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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    • "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.
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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.
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    ABSTRACT: Phormia regina (the black fly) is a common Holarctic blow fly species which serves as a primary indicator taxon to estimate minimal post mortem intervals. It is also a major research model in physiological and neurological studies on insect feeding. Previous studies have shown a sequence divergence of up to 4.3% in the mitochondrial COI gene between W European and N American P. regina populations. Here, we DNA barcoded P. regina specimens from six N American and 17 W European populations and confirmed a mean sequence divergence of ca. 4% between the populations of the two continents, while sequence divergence within each continent was a ten-fold lower. Comparable mean mtDNA sequence divergences were observed for COII (3.7%) and cyt b (5.3%), but mean divergence was lower for 16S (0.4-0.6%). Intercontinental divergence at nuclear DNA was very low (≤ 0.1% for both 28S and ITS2), and we did not detect any morphological differentiation between N American and W European specimens. Therefore, we consider the strong differentiation at COI, COII and cyt b as intraspecific mtDNA sequence divergence that should be taken into account when using P. regina in forensic casework or experimental research.
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