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

Department of Biological Sciences, Box 210006, University of Cincinnati, Cincinnati, OH 45221-0006. Department of Justice Sciences, University of Findlay, 1000 N. Main St., Findlay, OH 45840. Department of Biological Sciences, Northern Kentucky University, Nunn Drive, Highland Heights, KY 41099.
Journal of Forensic Sciences (Impact Factor: 1.16). 05/2012; 58(1). DOI: 10.1111/j.1556-4029.2012.02176.x
Source: PubMed


  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.

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    • "005 , Zhang et al . 2007 , Desmyter and Gosselin 2009 , Park et al . 2009 ) . Thus , the longer or more complete COI locus is more robust . However , COI sequencing for molecular identification has failed in discrimination between some closely related species within the genera Calli - phora ( Wallman et al . 2001 ) , Lucilia ( Wells et al . 2007 , DeBry et al . 2013 ) , and Chrysomya ( Harvey et al . 2008 ) . Multi - gene approaches are proposed alternatives to COI sequencing ( Zaidi et al . 2011 , Dai et al . 2012 ) ; however , the resulting phylogenetic trees based on COI fragments were similar to those based on three different gene fragments ( Zaidi et al . 2011 ) ."
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    ABSTRACT: Minimum postmortem interval estimations of a corpse using blow fly larvae in medicolegal investigations require correct identification and the application of appropriate developmental data of the identified fly species. Species identification of forensically relevant blow flies could be very difficult and time consuming when specimens are damaged or in the event of morphologically indistinguishable immature stages, which are most common at crime scenes. In response to this, an alternative, accurate determination of species may depend on sequencing and molecular techniques for identification. Chrysomyinae specimens (n = 158) belonging to three forensically important species [Chrysomya albiceps (Wiedemann), Chrysomya megacephala (F.), and Chrysomya marginalis (Wiedemann)] (Diptera: Calliphoridae) were collected from four locations in Egypt (Giza, Dayrout, Minya, and North Sinai) and sequenced across the mitochondrial cytochrome oxidase subunit I (COI) gene. Phylogenetic analyses using neighbor-joining, maximum likelihood and maximum parsimony methods resulted in the same topological structure and confirmed DNA based identification of all specimens. Interspecific divergence between pairs of species was 5.3% (C. marginalis-C. megacephala), 7% (C. albiceps-C. megacephala), and 8% (C. albiceps-C. marginalis). These divergences are sufficient to confirm the utility of cytochrome oxidase subunit I gene in the molecular identification of these flies in Egypt. Importantly, the maximum intraspecific divergence among individuals within a species was <1% and the least nucleotide divergence between species used for phylogenetic analysis was 3.6%. This study highlights the need for thorough and diverse sampling to capture all of the possible genetic diversity if DNA barcoding is to be used for molecular identification.
    Full-text · Article · May 2015 · Journal of Medical Entomology
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    • "We found that the TRA proteins from within the same genus were highly similar (92–94%) but less similar when comparing outside the genus (78–80%). These results are consistent with previous morphological and phylogenetic studies that showed that the two Lucilia species are very closely related, as are the two Cochliomyia species [19], [20]. Similar results were obtained previously comparing TRA proteins from different tephritid species [18]. "
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    ABSTRACT: Transformer (TRA) promotes female development in several dipteran species including the Australian sheep blowfly Lucilia cuprina, the Mediterranean fruit fly, housefly and Drosophila melanogaster. tra transcripts are sex-specifically spliced such that only the female form encodes full length functional protein. The presence of six predicted TRA/TRA2 binding sites in the sex-specific female intron of the L. cuprina gene suggested that tra splicing is auto-regulated as in medfly and housefly. With the aim of identifying conserved motifs that may play a role in tra sex-specific splicing, here we have isolated and characterized the tra gene from three additional blowfly species, L. sericata, Cochliomyia hominivorax and C. macellaria. The blowfly adult male and female transcripts differ in the choice of splice donor site in the first intron, with males using a site downstream of the site used in females. The tra genes all contain a single TRA/TRA2 site in the male exon and a cluster of four to five sites in the male intron. However, overall the sex-specific intron sequences are poorly conserved in closely related blowflies. The most conserved regions are around the exon/intron junctions, the 3' end of the intron and near the cluster of TRA/TRA2 sites. We propose a model for sex specific regulation of tra splicing that incorporates the conserved features identified in this study. In L. sericata embryos, the male tra transcript was first detected at around the time of cellular blastoderm formation. RNAi experiments showed that tra is required for female development in L. sericata and C. macellaria. The isolation of the tra gene from the New World screwworm fly C. hominivorax, a major livestock pest, will facilitate the development of a "male-only" strain for genetic control programs.
    Full-text · Article · Feb 2013 · PLoS ONE
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    ABSTRACT: Species of the fly genus Lucilia are commonly used in forensic investigations to estimate the postmortem interval (PMI). Two close-related species Lucilia caesar and L. illustris are difficult to identify. Previous studies showed that the mitochondrial cytochrome c oxidase subunit I (COI) marker could be used to identify many Lucilia species. However, mixed results were obtained for L. caesar and L. illustris due to some European specimens showing identical haplotypes. Here, we investigated 58 new European male specimens of L. illustris and L. caesar whose morphological identifications were checked and for which COI fragments were sequenced. In addition, two other mitochondrial (cytochrome c oxidase subunit II and 16S) and two nuclear (internal transcribed spacer 2 and 28S ribosomal RNA) markers were obtained for a subset of these samples. For each marker, genetic divergence within each species was in the same range as between species, confirming the close relationship between both species. Moreover, for each of the gene fragments, both species shared at least one haplotype/genotype. Hence, none of the molecular markers tested could be used, alone or in combination, to discriminate between L. illustris and L. caesar.
    No preview · Article · Sep 2012 · Forensic science international
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