Use of Cytochrome c Oxidase Subunit I (COI) Nucleotide Sequences for Identification of the Korean Luciliinae Fly Species (Diptera: Calliphoridae) in Forensic Investigations

Blowflies, especially species belonging to the subfamily Luciliinae, are the first insects to lay eggs on corpses in Korea. Fast and accurate species identification has been a key task for forensic entomologists. Because conventional morphologic identification methods have many limitations with respect to forensic practice, molecular methods have been proposed to identify fly species of forensic importance. To this end, the authors amplified and sequenced the full length of the cytochrome c oxidase subunit I (COI) gene of the Luciliinae fly species collected in Korea. The results showed the COI sequences are instrumental in identifying Luciliinae fly species. However, when compared with previously reported data, considerable inconsistencies were noted. Hemipyrellia ligurriens data in this study differed significantly from two of the five pre-existing data. Two closely related species, Lucilia illustris and Lucilia caesar, showed an overlap of COI haplotypes due to four European sequences. The results suggest that more individuals from various geographic regions and additive nuclear DNA markers should be analyzed, and morphologic identification keys must be reconfirmed to overcome these inconsistencies.

Optimization of Wet Fixation Methods for AFM Imaging of Human Fibroblast Cells

We investigated the effect by the chemical fixative on human fibroblast cells (HFCs) in order to make nano-scale images using by the atomic force microscopy (AFM). The cell fixation needed to be optimized as prerequisite step for the preparation before analysis. AFM imaging after optimal wet fixation can provide practical, simple and fast technique for scanning living cells. In this study, AFM images - topography and amplitude - and the optic images of HFCs which were fixed with phosphate buffered saline (PBS), 2:1 ethanol:acetic acid, 4% glutaraldehyde and 37% formaldehyde were compared respectively. The final effect by washing with PBS or distilled water (D.W.) was examined after 4% glutaraldehyde fixation. To determine the optimal fixation method for HFCs, we performed quantitative and qualitative analysis by the height profile, the presence of artifacts and the morphology of well-conserved fibroblastic topography image by AFM. From AFM image which showed fibroblastic cellular morphology and differential height value of cytoplasm (670+/-47 nm, n=10) and nucleus (847+/-32 nm, n=10) in HFCs, we proposed that wet fixation by 4% glutaraldehyde, followed by final washing with PBS, could be the most suitable preparation for AFM imaging of HFCs, which enable us to approach easily on living cells with the least shrinkage.

Molecular Identification of Four Muscidae Species Collected in Korea using the DNA Barcode Region of Mitochondrial Cytochrome Oxidase Subunit I / 대한법의학회지

DNA barcoding was recently introduced to molecular identification of forensically important fly species. So, we have analysed the barcode region (687 nucleotides) of mitochondrial cytochrome oxidase subunit I (COI) gene for four species of Muscidae flies collected from Korea. The sequences were aligned and analysed to construct a phylogenetic tree using DNA Star 5.01(DNAStar Inc) and MEGA 3.1 program(Kumar, Tamura, Nei 2004). Intraspecific variation was not noted between M.stabulans individual to each other. Intraspecific variation ranges of other species were 0.1%, 0.1~0.3% and 0.1~0.6% for O.leucostoma, M.angustifrons and M.domestica, respectively. Interspecific percent distance was minimal(9.7~10.0%) between M.stabulans and M.angustifrons. Other species showed above 10% distance from each other. The result showed that four species of Muscidae fly species (Muscina angustifrons, Muscina stabulans, Ophyra leucostoma and Musca domestica) were identifiable from each other with analysis of barcode region of COI gene. Therefore, we conclude that species identification of forensically important Muscidae flies used in this study is possible with percent distance of sequences of COI barcode region, but more species and individuals should be examined to be confident about the conclusion.

Identification Blow Fly Species in Korea by Mitochondrial DNA 'Barcodes' / 대한법의학회지

Recently many forensic scientists are trying to use the DNA 'barcode' region (upstream portion of mitochondrial cytochrome oxidase subunit I) to identify the species of forensically important fly species. We have analyzed to compare their sequences of the 'barcode' region for twelve blow fly species[A. grahami, C.lata, C. vicina, H.ligurriens, L. ampullaceal, L. Caesar, L. illustris, P. sericata, P. regina, T. calliphoroides, C. megacephala, C. pinguis] collected from the rural and urban regions in Korea. Intra- and interspecies sequence divergences were calculated as 0~0.9% and 0.9~11.4%, respectively. Phylogenetic trees were drawn with Mega 3.1 and Network 4.20 programs. The result illustrates that each genus is grouped as monophyletic group except for T. calliphoroides and all the same species were monophyletic group. This suggests that the 'Barcode' region of COI gene could be a marker for identification of necrophagous blow fly species. But the two closely related species, L.illustris and L.caesar show little differences from each other. Therefore more individuals of these species should be examined for population study.

DNA-based Identification of Forensically Important Blow Fly Species in Korea; Aldrichina grahami, Calliphora lata, Calliphora vicina and Phormia regina / 대한법의학회지

Entomological evidence, especially necrophagous flies, are important in estimating postmortem interval in a putrefied corpse. Accurate and rapid species identification of eggs, maggots and pupae is required because growth rates and ecological characteristics are different among different species. But species identification of these immature stages of insects is difficult or impossible to even an expert entomologist. We tried to identify the necrophagous fly species using molecular data. Adult specimens of four forensically important blow fly species [Aldrichina grahami, Calliphora lata, Calliphora vicina and Phormia regina] were used for DNA extraction and sequences analysis of mitochondrial cytochrome oxidase subunit I (CO1) in this study. A total of 560 base pairs(bp) of the CO1 region was recovered using the newly designed specific primer pairs and was sequenced to compare it with those of same fly species registered in NCBI GenBank. The results presented in Table 2 to 6 demonstrate not only the potential utility of the COI sequence in interspecific discrimination, but also indicate that this sequence is probably not suitable for use with intraspecific studies, especially for dividing different local populations within the same species.

Molecular Identification of Six Necrophagous Fly Species (Family: Muscidae) by Mitochondrial Cytochrome Oxidase Subunit I / 대한법의학회지

Estimation of postmortem interval (PMI) in a putrefied corpse has been a long theme in the forensic medicine. Insects, especially necrophagous fly species are now utilized as indicators of PMI because the first visitors to a dead body are usually known to be blow fly species (Family Calliphoridae). House flies (Family Muscidae) are later visitors but they are very significant in forensic entomology because of their worldwide distribution. Entomologic evidences recovered from the scene are often immature individuals such as eggs, maggots and pupae. Because growth rates and ecological characteristics are different among fly species, accurate species identification is essential. As species identification in immature stages is very difficult or even impossible to an expert entomologist, many researchers are trying to identify fly species by molecular techniques. Authors analyzed 400bp of mitochondrial COI gene sequences of six Muscidae fly species (Fannia prisca, Muscina angustifrons, Muscina stabulans, Musca domestica, Hydrotaea dentipes and Ophyra leucostoma). In spite of limited number of flies analyzed in this study, all six fly species have different haplotype of COI gene and shows minimal intraspecific variation. This result shows that six fly species analyzed in this study can be discriminated each other by COI gene sequence analysis. But, more individuals from various geographic region should be analyzed to apply this result to a forensic entomology practice.

Comparison of Mitochondrial Cytochrome Oxidase Subunit I (CO I) Sequences of Five Blow Fly Species in Korea / 대한법의학회지

In criminal investigations, the accurate identification of specimens must be a vary important procedure in the estimation of post-mortem interval(PMI) using insect specimens. However, the morphological-based identification method has many limitations, as species identification for immature stages of many species being difficult or impossible, and more convenient methods are needed to identify necrophagopus fly species. So, many authors have proposed DNA-based methods for identifying fly specimen because of identifying all life stages of carrion flies. In this study, we sequenced a part of mitochondrial cytochrome oxodase subunit I (COI) for each five blow fly species [Hemipyrellia ligurriens, Lucilia illustris, Phaenicia sericata, Chrysomya. megacephala, Chrysomya pinguis] collected from the rural and urban regions in Korea, and compared 400 nucleotides of one species with those of another species. Percent similarity of sequence compared was highest value between Ch. pinguis and Ch. megacephala (97.8%), and lowest one between H. ligurriens and Ch. pinguis(89.2%). Maximun intraspecific variation was observed in L. illustris (0.5%). In conclusion, the experimental results indicate that sequence analyses of more necrophagous flies collected from various regions of Korea should be performed to construct a database of mitochondrial CO I of necrophagous fly species.