Development of the new microsatellite markers of Lucilia sericata (Diptera: Calliphoridae) from Korea.

BACKGROUND: Lucilia sericata is a medical and veterinary important insect species because its larvae feed on tissues of vertebrates including humans. Very few microsatellite makers have been reported from the species to illuminate its genetic variability and population genetic structure. METHODS AND RESULTS: In this study, L. sericata samples were collected from four different localities in Korea to develop the microsatellite markers to provide basic information on the genetic variability and population genetic structure in Korea of this species. In total, ten new microsatellite markers were sequenced and analyzed. Genetic diversity was performed using these microsatellite markers. The observed heterozygosity varied from 0.205 to 0.824, with an average of 0.546. The expected heterozygosity ranged from 0.579 to 0.886, with an average of 0.804. PIC value varied from 0.553 to 0.876. CONCLUSIONS: The markers developed in the present study are expected as informative for estimating genetic diversity of L. sericata.

The Growth Model of Forensically Important Lucilia sericata (Meigen) (Diptera: Calliphoridae) in South Korea.

Development of forensically important Lucilia sericata (Meigen) was analyzed in South Korea. Rearing was replicated five times at seven constant temperatures between 20-35 °C to elucidate changes in accumulated degree hours, based on developmental stage and body length, and 2673 individuals were statistically analyzed. The results indicated that the optimum temperature, the base temperature, and the overall thermal constant were 22.31 °C (±1.21 °C, 95% CI), 9.07 °C, and 232.81 ± 23 (mean ± SD) accumulated degree days, respectively. In the minimum ADH models of each development stage, nonlinear regression graphs were parallel at the immature stages. Based on the scatter plot (n = 973) of immature stages using ADH values and body length, the logarithmic model using Log10ADH as the dependent variable was identified as the best fitting regression model. Additionally, the adjusted R2 value and mean square of error were 0.911 and 0.007, respectively. This is the first forensically focused study on the development of L.sericata for the estimation of minimum postmortem interval in South Korea. In future studies, we intend to study the development of other necrophagous fly species and to identify parameters for the determination of age at post-feeding and pupal stages.

First report of the forensically important fly, Stearibia nigriceps (Diptera: Piophilidae) in South Korea: Confirmation of specimens from human corpses based on cytochrome c oxidase subunit I barcodes.

Piophilidae are a relatively small family of Diptera that is frequently associated with cadavers at advanced stages of decomposition and are, therefore, considered potentially useful forensic indicators. However, their use in forensic investigations is typically hampered by a deficiency in reliable identification tools. This is particularly evident in countries such as South Korea, where forensic entomology is still in its infancy and the diversity of forensically relevant insect taxa remains largely undocumented. In the present study, we used cytochrome c oxidase subunit I (COI) barcodes to identify samples of piophilid larvae collected during medicolegal investigations performed in South Korea. A total of 174 COI sequences were obtained and have been made publicly available, thus augmenting the reference barcode library for forensically important Piophilidae species. Of the 174 sequenced samples, 172 were identified as Stearibia nigriceps (Meigen), whereas the two remaining samples may represent a previously unsequenced piophilid species. Stearibia nigriceps is recorded from South Korea for the first time, and our results suggest that it might be a particularly relevant forensic indicator in certain case types and scenarios in that country. The findings of this study highlight the utility of COI barcodes for achieving accurate identification of entomological samples, even by non-specialist forensic practitioners. They also contribute to the further development and consolidation of forensic entomology in South Korea and eastern Asia.

The Application of Mitochondrial COI Gene-Based Molecular Identification of Forensically Important Scuttle Flies (Diptera: Phoridae) in Korea.

Phoridae are a family of necrophagous flies commonly found in indoor death scene. They account for approximately 19.7% of the entomofauna in human cadavers in Korea. Additionally, this taxon is an indicator of indoor hygiene, and these flies appear in environments where access by other necrophagous insects is difficult, such as enclosed rooms. Thus, they are likely to be used as forensic evidence. Despite their importance in forensic investigations and environmental hygiene, detailed studies on the taxonomy and molecular barcoding for this family are scarce, including in Korea. Because accurate taxonomic information regarding necrophagous insects collected from a death-related scene is essential during medicolegal investigations, molecular barcoding data could be useful as well as reliable. In this paper, full-length nucleotide sequences of genes coding for the cytochrome c oxidase subunit I (COI) in 79 Phoridae larvae collected from 20 medicolegal autopsy cases in Korea were phylogenetically analyzed by comparing their sequences to the foreign barcoding data of Phoridae. Six mitochondrial haplogroups were identified, which two of them matched to foreign Phoridae fly species haplotypes, Megaselia scalaris (Loew, 1866) and M. spiracularis Schmitz 1938. Taxonomies of five other haplogroups, with nucleotide distances ranging from 1.68% to 2.26% from the M. scalaris group, could not be confirmed solely based on the molecular barcoding data. Further research should be performed to determine whether these five haplogroups are diverged conspecifics of M. scalaris or a closely related sister cryptic species of M. scalaris.

Extended model for estimation of ambient temperature for postmortem interval (PMI) in Korea.

One of the most important factors for calculating the accumulated degree days (ADD), which is used to estimate the postmortem interval (PMI) is the accurate estimation of the scene temperature after death. As the estimation method used up to now, they applied to the linear regression analysis using the temperature of the nearest weather station, but the prediction error becomes larger in the case that the ambient temperature do not follow the temperature of the station. In the present study applied we have more accurate methods such as the quadratic regression model and support vector machine (SVM) and have included weather factors such as wind capacity, wind speed and humidity, and regression method. Also, we investigate the optimal statistical method for estimating the ambient temperature in indoor and outdoor locations in Korea.

Molecular Identification of Necrophagous Dermestes Species in South Korea Using Cytochrome c Oxidase Subunit I Nucleotide Sequences (Genus Dermestes).

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.

Comparative analyses of the three complete mitochondrial genomes from forensic important beetle genus Dermestes with phylogenetic relationships.

Necrophagous Dermestes species have high forensic importance in relation to the estimation of elapsed time since death or death season. To further supplement the genome-level features for related species, the complete mitochondrial genome (mitogenome) of Dermestes species D. essellatocollis, D. frischii and D. coarctatus are amplified, sequenced, annotated, analyzed, and compared with other twelve species of the infraorder Bostrichoidea. The mitochondrial genomes were typical circular molecules with 16,218, 15,873 and 15,873 bp in length, respectively. They included 13 protein coding genes, two rRNAs, and 22 tRNAs, as well as the putative control region. The gene orders and orientations are identical to those of other recorded bostrichiformian species and had the ancestral insect gene composition. Furthermore, phylogenetic analyses based on all the mitochondrial protein coding genes for 13 Bostrichoidea and 16 outgroup taxa were performed using Bayesian and Maximum Likelihood analyses. The inferred trees indicate that the genus Dermestes is monophyletic. The monophyly of infraorder Bostrichiformia is not supported. This study provides genomic data for mitochondrial genome library of the genus Dermestes to investigate evolutionary and systematic studies.

SNP Typing Using Multiplex Real-Time PCR Assay for Species Identification of Forensically Important Blowflies and Fleshflies Collected in South Korea (Diptera: Calliphoridae and Sarcophagidae).

Medicolegal entomology-a subfield of forensic entomology-is mainly used in medicolegal investigations to estimate the postmortem interval (PMI). The minimum PMI of a corpse invaded by necrophagous immature insects can be estimated because the PMI is near to or earlier than the oviposition time of the larvae that hatched and fed on the corpse. As the growth speeds of larvae differ depending on temperature and species, species-specific growth data are used to estimate the minimum PMI. While morphological identification of adult necrophagous flies can be done by a well-trained entomologist, identification of larvae is relatively difficult. Larvae can only be identified up to the family level and developmental stage by observing the posterior spiracles. For these reasons, the molecular biology method of DNA barcoding has been developed. DNA barcoding that targets the mitochondrial cytochrome c oxidase subunit I (COI) gene is commonly used. COI sequences are currently acquired using polymerase chain reaction (PCR) and Sanger sequencing, which are too time-consuming and complex for practical use in medicolegal investigations. To compensate for these limitations and facilitate the use of entomology for medicolegal investigation, we designed a multiplex real-time PCR system to identify nineteen forensically important species of Calliphoridae and Sarcophagidae flies collected in South Korea. In contrast to the Sanger nucleotide sequencing process, this technology only requires a one-step real-time PCR with melt curve analysis of amplicons generated by primers targeting species-specific single nucleotide polymorphisms (SNPs). Multiplex real-time PCR was performed for twelve species of Calliphoridae (four reactions) and for seven species of Sarcophagidae (three reactions). This assay is expected to make it easier and faster for investigating authorities to identify major species of necrophagous flies at beginning of investigation and to increase the utilization of entomological evidence in forensic investigations.

Characterization of the complete mitochondrial genome of Triceratopyga calliphoroides (Rohdendorf, 1931) (Insecta: Diptera: Calliphoridae).

Triceratopyga calliphoroides is a blowfly species which is the only member of the genus Triceratopyga. Because of their forensic importance, we sequenced the complete mitogenome of the T. calliphoroides and analyzed phylogenetic relationships. According to data, it has the longest mitogenome in the family with 16,529 bp in length. In the phylogenetic tree, T. calliphoroides were positioned in the subfamily Calliphorinae, and the closest species is Caliphora vomitoria. This is the first complete mitogenome record for the species.

The complete mitochondrial genome of a blowfly Calliphora nigribarbis (Vollenhoven, 1863) (Insecta: Diptera: Calliphoridae).

In the present study, the complete mitochondrial genome of a blowfly Calliphora nigribarbis has been sequenced and analyzed. The length of complete the mitochondrial genome is 16,279 bp, with 39.50% A, 13.20% C, 9.30% G, and 38.0% T nucleotide distribution. The complete mitochondrial genome consists of 13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs likewise the most metazoan mitochondrial genomes. Furthermore, phylogenetic relationships of C. nigribarbis in the subfamily Calliphorinae investigated. The results suggested that C. vomitoria is the most related species to C. nigribarbis and the genus Calliphora is not monophyletic. This study provides the first complete mitochondrial genome sequence for the species.

Comprehensive transcriptome analysis of Sarcophaga peregrina, a forensically important fly species.

Sarcophaga peregrina (flesh fly) is a frequently found fly species in Palaearctic, Oriental, and Australasian regions that can be used to estimate minimal postmortem intervals important for forensic investigations. Despite its forensic importance, the genome information of S. peregrina has not been fully described. Therefore, we generated a comprehensive gene expression dataset using RNA sequencing and carried out de novo assembly to characterize the S. peregrina transcriptome. We obtained precise sequence information for RNA transcripts using two different methods. Based on primary sequence information, we identified sets of assembled unigenes and predicted coding sequences. Functional annotation of the aligned unigenes was performed using the UniProt, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes databases. As a result, 26,580,352 and 83,221 raw reads were obtained using the Illumina MiSeq and Pacbio RS II Iso-Seq sequencing applications, respectively. From these reads, 55,730 contigs were successfully annotated. The present study provides the resulting genome information of S. peregrina, which is valuable for forensic applications.

Identification of Forensically Important Calliphoridae and Sarcophagidae Species Collected in Korea Using SNaPshot Multiplex System Targeting the Cytochrome c Oxidase Subunit I Gene.

Estimation of postmortem interval (PMI) is paramount in modern forensic investigation. After the disappearance of the early postmortem phenomena conventionally used to estimate PMI, entomologic evidence provides important indicators for PMI estimation. The age of the oldest fly larvae or pupae can be estimated to pinpoint the time of oviposition, which is considered the minimum PMI (PMImin). The development rate of insects is usually temperature dependent and species specific. Therefore, species identification is mandatory for PMImin estimation using entomological evidence. The classical morphological identification method cannot be applied when specimens are damaged or have not yet matured. To overcome this limitation, some investigators employ molecular identification using mitochondrial cytochrome c oxidase subunit I (COI) nucleotide sequences. The molecular identification method commonly uses Sanger's nucleotide sequencing and molecular phylogeny, which are complex and time consuming and constitute another obstacle for forensic investigators. In this study, instead of using conventional Sanger's nucleotide sequencing, single-nucleotide polymorphisms (SNPs) in the COI gene region, which are unique between fly species, were selected and targeted for single-base extension (SBE) technology. These SNPs were genotyped using a SNaPshot® kit. Eleven Calliphoridae and seven Sarcophagidae species were covered. To validate this genotyping, fly DNA samples (103 adults, 84 larvae, and 4 pupae) previously confirmed by DNA barcoding were used. This method worked quickly with minimal DNA, providing a potential alternative to conventional DNA barcoding. Consisting of only a few simple electropherogram peaks, the results were more straightforward compared with those of the conventional DNA barcoding produced by Sanger's nucleotide sequencing.

Molecular identification of forensically important calliphoridae and sarcophagidae species using ITS2 nucleotide sequences.

The application of insect evidence to forensic investigations is mainly based on the estimation of postmortem interval and the identification of insect species from samples that are collected from the crime scene. Due to the limited number of expert taxonomists, species identification is one of the major barriers for crime scene investigators to utilize forensic entomology. Therefore, the molecular identification of species, using mitochondrial cytochrome c oxidase subunit I (COI) gene, has been suggested as an alternative strategy. However, in some cases, these maternally inherited markers cause confusion; hence, nuclear DNA markers such as ITS2 are also required as supporting tools. Eleven Calliphoridae and 5 Sarcophagidae fly species collected from Korea were utilized for PCR amplification and nucleotide sequencing of ITS2 locus. Species Identifier software was used for sequence analysis and comparison. The results demonstrated that 11 Korean Calliphoridae and 5 Korean Sarcophagidae fly species could be distinguished using ITS2 nucleotide sequences. In particular, the sister species, Lucilia illustris and Lucilia caesar were also distinguished, despite the very low level of interspecific diversity. However, when compared with previously reported ITS2 nucleotide sequences, several identification failures were noted. This is the first study that widely analyzed nucleotide sequences of the ITS2 locus from Calliphoridae and Sarcophagidae fly species collected in Korea.

Sequencing and analyzing complete mitochondrial DNA of Muscina angustifrons (Insecta, Diptera, Muscidae).

In this study, the complete mitochondrial genome sequenced and analyzed from a fly, Muscina angustifrons which collected from South Korea. The size of mitochondrial genome is 16,316 bp with 40.9% A, 12.3% C, 8.4% G and 38.4% T distribution. Furthermore, phylogenetic relationships of the superfamily Muscoidea evaluated due to mitochondrial protein coding genes. The results showed that the family Muscidae is a paraphyletic group and the closest species to M. angustifrons is M. levida. This is the third complete mitochondrial genome for the genus Muscina and the first genus record from South Korea.

Complete mitochondrial DNA analysis of Hydrotaea ignava (Insecta, Diptera, Muscidae).

The complete mitochondrial genome sequenced and analyzed from a black garbage fly, Hydrotaea ignava which is critically important species for forensic investigations. The size of mitochondrial genome is 17,026 bp with 40.8% A, 11.2% C, 8.2% G and 39.8% T distribution. This is the longest within complete mitochondrial genome records of the Muscidae species. The mitochondrial genome is composed of 13 protein coding, two ribosomal RNA and 22 tRNA genes and a putative control region. Furthermore, phylogenetic relationships of the superfamily Muscoidea evaluated due to mitochondrial protein coding genes. The results showed that the H. ignava placed in the paraphyletic Muscidae family and early diverged from a clade including Muscidae, Anthomyiidae and Scathophagidae species. This is the first complete mitochondrial genome for the genus Hydrotaea.

Complete mitogenome and phylogenetic analysis of hide beetle Dermestes maculatus (Insecta, Coleoptera, Dermestidae).

In this study, the complete mitochondrial genome of hide beetle Dermestes maculatus which was collected from Seoul, South Korea was sequenced by next-generation sequencing. The size of mitochondrial genome is 17,026 bp that composed of 13 protein coding, two ribosomal RNA and 22 tRNA genes which has the identical gene orientation with the other Bostrichiformia species. Additionally, the phylogenetic tree of the D. maculatus in the infraorder Bostrichiformia was reconstructed by using 13 protein-coding genes of complete mitochondrial genome. The results showed that the family Dermestidae is positioned in the infraorder Bostrichiformia early branched than family Bostrichidae. This study provides the first complete mitochondrial genome from the genus Dermestes.

The First Survey of Forensically Important Entomofauna Collected from Medicolegal Autopsies in South Korea.

Forensic entomology applies insect evidence to legal problems such as the estimation of minimum postmortem interval (mPMI). For this purpose, knowledge of the insect fauna that are attracted to human cadavers in each geographic region is a prerequisite. Despite many studies investigating the insect fauna attracted to meat, there has been no survey of the entomofauna on human cadavers in the East Asian temperate climate zone, particularly in Korea. Therefore, this study reports the entomofauna collected from medicolegal autopsies in northeastern Seoul and its suburbs. Insect samples were collected from 35 medicolegal autopsies in 2010, 2011, and 2013. Molecular and morphological methods were utilized for taxonomic identification. Among 1398 individual samples belonging to 3 orders, 13 families, 18 genera, and 32 species, the dominant family and species were Calliphoridae and Lucilia sericata, respectively. Despite its limited scale, this study provides a snapshot of the general entomofauna that are attracted to human cadavers in this region.

First report of external ophthalmomyiasis caused by Lucilia sericata Meigen in a healthy patient without predisposing risk factors.

A 72-year-old man with no medical history initially presented to the emergency room with severe tearing, redness, foreign body sensation, and pain in the left eye. He reported no previous history of any periocular trauma, malignancy, surgery, or systemic illness. On presentation, the patient only showed left periorbital edema and erythema in the left eyelid with no evidence of any skin malignancy. On slit lamp examination, multiple small whitish motile organisms were observed on the left conjunctival fornices. The organisms were removed, preserved, and identified as the third-stage larvae of Lucilia sericata (green bottle fly). The patient was treated with topical antibiotic and steroid eye drops and the inflammation resolved 1 week after treatment initiation. This is the first report of external ophthalmomyiasis caused by facultative parasite, L. sericata maggots in a healthy patient without any predisposing factors.

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.