensic science application. PART II: Effect of feeding on different tissues on larva developmentof Chrysomya megacephala (Diptera: Calliphoridae)Objective To observe the effect of dietary factors on the developmentof Chrysomya megacephala larvae and provide assistance for the entomology research and estimating of post mortem interval (PMI) inforensic science application.Methods The pig’s brain, heart, liver and kidney were collected tomake different substrates for the subsequent experiment. Fresh pure leanpork and subcutaneous fat was minced and mixed for5groups substratesand the proportion of fat in the mixture was0%,10%,30%,50%,80%byweight. The Chrysomya megacephala larvae were reared on the differentsubstrates at a constant temperature of25℃. Length and weight of larvaeand pupae were measured at12h interval16h after eclosion.10larvae orpupae were collected each time. The time of development, mortality, andsex ratio of adults were recorded for statistical analysis of the5replicatedexperiments.Results Variation in the type of tissue that larvae feed on canproduce marked differences in developmental rate (P<0.01). The larvaefed on liver grew slowly and the time of reaching maximum length andweight was delayed for about36hours compared with other groups(P<0.01). Duration of second instar, third instar and total larvadevelopment was longer than that of other groups (P<0.01). More dietaryfat content was beneficial for development of larvae in the early stage butit was adverse in the later larval stages (P<0.01). Larvae fed on high-fatdiet reached the wandering stage earlier and the mortality of larvae andpupae was also hiher (P<0.01). There was no significant difference in the sex ratio between the four groups (P﹥0.05).Conclusion The body size, development duration and mortality ofChrysomya megacephala were different due to feed on the different tissuesor diets. This results should be considered in the entomology experimentsand forensic science application. PART III: Determination of clozapine in samples of Chrysomyamegacephala by SPME-GC-MS methodObjective To explore and establish effective and reliably quantitativedetection methods of clozapine in samples of necrophagous flies so as tooffer a new detection methods and forensic medical analysis strategies forthis kind of poisoning cases.Methods Method including standard curve of Solid-phasemicroextraction (SPME)-Gas Chromatography-Mass Spectrometry (GC-MS) was established to determine clozapine concentration in samples.Clozapine was extracted by100m polydimethylsiloxane (PDMS) fiber.Loxapine was used as internal standard and detected by GC-MS inselected ion monitoring (SIM). Samples of Chrysomya megacephala fedon different diet substrate which added a certain amount of clozapine wasdetected and statistical analyzed at different developmental stages including larvae and pupae.Results The analytical results showed that SPME-GC-MS can beused in the samples detection and the detection limit of clozapine was0.1ng/mL. The standard curve was linear in the range of5～5000ng/mL. Theaverage recovery of clozapine in the range of93%～104%in differentconcentration. The precisions (RSD) of within-day and between-day wasless than8%. The concentration of CLP in samples was related with theconcentration of diet which Chrysomya megacephala fed on.Conclusion This method was validated with respect to simplicity,accuracy and precision and provided a new potential effective detectionstrategies. It can be used for in necrophagous flies samples in this kind oftoxic poisoning cases.