Molecular Identification of Necrophagous Muscidae and Sarcophagidae Fly Species Collected in Korea by Mitochondrial Cytochrome c Oxidase Subunit I Nucleotide Sequences

Identification of insect species is an important task in forensic entomology. For more convenient species identification, the nucleotide sequences of cytochrome c oxidase subunit I (COI) gene have been widely utilized. We analyzed full-length COI nucleotide sequences of 10 Muscidae and 6 Sarcophagidae fly species collected in Korea. After DNA extraction from collected flies, PCR amplification and automatic sequencing of the whole COI sequence were performed. Obtained sequences were analyzed for a phylogenetic tree and a distance matrix. Our data showed very low intraspecific sequence distances and species-level monophylies. However, sequence comparison with previously reported sequences revealed a few inconsistencies or paraphylies requiring further investigation. To the best of our knowledge, this study is the first report of COI nucleotide sequences from Hydrotaea occulta, Muscina angustifrons, Muscina pascuorum, Ophyra leucostoma, Sarcophaga haemorrhoidalis, Sarcophaga harpax, and Phaonia aureola.


Introduction
The postmortem interval (PMI) is a key piece of information that needs to be determined in the investigation of a death. In fresh bodies, early postmortem changes such as body cooling, rigidity, and lividity are used for the estimation of PMI [1]. In putrefied bodies, however, these early changes cannot be used for PMI estimation, and it is not possible to estimate PMI from the degree of putrefaction [1]. As a result, PMI estimation in putrefied bodies is one of the most difficult tasks for forensic scientists and pathologists.
Many kinds of arthropods, especially insects belonging to the orders Diptera (flies) and Coleoptera (beetles), are attracted to the bodies of dead animals. Flies, particularly blow flies (Family Calliphoridae), are typically the first to arrive and oviposit into animal carcasses [2]. In addition to blow flies, 2 other families, Muscidae (house flies and allies) and Sarcophagidae (flesh flies), are important in forensic entomology. Although house flies are not commonly attracted to putrefied meat as blow flies and flesh flies are, they are often important indicators of PMI particularly in indoor deaths [2]. When larvae or pupae in various stages of development are collected from the site of investigation and the growth rates of samples are known, an approximate time of oviposition or larviposition can be estimated [3]. Species identification is essential for determining growth rates, as these rates are species-specific [2]. Therefore, species identification is a key step in estimating the PMI from entomological evidence. The traditional species identification method is dependent on the morphological features of insects and is not easily applicable to immature samples such as eggs, larvae, and pupae [4][5][6][7][8][9]. Moreover, only a few expert taxonomists specialize in forensically important insect species, not only in Korea but also worldwide. DNA-based approaches have been developed in an effort to improve accessibility to methods of species identification. Sperling et al. developed   [15] and Kano et al. (1967) [4] and by Pape (1996) [17], respectively. a method to identify 3 forensically important fly species by using the mitochondrial cytochrome c oxidase subunit I (COI) gene and its flanking loci [10]. Although mitochondrial COI nucleotide sequence analysis frequently yields specieslevel or even genus-level paraphylies in forensically important flies, this locus is still used as the standard method of identification [11,12]. Two previously reported studies have used the full-length DNA of the COI gene for Calliphoridae species in Korea [13,14]. However, there has been little effort to characterize the COI haplotypes of Korean Muscidae and Sarcophagidae fly species. This study examined the fulllength nucleotide sequences of the COI gene of 10 Muscidae and 6 Sarcophagidae fly species collected in Korea.  [4,[15][16][17]. Taxonomic information and the sample sizes of the flies analyzed are listed in Table 1. Flies were first frozen in liquid nitrogen, and the whole bodies were ground using a SKMILL-200 (Tokken, Chiba, Japan). Genomic DNA was extracted from the ground samples by using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Polymerase Chain Reaction (PCR) and Automatic
Sequencing. Universal primer sequences for the COI gene were taken from the literature (Table 2) [13,14,[24][25][26], and PCRs were performed using a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The PCR reaction conditions consisted of an initial denaturation step at 95 ∘ C for 11 min, followed by 35 cycles at 95 ∘ C for 30 s, 50 ∘ C for 1 min, and 72 ∘ C for 1 min, and then a final elongation step at 72 ∘ C for 15 min. Each reaction mixture was The Scientific World Journal 3  The Scientific World Journal  The sequencing products were analyzed using an ABI 3730xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Assembled sequences were deposited into the NCBI GenBank database (JX861406-JX861482).

Phylogenetic Analysis and Sequence Comparison.
Phylogenetic trees were generated for 2 fly families by using the maximum likelihood method with 1,000 replicates of bootstrapping based on the Tamura-Nei model using MEGA6 software [27]. Initial trees for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. To make a root for each tree, COI sequences for Lucilia sericata (NCBI accession number EU880212), Calliphora vicina (EU880188), and Drosophila melanogaster (NC 001709) were introduced as outgroup taxa. Average intraspecific and interspecific sequence distances were calculated for sequence comparison. Sequences obtained in this study were also compared to previously announced sequence data (Table 3).

Nucleotide Sequence Distances.
A pairwise percentage distance matrix of 10 Muscidae fly species is shown in Table 4. Because only 1 individual COI sequence was obtained for H. occulta, intraspecific variation was not estimated for this species. Interspecific distance was the lowest between O. chalcogaster and O. leucostoma (6.3%) and the highest between Musca domestica and Phaonia aureola (15.3%). Intraspecific distances were 0.3% or less. A pairwise percentage distance matrix for the 6 Sarcophagidae fly species is shown in Table 5. Interspecific distance was the lowest between Sarcophaga similis and Sarcophaga peregrina (6.4%), whereas it was the highest between Sarcophaga haemorrhoidalis and S. peregrina (8.9%). Intraspecific distances were 0.3% or less.

Phylogenetic Analysis.
Maximum likelihood phylogenetic trees were generated from COI nucleotide sequences of 10 Muscidae and 6 Sarcophagidae fly species. All taxa were clustered according to species and genera, without any species-or genus-level paraphyly (Figures 1 and 2). Although a few internal nodes display low bootstrap values under 50%, every bootstrap value at the species level was 100%.
Because only 1 H. occulta COI sequence was identified in this study, and there are currently no COI sequences from this species in the NCBI GenBank, it is impossible to determine the validity of this sequence. As expected, however, H. occulta formed a genus Hydrotaea clade with H. dentipes (Figure 1). Previously reported sequences from H. cyrtoneurina, H. irritans, and H. dentipes in the NCBI GenBank (Table 3) showed interspecific distances of at least 7.4% compared with the H. occulta sequence determined in this study [18].
M. domestica, the common house fly, exhibits a cosmopolitan distribution [6]. The COI gene has been widely studied in this species, and 28 COI sequences of this species from the NCBI GenBank (Table 3) are highly homologous to conspecific sequences in this study (average distance = 0.2%) [19,20].
As reported by Shinonaga, 5 species of the genus Muscina have been identified in Japan [6]. Three of these species were analyzed in this study. Of these, M. stabulans (stable fly) is the most forensically important species, and it is more often attracted to decaying animals than are other Muscina flies [6]. relatively straightforward. Compared to previously reported conspecific data, in this study, M. stabulans sequences were very similar to 2 previously reported conspecific sequences (EU627711 and AJ879595; sequence distance 0.1-0.3%) but very divergent from another reported sequence (EF531210; sequence distance 5.0-5.1%) [21]. Because only EF531210 is inconsistent with other conspecific sequences, the validity of this sequence should be reviewed by analysis of the voucher specimen and the morphological features used for identification. The M. assimilis sequence (EU627712) does not match any Muscina sequences reported in this study.
Three Ophyra species were analyzed in this study, each with low intraspecific distances and at least 6.3% interspecific distances. Therefore, identification of these 3 Korean Ophyra species is plausible. Compared to previously reported conspecific sequences, the O. nigra sequence obtained in this study was monomorphic with EU627714 (distance 0.3%), whereas O. chalcogaster showed distances of 1.2-1.3% from EU627715. Since the O. leucostoma COI gene has not previously been analyzed, conspecific comparison is not possible at this time.
There are no nucleotide sequences in the NCBI GenBank database that match the O. leucostoma sequences reported in this study.
S. haemorrhoidalis showed a very low intraspecific average sequence distance (0.1%) and interspecific distances of at least 6.8% (Table 5). There are currently no other COI nucleotide sequences in the NCBI GenBank for this species name. However, a COI sequence of a synonymous species, Sarcophaga africa (GQ223343), is available [17]. Since the sequence distance between S. haemorrhoidalis and S. africa is only 0.8%, the DNA result also supports that they are conspecific.
S. peregrina sequences in this study showed a very low intraspecific average sequence distance (0.1%) and interspecific distances of at least 6.4% (Table 5). Because S. peregrina was once categorized in the old genus Boettcherisca, a phylogenetic tree was generated from S. peregrina sequences in this study and the COI sequences of old genus Boettcherisca submitted by other authors. The phylogenetic tree showed a species-level paraphyly of S. peregrina, with 2 Malaysian S. peregrina sequences, submitted by Tan et al., clustering with 2 Malaysian S. javanica sequences (Figure 3) [22]. Because these 2 Malaysian S. peregrina sequences are divergent from other conspecific sequences from Korea and China (sequence distance 2.4-3.0%), further consideration, such as a review of the voucher specimens, would be necessary.
Sarcophaga melanura showed a very low intraspecific average sequence distance (0.1%) and interspecific distances of at least 6.5% (Table 5). Compared with the 6 short S. melanura COI sequences shown in Table 3, the S. melanura COI sequences reported in this study showed intraspecific distances of only 0.0-0.7% [23].
In conclusion, 10 Muscidae and 6 Sarcophagidae fly species collected in Korea were identifiable using COI sequence analysis. However, a few inconsistencies with previously reported sequences require further evaluation. To our knowledge, the present study provides the first report of the COI nucleotide sequences of H. occulta, M. angustifrons, M. pascuorum, O. leucostoma, S. haemorrhoidalis, P. harpax, and P. aureola.