Dataset Information

Using the developmental gene bicoid to identify species of forensically important blowflies (Diptera: calliphoridae).

ABSTRACT: 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.

SUBMITTER: Park SH

PROVIDER: S-EPMC3613069 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Using the developmental gene bicoid to identify species of forensically important blowflies (Diptera: calliphoridae).

Park Seong Hwan SH Park Chung Hyun CH Zhang Yong Y Piao Huguo H Chung Ukhee U Kim Seong Yoon SY Ko Kwang Soo KS Yi Cheong-Ho CH Jo Tae-Ho TH Hwang Juck-Joon JJ

BioMed research international 20130318

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 l ...[more]

PMID: 23586044

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Project description: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.

Project description:Correct identification of forensically important insects, such as flies in the family Calliphoridae, is a crucial step for them to be used as evidence in legal investigations. Traditional identification based on morphology has been effective, but has some limitations when it comes to identifying immature stages of certain species. DNA-barcoding, using COI, has demonstrated potential for rapid and accurate identification of Calliphoridae, however, this gene does not reliably distinguish among some recently diverged species, raising questions about its use for delimitation of species of forensic importance. To facilitate DNA based identification of Calliphoridae in the Caribbean we developed a vouchered reference collection from across the region, and a DNA sequence database, and further added the nuclear ITS2 as a second marker to increase accuracy of identification through barcoding. We morphologically identified freshly collected specimens, did phylogenetic analyses and employed several species delimitation methods for a total of 468 individuals representing 19 described species. Our results show that combination of COI + ITS2 genes yields more accurate identification and diagnoses, and better agreement with morphological data, than the mitochondrial barcodes alone. All of our results from independent and concatenated trees and most of the species delimitation methods yield considerably higher diversity estimates than the distance based approach and morphology. Molecular data support at least 24 distinct clades within Calliphoridae in this study, recovering substantial geographic variation for Lucilia eximia, Lucilia retroversa, Lucilia rica and Chloroprocta idioidea, probably indicating several cryptic species. In sum, our study demonstrates the importance of employing a second nuclear marker for barcoding analyses and species delimitation of calliphorids, and the power of molecular data in combination with a complete reference database to enable identification of taxonomically and geographically diverse insects of forensic importance.

Project description:BackgroundThe time-length between the first colonization of necrophagous insect on the corpse and the beginning of investigation represents the most important forensic concept of minimum post-mortem inference (PMImin). Before colonization, the time spent by an insect to detect and locate a corpse could significantly influence the PMImin estimation. The olfactory system plays an important role in insect food foraging behavior. Proteins like odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs) and sensory neuron membrane proteins (SNMPs) represent the most important parts of this system. Exploration of the above genes and their necrophagous products should facilitate not only the understanding of their roles in forging but also their influence on the period before PMImin. Transcriptome sequencing has been wildly utilized to reveal the expression of particular genes under different temporal and spatial condition in a high throughput way. In this study, transcriptomic study was implemented on antennae of adult Aldrichina grahami (Aldrich) (Diptera: Calliphoridae), a necrophagous insect with forensic significance, to reveal the composition and expression feature of OBPs, CSPs, ORs, IRs and SNMPs genes at transcriptome level.MethodAntennae transcriptome sequencing of A. grahami was performed using next-generation deep sequencing on the platform of BGISEQ-500. The raw data were deposited into NCBI (PRJNA513084). All the transcripts were functionally annotated using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Differentially expressed genes (DEGs) were analyzed between female and male antennae. The transcripts of OBPs, CSPs, ORs, IRs and SNMPs were identified based on sequence feature. Phylogenetic development of olfactory genes of A. grahami with other species was analyzed using MEGA 5.0. RT-qPCR was utilized to verify gene expression generated from the transcriptome sequencing.ResultsIn total, 14,193 genes were annotated in the antennae transcriptome based on the GO and the KEGG databases. We found that 740 DEGs were differently expressed between female and male antennae. Among those, 195 transcripts were annotated as candidate olfactory genes then checked by sequence feature. Of these, 27 OBPs, one CSPs, 49 ORs, six IRs and two SNMPs were finally identified in antennae of A. grahami. Phylogenetic development suggested that some olfactory genes may play a role in food forging, perception of pheromone and decomposing odors.ConclusionOverall, our results suggest the existence of gender and spatial expression differences in olfactory genes from antennae of A. grahami. Such differences are likely to greatly influence insect behavior around a corpse. In addition, candidate olfactory genes with predicted function provide valuable information for further studies of the molecular mechanisms of olfactory detection of forensically important fly species and thus deepen our understanding of the period before PMImin.

Dataset Information

Using the developmental gene bicoid to identify species of forensically important blowflies (Diptera: calliphoridae).

Publications

Using the developmental gene bicoid to identify species of forensically important blowflies (Diptera: calliphoridae).

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