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.     

洛阳地区5种嗜尸性麻蝇分子鉴定研究

目的应用经典DNA条形码和28S r RNA基因鉴定洛阳地区5种常见嗜尸性麻蝇种属,评价其在常见嗜尸性麻蝇种属鉴定中应用的可行性。方法收集洛阳地区常见嗜尸性麻蝇类标本18只,经形态学分类鉴定后,Chelex-100法提取足部DNA,扩增并测序线粒体细胞色素C氧化酶亚基I(COI)和28S核糖体核糖核酸(28S r RNA)基因片段,BLAST比对后,与来自DNA条形码数据库(Barcode of Life Data,BOLD)的中国和韩国地区20条相应蝇种序列进行比对,用MEGA 7.0软件整理分析所得序列,计算碱基组成、种内及种间进化分歧率,并构建系统发育树。结果形态学鉴定18只嗜尸性麻蝇归于3属5种。每个样本获得COI和28S r RNA的扩增长度分别为646bp和721bp。在线BLAST比对结果显示样本COI序列相似度99%以上,邻近法构建发育树Bootstrap值为1 000,5种麻蝇可以较好聚类,与形态学鉴定结果一致。38个样本COI种内范围为0~0.022,棕尾别麻蝇、酱亚麻蝇和急钩(亚)麻蝇种间范围在0.057~0.090;赤尾麻蝇和红尾粪麻蝇种间范围在0~0.086。5种麻蝇的28S r RNA序列发育树显示,棕尾别麻蝇和急钩(亚)麻蝇明显各自聚类,其余3种成一类。结论对于本研究中的5种嗜尸性麻蝇,基于COI基因的DNA条形码可以有效区分棕尾别麻蝇、酱亚麻蝇和急钩(亚)麻蝇,28S r RNA基因只可以区分棕尾别麻蝇,二者可作为形态学鉴定之外的重要方法补充。     

The utility of mitochondrial DNA sequences for the identification of forensically important blowflies (Diptera: Calliphoridae) in southeastern Australia.

The applicability of mitochondrial DNA (mtDNA) sequencing was investigated for the identification of the following forensically important species of blowflies from southeastern Australia: Calliphora albifrontalis, C. augur, C. dubia, C. hilli hilli, C. maritima, C. stygia, C. vicina, Chrysomya rufifacies, Ch. varipes and Onesia tibialis. All breed in carrion except O. tibialis, which is an earthworm parasitoid. Emphasis was placed on Calliphora species because they predominate among the carrion-breeding blowfly fauna of southern Australia and their immatures are difficult to identify morphologically. A partial sequence of the mitochondrial COII gene was determined for all species and for COI for C. albifrontalis, C. augur, C. dubia and C. stygia only. Five other species of blowflies, Chrysomya albiceps, Ch. rufifacies, Protophormia terraenovae, Lucilia illustris and L. sericata, for which sequence data were already available, were also included. Analysis of the COI and COII sequences revealed abundant phylogenetically informative nucleotide substitutions that could identify blowfly species to species group. In contrast, because of the low level of sequence divergence of sister species, the data could not distinguish among taxa from the same species group, i.e. the species within the C. augur and C. stygia groups. The molecular data support the existing species group separation of the taxa within Calliphora. Because of the speed and accuracy of current nucleotide sequencing technology and the abundant apomorphic substitutions available from mtDNA sequences, this approach, with the analysis of additional taxa and genes, is likely to enable the reliable identification of carrion-breeding blowflies in Australia.     

Use of PCR-RFLP for differentiation of calliphorid larvae (Diptera,Calliphoridae) on human corpses

Blowfly larvae found on human corpses are important for the estimation of the postmortem interval (PMI) and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, therefore a molecular method was elaborated for species identification. Specific fragments of the COI and COII region of the mitochondrial DNA (mtDNA) were amplified followed by digestion with different restriction enzymes. Using a 1.3 kb fragment, identification of Lucilia sericata, Calliphora vicina and Calliphora vomitoria was possible by digestion with only one restriction enzyme using either DraI or HinfI. Furthermore, we sequenced 349 bp (a part of the COI and COII regions) from the same three species and found 34 nucleotide distinctions between C. vicina and L. sericata, 30 between C. vomitoria and L. sericata and 15 between the two Calliphora species. These results aid in quick identification of species used for estimation of PMI.     

The use of mitochondrial cytochrome oxidase I gene (COI) to differentiate two UK blowfly species -- Calliphora vicina and Calliphora vomitoria

Traditionally identification of forensically important insects has been carried out based upon morphological differences between species. However insect evidence found at a crime scene may on occasion be difficult to distinguish by morphological techniques and under these circumstances another method of accurate identification is required. This work utilises a cytochrome oxidase I partial mitochondrial gene region (COI) to distinguish the two of the main UK blowfly species -- Calliphora vicina (Robineau Desvoidy) and Calliphora vomitoria (Linnaeus) (Diptera:Calliphoridae). Seventeen interspecific differences in COI sequence were located. Use of the restriction enzyme SfcI on this gene region provides a simple method for distinguishing between C. vicina and C. vomitoria.     

应用mtDNA COI基因序列鉴别常见嗜尸性蝇类种属

目的探讨mtDNA基因序列对常见嗜尸性蝇类的种属鉴别应用价值。方法收集不同区域2科4属6个种30个蝇类样本,提取样本线粒体DNA后扩增COI基因序列,以琼脂糖电泳检测扩增产物并测序,以DNAMAN6.0分析软件分别截取498bp序列,用MEGA5.2软件分别进行序列分析,然后构建系统发育树,比较各地区不同种属样本的序列差异。结果 6个种属的嗜尸性蝇类30个样本mtDNA的COI基因具有一定的序列差异,种内进化分歧均数在0.1%~1.6%之间,种间进化分歧均数在2.2%~11.2%之间,6个种属通过系统发育树均可明确区分。结论 COI基因序列分析和系统发育树对嗜尸性蝇类的种属检验具有重要帮助作用,可用于现场样本的准确、快速种属鉴定。     

利用COI序列序列鉴定常见嗜尸性蝇种

目的 检测嗜尸性蝇类线粒体DNA(mtDNA)细胞色素氧化酶辅酶Ⅰ(COI)中278bp序列,鉴定蝇科3属5种嗜尸性蝇,解决其形态学种类鉴定的难题,为死亡时间推断提供技术支持.方法 从15省、市(地区)室外草地家兔尸体上采集蝇科3属5种共计18个蝇类成蝇样本,提取mtDNA进行PCR扩增,序列测定且上传GENBANK,...     

Internal Morphological Analysis for Age Estimation of Blow Fly Pupae (Diptera: Calliphoridae) in Postmortem Interval Estimation*

Abstract: Larvae and pupae of blow fly species are frequently used in postmortem interval estimation, their age indicating minimum time since death. Most studies have considered age estimation of larvae, neglecting study of pupae. Relative development of external pupal features is useful, but there are also internal changes during metamorphosis that may be indicators of age, utilizing histological techniques. This study aimed to optimize preservation and histological analysis of blow fly pupae, specifically Calliphora vicina Robineau‐Desvoidy and Lucilia sericata (Meigen), and to examine internal features with potential for age estimation. Effect of hot‐water‐killing and different preservatives were examined. It was determined that blow fly pupae should be pierced through the three tagma, hot‐water‐killed, and preserved in 80% ethanol as the optimal preservation for subsequent analyses. Hematoxylin and eosin stained pupal sections revealed differences in brain and thoracic muscle development throughout the pupal stage with potential for age estimation.     

Correlation of Molecular Expression with Diel Rhythm of Oviposition in Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) and Implications for Forensic Entomology

This study explored the molecular mechanisms potentially underlying blow fly nocturnal oviposition. A behavioral study revealed that Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) possesses a diel rhythm of oviposition in light under 12:12 light/dark conditions. Reversal to 12:12 dark/light resulted in oviposition behavior changing to align with the adjusted regime in most females, but four of 59 experimental females lacked a diel rhythm of oviposition (were arrhythmic). Real-time PCR was used to monitor the molecular expression levels of known circadian genes per and tim in C. vicina to determine whether gene expression and behavior correlated. As with behavior, reversing light/dark conditions changed rhythmic gene expression to align with an adjusted light regime. This suggests that although it is unlikely that C. vicina will colonize dead bodies at night, arrhythmic females and oviposition in the dark was demonstrated.     

Identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China, based on COI and 16S rDNA gene sequences

Insects attracted to cadavers may provide important indications of the postmortem interval (PMI). However, use of the flesh flies (Diptera: Sarcophagidae) for PMI estimation is limited as the species are often not morphologically distinct, especially as immatures. In this study, 23 forensically important flesh flies were collected from 13 locations in 10 Chinese provinces. Then, a 278-bp segment of the cytochrome oxidase subunits one (COI) gene and a 289-bp segment of the 16S rDNA gene of all specimens were successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into four species (Boerttcherisca peregrina [Robineau-Desvoidy, 1830], Helicophagella melanura [Meigen, 1826], Parasarcophaga albiceps [Meigen, 1826], and Parasarcophaga dux [Thompson, 1869]) with relatively strong supporting values, thus indicating that the COI and 16S rDNA regions are suitable for identification of sarcophagid species. The difference between intraspecific threshold and interspecific divergence confirmed the potential of the two regions for sarcophagid species identification.     

DNA Barcoding Identifies all Immature Life Stages of a Forensically Important Flesh Fly (Diptera: Sarcophagidae)

Carrion‐breeding insects, such as flesh flies (Diptera: Sarcophagidae), can be used as evidence in forensic investigations. Despite their considerable forensic potential, their use has been limited because morphological species identification, at any life stage, is very challenging. This study investigated whether DNA could be extracted and cytochrome oxidase subunit I (COI) barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic DNA extracts were prepared from all larval instars and puparia. Amplification of the barcoding region was successful from all extracts, but puparia amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcoding of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from late second and early third instars. Similar results are expected for immatures of other carrion‐breeding species, enhancing the use of evidence from immature flies in forensic investigations.     

Minimum and maximum development rates of some forensically important Calliphoridae (Diptera)

Blow fly development rates are frequently used to estimate elapsed time since death in homicide investigations in the first few weeks after death. However, in order to make more precise estimates of time since death, accurate developmental data must be generated for all carrion species, and at temperatures that are comparable with those found at crime scenes. This paper presents developmental rates determined for five forensically important species in British Columbia, Phormia regina (Meigen), Phaenicia sericata (Meigen), Eucalliphora latifrons (Hough), Lucilia illustris (Meigen), and Calliphora vicina Robineau-Desvoidy, at several temperatures.     

Calliphora vicina (Diptera: Calliphoridae) collected from a human corpse above 3400 m in elevation

In the summer of 2005 an adult hiker was killed in a fall in Rocky Mountain National Park, Colorado (U.S.A.). His body was subsequently colonized by the blowfly Calliphora vicina (Diptera: Calliphoridae). The body was discovered 9 days later at an elevation of 3475 m (11,400 feet). This is the first report of C. vicina on a human corpse at this elevation in Colorado. This paper provides additional confirmation of this fly colonizing a human corpse at high elevations in Colorado.     

潍坊地区六种常见嗜尸性蝇类mtDNA COI区序列研究

目的解决蝇类成虫、蛹和蛆快速、准确鉴定的难题。方法随机采集潍坊地区人尸周围环境中的蝇类成虫、蛹和蛆,提取其mtDNA,经PCR扩增、产物检测与纯化、测序等,比较其序列差异。结果六种常见嗜尸性蝇类相互之间序列差异明显,同一种蝇的成虫、幼虫、蛹之间未见差异。结论mtDNACOI区序列分析可作为法医学嗜尸性蝇类种属鉴别的可靠方法。     

Identification of necrophagous fly species using ISSR and SCAR markers

Necrophagous fly is the most common insect evidence collected during a death investigation. We analyzed the DNA polymorphism among the five forensically important fly species, namely, Lucilia sericata, Aldrichina grahami, Chrysomya megacephala, Parasarcophaga crassipalpis and Musca domestica using inter simple sequence repeat (ISSR) method. Nine ISSR primers selected from 18 primers could amplify 105 clear and stable bands, of which 95 bands were polymorphic. Some primers produced completely different band pattern in different species, indicating that they can be used to identify these species. Aiming at obtaining more reliable markers that might be universally used, we started an effort to convert species-specific ISSR fragments into the sequence-characterized amplified region (SCAR) markers that can be used for the molecular diagnosis of the five species.     

Morphological Features of Regurgitate and Defecatory Stains Deposited by Five Species of Necrophagous Flies are Influenced by Adult Diets and Body Size

The morphological characteristics of artifacts from five species of necrophagous flies were examined following feeding on several types of diets. Four types of insect stains were produced by each species: regurgitate, defecatory, translocation, and tarsal tracks. Regurgitate was the most frequent type deposited (70.9 ± 2.4%), followed by defecatory (19.8 ± 4.0%), tarsal tracks (8.6 ± 1.2%), and translocation (0.7 ± 0.1%). Artifact shapes, sizes, and color were highly variable and species and diet specific. Calliphora vicina and Sarcophaga bullata consistently deposited the largest artifacts after feeding, whereas Chrysomya rufifacies and Ch. megacephala produced more tarsal tracks than the other species examined. Artifacts with tails were infrequently observed (4.1 ± 0.6% of all stains) but occurred as either defecatory or regurgitate stains. The widely variable morphologies of all types of fly artifacts underscores the view that insect stains cannot be distinguished from human bloodstains based on morphology alone.     

mtDNA COI和ND5基因用于鉴别常见嗜尸性蝇类

目的对蝇类mtDNA 523bp COI和347bp ND5基因片段进行序列分析,评价其在以嗜尸性蝇类种属鉴定中应用的可行性。方法从广州(广东省)、湛江(广东省)、韶关(广东省)、沈丘(河南省)及蜂蛹寨(四川省)采集7种嗜尸性蝇类标本,进行形态学种属鉴定,取其腹部肌肉提取DNA,利用基因特异性引物对线粒体COI、ND5基因进行PCR扩增,产物经纯化后进行测序,MEGA 3.0软件对DNA序列进行碱基组成、进化分歧率和系统发育分析。结果进化分歧率ND5基因种内小于1.83%,种间大于2.62%;种间与种内进化分歧率范围间没有交叉;COI基因种间在0.48%～14.8%之间,种内在0.24%～8.3%之间,种内进化分歧范围与种间进化分歧范围存在交叉。结论 ND5基因片段可在种水平有效鉴别常见嗜尸性蝇类,也可鉴别近缘种。而单独运用COI基因不能有效进行种属鉴定。     

Three species of insects collected from an adult human corpse above 3300 m in elevation: a review of a case from Colorado

We report on the colonization of an adult human corpse by three insect species at 3350 m (11,000 ft) in elevation. The adult silphid Thanatophilus coloradensis (Wickham), adults of the blow fly Calliphora coloradensis (Hough), and larvae and adults of Lucilia silvarum (Meigen) were all collected from the victim's body which had been wrapped in plastic. The victim was found in late June in the Rocky Mountains of Colorado. This paper provides additional confirmation of the taxa utilizing a human corpse at high elevations in Colorado.     

DNA‐Based Identification of Forensically Important Lucilia (Diptera: Calliphoridae) in the Continental United States*

Abstract: Correct species identification is critical when dipteran larvae are used for inference of the postmortem interval. To facilitate DNA‐based identification of forensically important flies of the genus Lucilia in the continental United States, we develop a vouchered reference collection and DNA sequence database. A total of 122 specimens were collected for nine of the 10 species of Lucilia reported to occur in the continental United States. Using the polymerase chain reaction and DNA sequencing, data were obtained for an 1100‐bp region of the mitochondrial gene encoding cytochrome oxidase I (COI). We consider a species suitable for DNA‐based identification if it is exclusively monophyletic in >95% of bootstrap pseudoreplicate phylogenetic analyses. Seven of the nine species meet that criterion. Two species (Lucilia coeruleiviridis and Lucilia mexicana) share COI sequence and cannot be distinguished using our reference database. We conclude that DNA‐based identification is likely to be successful for the other seven species.     

Determination of Calliphoridae (Diptera) Fauna and Seasonal Distribution on Carrion in Ankara Province*

Abstract: This study was conducted from March 2006 to 2007. The aim of the study was to determine the forensically significant Calliphoridae (Diptera) species and their seasonal distribution in Ankara province at Beytepe Campus. Pig carcasses were killed by a veterinary using pentobarbital sodium with intervals from 2 to 3 weeks to a month. Samples were collected from the carcass everyday in the morning, in the afternoon, and at sunset. Temperature and parameters of weather, such as cloudy, sunny, rainy, and snowy, were recorded at each visit. Phaenicia sericata (Meigen), Chrysomya albiceps (Wiedemann), Calliphora vomitoria (Linnaeus), and Calliphora vicina (Robineau‐Desvoidy) species, which belong to Calliphoridae family, were collected. It was shown that the seasonal distribution of the collected species was different from each other. The species were determined on carcass between the following times; P. sericata, from April to November; C. albiceps, from May to November; Ca. vomitoria, from February to June and from September to December; and Ca. vicina, from February to May and in June and September. Calliphoridae fauna and its annual seasonal distribution in Turkey on 12 pig carcasses were reported for the first time in this study.