Identification of Forensically Important Flesh Flies Using the Cytochrome C Oxidase Subunits I and II Genes.

Forensically important flesh flies (Diptera: Sarcophagidae) often are not morphologically distinguishable, especially at the immature stage. In addition, female flies are quite similar in general morphology, making accurate identifications difficult. DNA-based technologies, particularly mitochondrial DNA (mtDNA), have been used for species-level identification. The cytochrome oxidase subunits I and II (COI-COII) sequences of Iranian Sarcophagidae are still unavailable in GenBank. In this study as many as 648 (540 males and 106 females) fly specimens from family Sarcophagidae, representing 10 sarcophagid species, including eight forensically important species were collected from seven locations in five Iranian provinces. Of these, 150 male specimens were identified based on both morphology of male genitalia and DNA sequencing analysis. Sequence data from the COI-COII regions for 10 flesh fly species collected in Iran were generated for the first time. Digestion of COI-COII region by restriction enzymes RsaI, EcoRV, and HinfI provided distinct restriction fragment length polymorphism profiles among the species and can serve as molecular markers for species determination. Phylogenetic analysis represented that the COI-COII sequences are helpful for delimitation of sarcophagid species and implementation in forensic entomology. However, the application of the COI-COII fragment as a species identifier requires great caution and additional species and markers should be studied to ensure accurate species identification in the future.

[Identification of common Sarcophagidae by short cytochrome oxidase I fragment].

OBJECTIVE: To identify the common Sarcophagidae with a 278 bp fragment of cytochrome oxidase I in mitochondrial DNA and to obtain an unambiguous and rapid identification method for Sarcophagidae in forensic investigations. METHODS: Nineteen Sarcosaprophagous flies were collected from 16 locations in 12 Chinese provinces. All specimens were comprised of 4 species. The mtDNA of flies was extracted with SDSPK extraction method. Polymerase chain reaction was conducted in an Eppendorf 5331 thermal cycler. The PCR products were purified and sequenced and the obtained sequences were uploaded to GenBank. A neighbor-joining tree was constructed with MEGA4.0 package. RESULTS: The 19 Sarcosaprophagous flies were well clustered. The intraspecific variation within species varied from 0% to 3%, while the interspecific variations between species varied from 8% to 12%. CONCLUSION: Congeneric species can be separated by the short fragment (278 bp region in the cytochrome oxidase subunit I gene), which will be instrumental for implementation of the Chinese Sarcophagidae database and lay a foundation for post mortem interval estimation in future forensic cases.

Rapid cold-hardening blocks cold-induced apoptosis by inhibiting the activation of pro-caspases in the flesh fly Sarcophaga crassipalpis.

Apoptosis plays important roles in the selective elimination of sub-lethally damaged cells due to various environmental stresses. The rapid cold-hardening (RCH) response protects insects from the otherwise lethal consequences of injury due to cold-shock. We recently demonstrated that cold shock induces apoptotic cell death in insects and that RCH functions to specifically block cold-shock-induced apoptosis. In the present study we used isolated fat body, midgut, muscle, and Malpighian tubules from adult flesh flies Sarcophaga crassipalpis to test the following hypotheses: (1) cold-induced apoptosis varies among different tissues and (2) RCH blocks the apoptotic pathway by preventing the activation of pro-caspases. Cold-shock induced substantial amounts of apoptotic cell death that matched with tissue damage as determined using vital dyes. RCH treatment significantly reduced apoptotic cell death in all tested tissues. Caspase-3 (executioner) activity was 2-3 times higher in the cold- and heat-shocked groups than in control and RCH groups. Likewise, the activity of caspase-9 (initiator) showed a similar trend as for caspase-3 in all tissues but midgut. In addition, cold-shock and heat-shock treatments also increased caspase-2 activity 2-3 folds in both soluble and membrane fractions of fat body and muscle extracts compared to controls.