Molecular Analysis of Forensically Important Blow Flies in Thailand.
ABSTRACT: Blow flies are the first insect group to colonize on a dead body and thus correct species identification is a crucial step in forensic investigations for estimating the minimum postmortem interval, as developmental times are species-specific. Due to the difficulty of traditional morphology-based identification such as the morphological similarity of closely related species and uncovered taxonomic keys for all developmental stages, DNA-based identification has been increasing in interest, especially in high biodiversity areas such as Thailand. In this study, the effectiveness of long mitochondrial cytochrome c oxidase subunit I and II (COI and COII) sequences (1247 and 635 bp, respectively) in identifying 16 species of forensically relevant blow flies in Thailand (Chrysomya bezziana, Chrysomya chani, Chrysomya megacephala, Chrysomya nigripes, Chrysomya pinguis, Chrysomya rufifacies, Chrysomya thanomthini, Chrysomya villeneuvi, Lucilia cuprina, Lucilia papuensis, Lucilia porphyrina, Lucilia sinensis, Hemipyrellia ligurriens, Hemipyrellia pulchra, Hypopygiopsis infumata, and Hypopygiopsis tumrasvini) was assessed using distance-based (Kimura two-parameter distances based on Best Match, Best Close Match, and All Species Barcodes criteria) and tree-based (grouping taxa by sequence similarity in the neighbor-joining tree) methods. Analyses of the obtained sequence data demonstrated that COI and COII genes were effective markers for accurate species identification of the Thai blow flies. This study has not only demonstrated the genetic diversity of Thai blow flies, but also provided a reliable DNA reference database for further use in forensic entomology within the country and other regions where these species exist.
Project description:This is the first study to report Chrysomya pinguis (Walker) and Lucilia porphyrina (Walker) (Diptera: Calliphoridae) as forensically important blow fly species from human cadavers in Thailand, in addition to Chrysomya villeneuvi (Patton) already known in Thailand. In 2016, a fully decomposed body of an unknown adult male was discovered in a high mountainous forest during winter in Chiang Mai province. The remains were infested heavily with thousands of blow fly larvae feeding simultaneously on them. Morphological identification of adults reared from the larvae, and molecular analysis based on sequencing of 1,247 bp partial mitochondrial cytochrome c oxidase subunit 1 gene (CO1) of the larvae and puparia, confirmed the above mentioned 3 species. The approving forensic fly evidence by molecular approach was described for the first time in Thailand. Moreover, neighbor-joining phylogenetic analysis of the CO1 was performed to compare the relatedness of the species, thereby affirming the accuracy of identification. As species of entomofauna varies among cases in different geographic and climatic circumstances, C. pinguis and L. porphyrina were added to the list of Thai forensic entomology caseworks, including colonizers of human remains in open, high mountainous areas during winter. Further research should focus on these 3 species, for which no developmental data are currently available.
Project description:The subfamily Luciliinae is diverse and geographically widespread. Its four currently recognised genera (Dyscritomyia Grimshaw, 1901, Hemipyrellia Townsend, 1918, Hypopygiopsis Townsend 1916 and Lucilia Robineau-Desvoidy, 1830) contain species that range from saprophages to obligate parasites, but their pattern of phylogenetic diversification is unclear. The 28S rRNA, COI and Period genes of 14 species of Lucilia and Hemipyrellia were partially sequenced and analysed together with sequences of 11 further species from public databases. The molecular data confirmed molecular paraphyly in three species-pairs in Lucilia that hamper barcode identifications of those six species. Lucilia sericata and Lucilia cuprina were confirmed as mutual sister species. The placements of Dyscritomyia and Hypopygiopsis were ambiguous, since both made Lucilia paraphyletic in some analyses. Recognising Hemipyrellia as a genus consistently left Lucilia s.l. paraphyletic, and the occasionally-recognised (sub)genus Phaenicia was consistently paraphyletic, so these taxa should be synonymised with Lucilia to maintain monophyly. Analysis of a matrix of 14 morphological characters scored for adults of all genera and for most of the species included in the molecular analysis confirmed several of these findings. The different degrees of parasitism were phylogenetically clustered within this genus but did not form a graded series of evolutionary stages, and there was no particular relationship between feeding habits and biogeography. Because of the ubiquity of hybridization, introgression and incomplete lineage sorting in blow flies, we recommend that using a combination of mitochondrial and nuclear markers should be a procedural standard for medico-criminal forensic identifications of insects.
Project description:Identifying species of insects used to estimate postmortem interval (PMI) is a major subject in forensic entomology. Because forensic insect specimens are morphologically uniform and are obtained at various developmental stages, DNA markers are greatly needed. To develop new autosomal DNA markers to identify species, partial genomic sequences of the bicoid (bcd) genes, containing the homeobox and its flanking sequences, from 12 blowfly species (Aldrichina grahami, Calliphora vicina, Calliphora lata, Triceratopyga calliphoroides, Chrysomya megacephala, Chrysomya pinguis, Phormia regina, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Hemipyrellia ligurriens and Lucilia sericata; Calliphoridae: Diptera) were determined and analyzed. This study first sequenced the ten blowfly species other than C. vicina and L. sericata. Based on the bcd sequences of these 12 blowfly species, a phylogenetic tree was constructed that discriminates the subfamilies of Calliphoridae (Luciliinae, Chrysomyinae, and Calliphorinae) and most blowfly species. Even partial genomic sequences of about 500 bp can distinguish most blowfly species. The short intron 2 and coding sequences downstream of the bcd homeobox in exon 3 could be utilized to develop DNA markers for forensic applications. These gene sequences are important in the evolution of insect developmental biology and are potentially useful for identifying insect species in forensic science.
Project description:Contemporary studies in forensic entomology exhaustively evaluate gene sequences because these constitute the fastest and most accurate method of species identification. For this purpose single gene segments, cytochrome oxidase subunit I (COI) in particular, are commonly used. However, the limitation of such sequences in identification, especially of closely related species and populations, demand a multi-gene approach. But this raises the question of which group of genes can best fulfill the identification task? In this context the utility of five gene segments was explored among blowfly species from two distinct geographic regions, China and Pakistan. COI, cytochrome b (CYTB), NADH dehydrogenase 5 (ND5), nuclear internal transcribed spacers (ITS1 and ITS2), were sequenced for eight blowfly species including Chrysomya megacephala F. (Diptera: Calliphoidae), Ch. pinguis Walker, Lucilia sericata Meigen L. porphyrina Walker, L. illustris Meigen Hemipyrellia ligurriens Wiedemann, Aldrichina grahami Aldrich, and the housefly, Musca domestica L. (Muscidae), from Hangzhou, China; while COI, CYTB, and ITS2 were sequenced for four species, i.e. Ch. megacephala, Ch. rufifacies, L. cuprina, and the flesh fly, Sarcophaga albiceps Meigen (Sarcophagidae), from Dera Ismail Khan Pakistan. The results demonstrate a universal utility of these gene segments in the molecular identification of flies of forensic importance.
Project description:Blow flies of the subfamily Luciliinae (Diptera: Calliphoridae) are one of the main forensically important subfamilies globally. In addition to being used to estimate the minimum post-mortem interval (PMImin), assuming colonization occurred after death, blow fly specimens found infesting a human corpse are used to determine if the corpse was relocated or if the individual ingested narcotics prior to death. The presence of these blow flies in a given area is strongly influenced by abiotic and biotic factors, such as temperature, elevation, and habitat. Having this information, along with geographical distributions and the characteristics of preferred habitats, is necessary to better understand the biology of this group. This study aimed to characterize the spatial distribution of Luciliinae throughout 18 sampling sites within six ecozones (disturbed mixed deciduous forest, mixed deciduous forest, mixed orchard, paddy field, lowland village, and city/town) in central Chiang Mai Province, northern Thailand over one year (May 2009⁻May 2010). The purpose of the study was to elucidate the relationship of blow fly species composition with environmental abiotic factors (e.g., temperature, relative humidity, light intensity), and to predict the distribution of the common species within this subfamily using GIS. Adult collections were performed biweekly, baited with one-day-old beef offal. A total of 2331 Luciliinae flies trapped, comprising eight species, of which the four predominant species were Hemipyrellia ligurriens (Wiedemann) (n = 1428; 61.3%), Lucilia porphyrina (Walker) (n = 381; 16.3%), Hemipyrellia pulchra (Wiedemann) (n = 293; 12.6%), and Lucilia papuensis Macquart (n = 129; 5.5%). Population density across species varied seasonally, peaking in August 2009 coinciding with the rainy season. Predicting population composition was based on a model developed with ArcGIS 9.2, which utilized environmental variables (temperature, relative humidity, and light intensity) in conjunction with abundance data. Models indicated H. ligurriens had the most widespread geographic distribution, while H. pulchra was predicted to occur largely in mixed orchards and lowland villages. Lucilia porphyrina and L. papuensis were less widespread, restricted mainly to mixed deciduous forest. This model, along with knowledge of forensic information, may be useful under certain investigations where the corpse may have been relocated.
Project description:Blow flies are worldwide the most important insects from a forensic point of view. In Thailand, aside from the two most common species, Chrysomya megacephala (F.) and Chrysomya rufifacies (Macquart), Chrysomya chani Kurahashi was also found to be of forensic importance. We present a case of a human female cadaver in its bloated stage of decomposition, discovered at Pachangnoi Subdistrict, northern Thailand. Entomological sampling during the autopsy displayed an assemblage of numerous dipteran larvae. Macroscopic observations showed the coexistence of third instar larvae of the three blow flies C. megacephala, Chrysomya villeneuvi Patton, an unknown blow fly species and one muscid, Hydrotaea sp. The minimum post-mortem interval was estimated to be six days, based on the developmental rate of C. megacephala. The ID of the unknown larva, which is the focus of this report, was revealed later as C. chani by DNA sequencing, using a 1205 bp of cytochrome c oxidase subunit I (COI). The occurrence of C. chani on a human body revealed the need to analyse and describe the morphology of its immature stage, to enable forensic entomologists to identify this fly species in future cases. The morphological examination of the third instar was performed, revealing peculiar characteristics: protuberant tubercles encircling abdominal segments; 9-11 lobes on the anterior spiracle; six prominent pairs of tubercles along the peripheral rim of the eighth abdominal segment; a heavily sclerotized complete peritreme of the posterior spiracles. A key to differentiate the third instar of blow flies of forensic importance in Thailand is provided.
Project description:Regional surveys were carried out in different parts of North West Pakistan among domestic animals (N=57,921) including pets and livestock identifying cases of traumatic myiasis (n=1037). A total of four surveys focused general livestock population during Eid ul Adha (Eid surveys; incidence=1.21%) while another four surveys (Miscellaneous surveys; incidence=7.34%) targeted animal population brought to veterinary hospitals and dispensaries. Timeframe spanned four years from 2012 to 2015. Maggots were sampled and location of the wound was recorded for each host. Taxonomic identification used light and electron microscopic techniques. Our dataset shows three species as principle agents of myiasis (n=882) including Chrysomya bezziana Villeneuve (n=394), Wohlfahrtia magnifica (n=244) and Lucilia cuprina Wiedemann (n=244). Others (n=155) including Chrysomya megacephala (Fabricius), Chrysomya rufifacies (Macquart), Lucilia sericata (Meigen), Lucilia illustris (Meigen), Lucilia porphyrina (Walker), Hemipyrellia ligguriens (Wiedemann), Calliphora vicina (Robineau-Desvoidy), Sarcophaga crassipalpalis (Macquart) and Sarcophaga species were identified as species of minor importance. The obligatory screwworm species W. magnifica is a first report from Pakistan. The results based on this dataset are presented in a recent publication "Distribution Modeling of three screwworm species in the ecologically diverse landscape of North West Pakistan" (Zaidi et al., 2016) .
Project description: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.
Project description:Species identification is a crucial step in forensic entomology. In several cases the calculation of the larval age allows the estimation of the minimum Post-Mortem Interval (mPMI). A correct identification of the species is the first step for a correct mPMI estimation. To overcome the difficulties due to the morphological identification especially of the immature stages, a molecular approach can be applied. However, difficulties in separation of closely related species are still an unsolved problem. Sequences of 4 different genes (COI, ND5, EF-1?, PER) of 13 different fly species collected during forensic experiments (Calliphora vicina, Calliphora vomitoria, Lucilia sericata, Lucilia illustris, Lucilia caesar, Chrysomya albiceps, Phormia regina, Cynomya mortuorum, Sarcophaga sp., Hydrotaea sp., Fannia scalaris, Piophila sp., Megaselia scalaris) were evaluated for their capability to identify correctly the species. Three concatenated sequences were obtained combining the four genes in order to verify if longer sequences increase the probability of a correct identification. The obtained results showed that this rule does not work for the species L. caesar and L. illustris. Future works on other DNA regions are suggested to solve this taxonomic issue.
Project description:<i>Chrysomya nigripes</i> (Diptera: Calliphoridae) is a blow fly species of forensic importance. Here we demonstrated the complete mitochondrial genome of this species for the first time. Phylogenetic analyses indicated that entire mitochondrial genome sequences can provide more useful information for distinguishing <i>C. nigripes</i> from the other species.