Forensic Science International 164 (2006) 179182 www.elsevier.

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The use of mitochondrial cytochrome oxidase I gene (COI) to differentiate two UK blowy species Calliphora vicina and Calliphora vomitoria
Carole Ames a, Bryan Turner b, Barbara Daniel a,*
a

Kings College London, Department of Forensic Science and Drug Monitoring, 150 Stamford Street, London SE1 9NH, UK b Department of Life Science, Kings College London, SE1 9NN England, UK Received 27 October 2004; received in revised form 10 January 2006; accepted 11 January 2006 Available online 28 February 2006

Abstract Traditionally identication 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 difcult to distinguish by morphological techniques and under these circumstances another method of accurate identication is required. This work utilises a cytochrome oxidase I partial mitochondrial gene region (COI) to distinguish the two of the main UK blowy species Calliphora vicina (Robineau Desvoidy) and Calliphora vomitoria (Linnaeus) (Diptera:Calliphoridae). Seventeen interspecic 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. # 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic entomology; Cytochrome oxidase I (COI); Calliphora vicina; Calliphora vomitoria; Mitochondrial gene

1. Introduction Insect evidence is used in forensic science, amongst other things, to aid in estimating time of death of a corpse (postmortem interval, PMI). This estimation is based upon the time taken for insects developing on a corpse to reach the stage present when the body is found. Insect species have different developmental lifecycle timings and therefore, to utilise the correct developmental information, species need to be accurately identied. This can be done based upon morphological differences [1]. However under the far from ideal circumstances of a crime scene the larval forms and eggs of many forensically important dipteran species are difcult to distinguish as their morphological differences may be obscured or badly preserved. [2]. Denitive identication may be achieved by rearing larvae to adults but this can be time consuming and require the larvae to be collected live and kept in conditions suitable for continued development.

* Corresponding author. Tel.: +44 20 7848 3841; fax: +44 20 7848 3843. E-mail address: barbara.daniel@kcl.ac.uk (B. Daniel). 0379-0738/$ see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2006.01.005

The use of DNA techniques can not only provide a suitable alternative but may also give information on population substructures [3]. This identication can be carried out on any lifecycle stage without further rearing and on dead, preserved or live samples. DNA techniques are also relatively insensitive to the methods of preservation or age of samples [4]. Recent use of molecular markers for forensic species has focussed on the use of mitochondrial DNA [2]. Mitochondria are present in high numbers within cells and hence mitochondrial DNA is present in a much higher copy number than nuclear DNA [5]. It is therefore the preferred target when dealing with incomplete or aged tissue samples [57]. Previous work on mitochondrial DNA has used regions of the cytochrome oxidase gene (COI) to differentiate Calliphoridae. Wallman and Donnellan [8] have demonstrated that COI can be used for identication of the most forensically important species of blowies from south-eastern Australia. Another recent study using a region of the COI gene (278 bp) [9] identied 45 regions that distinguished species from three genera Lucilla, Calliphora and Chrysomya. These researchers used two species of Calliphora (Calliphora augur (Fabricius) and Calliphora dubae (Acquart)) and located three areas in the COI gene that showed variation between these two species.

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In the UK the rst arrivals at a corpse are often the blowies, Calliphora vicina (Robineau-Desvoidy) and/or Calliphora vomitoria (Linnaeus). The aim of this study was to use the COI gene to differentiate these two species of Calliphoridae. 2. Materials and methods 2.1. Samples Wild adult specimens were obtained from various locations across England and Wales. Flies were immediately killed and placed in 99% ethanol and frozen until extraction. Laboratory populations of both species were used as a source for all other samples used in this work. These have been maintained in cages for several generations within the laboratory. Sequence analysis was carried out on 100 adult ies from each species. In addition for each species three replicates of each developmental stage (eggs, each larval stage and pupae). Also included were triplicate DNA extractions from single adult legs and wings and from empty pupal cases. 2.2. DNA extraction

Analyser according to manufacturers protocols. Both forward and reverse samples were sequenced. 2.5. Comparison of sequences Sequences were analysed using BioEdit Sequence Alignment Editor Software. Initially, forward and reverse sequences from the same sample were compared and any discrepancies resolved. Samples were then compared using CLUSTAL X [11] to nd any interspecic differences. 2.6. Restriction enzyme digests From the consensus sequences, theoretical restriction sites within the sequences were established. The restriction enzyme SfcI (New England Biolabs) was chosen to digest the samples. Each reaction mix consisted of 0.5 ml SfcI (1U), 2 ml 10 NEB Buffer 4 (50 mM potassium acetate; 20 mM Trisacetate; 10 mM magnesium acetate; 1 mM DTT), 0.2 ml 100 BSA, approximately 1 mg DNA with ddH2O to make the total volume 20 ml. Reactions were incubated for 1 h at 37 8C. Samples (5 ml) were subjected to electrophoresis on a 2% agarose gel and visualised with ethidium bromide. 3. Results

DNA was extracted using the QIAamp DNA Mini Kit (QIAGEN, UK) according the manufacturers Tissue protocol. DNA was eluted in 100 ml ddH2O. DNA was then quantied using PicoGreen (Molecular Probes, OR, USA) according to manufacturers protocol. 2.3. Polymerase chain reaction (PCR) A region of the COI gene was amplied using primers described in Simon et al. [10]. C1-N-2191 (50 -CCCGGTAAAATTAAAATATAAACTTC-30 ) and C1-J-1718 (50 -GGAGGATTTGGAAATTGATTAGTTCC-30 ). PCR reactions were carried out using a total reaction volume of 50 ml. Fifty picomolar of each primer were added to a nal concentration of 1 mM. Approximately 1 ng of extracted DNA was added to each reaction. Sterile water was added to a volume of 25 ml. Samples were then heated for 1 min at 94 8C before 25 ml of ReadymixTM Red TaqTM PCR Reaction Mix (Sigma, UK) was added. This reaction mix contains 1.5U Taq DNA polymerase, 1.5 mM MgCl2, 0.2 mM dNTPs and buffer (10 mM TrisHCl, 50 mM KCl) in the 50 ml nal volume. Samples were then loaded onto the GeneAmp1 PCR System 9700 Thermal Cycler (PE Applied Biosystems, USA). The following cycles were run 94 8C for 2 min; 30 cycles of [94 8C for 30 s; 60 8C for 30 s; 72 8C for 60 s]; 72 8C for 15 min; 4 8C to nish. 2.4. Sequencing Sequencing of the puried PCR products was carried out on an Applied Biosystems (Foster City, CA, USA) 310 Genetic

This DNA extraction technique (QIAGEN spin column) yielded adequate DNA for cytochrome oxidase amplication in all samples tested. DNA concentrations ranged from 0.2 ng/ml from a single wing to 30 ng/ml for a complete adult y. For comparable developmental stages or structures there was little variation in DNA yield between the two species. The PCR amplication of the cytochrome oxidase region produced a fragment of 523 bp for all samples. Approximately 400 bp were sequenced and these were compared by pairwise alignment (CLUSTAL X). Consensus sequences for C. vicina and C. vomitoria mitochondrial COI partial gene regions can be located in GenBank (accession numbers AY536642 and AY536643). There are 17 differences between the species sequences for COI and these are highlighted in Fig. 1. The base numbering is relative to the consensus sequence established and is not an indication of location within the mitochondrial genome. The differences consist of 17 base substitutions. From these results a level of 4% variation between species can be calculated for this part of the gene. All substitutions produced synonymous changes in the amino acid sequence for this part of the COI gene. There were single intraspecic differences observed in 17 samples. One sample had two intraspecic nucleotide differences. All these were located at positions distinct from the 17 interspecic variations in sequence. Samples were then digested with the restriction enzyme SfcI. Digestion of this C. vicina COI region with this enzyme produces three fragments (63, 239 and 273 bp) and digestion of the C. vomitoria COI region produces fragments (250 and 273 bp). Fig. 2 illustrates the banding pattern of the two blowy

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Fig. 1. Sequence differences between C. vicina and C. vomitoria partial cytochrome oxidase I gene region. Base positions are relative to the sequence established in this work and not the insect mitochondrial genome. Symbol . in the C. vomitoria sequence represents an identical base to the C. vicina sequence.

Fig. 2. Restriction enzyme SfcI digest. Lane 1 contains a 100 bp ladder. Numbers represent number of DNA basepairs. Lanes 2 and 6 undigested and digested Calliphora vicina replicate one COI partial gene region. Lanes 3 and 7 undigested and digested Calliphora vicina replicate two COI partial gene region. Lanes 4 and 8 undigested and digested Calliphora vomitoria replicate one COI partial gene region. Lanes 5 and 9 undigested and digested Calliphora vomitoria replicate two COI partial gene region.

species when run on an agarose gel and stained with ethidium bromide. The C. vomitoria digestion fragments run as an unresolved doublet on this 2% agarose gel system. However, the two species can be easily distinguished as the C. vicina digestion fragments (239 and 273 bp) appear as two distinct bands. 4. Discussion Use of QIAGEN spin columns provides a DNA extraction method capable of extracting ampliable mitochondrial DNA from all the lifecycle stages including single eggs, adult legs and wings and empty pupal cases. After sequencing this 414 bp mitochondrial COI gene region there appears to be 17 species-specic differences present. As expected, the sequence did not vary between adults and immature forms. These data would therefore allow an

entomologist to distinguish between specimens of C. vicina and C. vomitoria, in cases where morphological differentiation was difcult. This agrees with Sperling et al. [12], who demonstrated that mtDNA COI sequences (along with COII and tRNAleu) could be used to identify the immature larvae of other forensically interesting Diptera (Lucilla illustris, Phormia regina and Phaenicia sericata). This study showed interspecic variation of 4% between C. vicina and C. vomitoria. This is similar to the work of Wells and Sperling [13] that found greater than 3% variation between Chrysomyinae y species. When the region sequenced in this study is compared with other forensically important UK dipteran species already deposited in GenBank there is 2.3% variation between the Calliphora samples in this study and the important Lucilia species. This partial COI sequence also provides unique nucleotides to differentiate between Lucilia species (ranging from one unique site in Lucilia sericata and Lucilia caesar to seven in Lucilia ampullacea). The DNA sequence from 212 individuals matched the consensus sequence (GenBank accession numbers AY536642 and AY536643). Seventeen individuals displayed different single base variations in this partial gene region. There was one individual sample with 2 bp differences to the consensus sequence (0.48% variation). These intra specic results are similar to those found by other researchers. Wells and Sperling [13] reported intraspecic level of variation within the COI and COII sequence for chrysomyinae ies of below 1%. Likewise, Otranto et al. [14], who used COI to distinguish between Hypoderma larvae, reported a rate of intraspecic variation ranging from 0.14% to 1.59%. None of the samples used in this study demonstrated intraspecic variation at any of the 17 interspecic base

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C. Ames et al. / Forensic Science International 164 (2006) 179182 [5] J.M. Butler, Forensic DNATyping, Academic Press, London, 2001, p. 322. [6] Y. Malgorn, R. Coquoz, DNA typing for identication of some species of Calliphoridae. An interest in forensic entomology, For. Sci. Int. 102 (1999) 111119. [7] S. Vincent, J.M. Vian, M.P. Carlotti, Partial sequencing of the cytochrome oxydase b subunit gene I: a tool for the identication of European species of blow ies for postmortem interval estimation, J. For. Sci. 45 (4) (2000) 820823. [8] J.F. Wallman, S.C. Donnellan, The utility of mitochondrial DNA sequences for the identication of forensically important blowies (Diptera: Calliphoridae) in southeastern Australia, For. Sci. Int. 120 (12) (2001) 6067. [9] M.L. Harvey, I.R. Dadour, S. Gaudieri, Mitochondrial DNA cytcochrome oxidase I gene: potential for distinction between immature stages of some forensically important y species (Diptera) in western Australia, For. Sci. Int. 131 (2003) 134139. [10] C. Simon, F. Frati, A. Beckenbach, B. Crespi, H. Liu, P. Flook, Evolution, weighting, and phylogenetic utility of mitochondrial gene-sequences and a compilation of conserved polymerase chain-reaction primers, Ann. Ent. Soc. Am. 87 (6) (1994) 651701. [11] J.D. Thompson, D.G. Higgins, T.J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specic gap penalties and weight matrix choice, Nucleic Acids Res. 22 (1994) 46734680. [12] F.A.H. Sperling, G.S. Anderson, D.A. Hickey, A DNA-based approach to the identication of insect species used for postmortem interval estimation, J. For. Sci. 39 (2) (1994) 418427. [13] J.D. Wells, F.A.H. Sperling, DNA-based identication of forensically important Chrysomyinae (Diptera: Calliphoridae), For. Sci. Int. 120 (12) (2001) 110115. [14] D. Otranto, D. Traversa, B. Guida, E. Tarsitano, P. Fiorente, J.R. Stevens, Molecular characterization of the mitochondrial cytochrome oxidase I gene of Oestridae species causing obligate myiasis, Med. Vet. Ent. 17 (3) (2003) 307315.

substitution positions and therefore not at the SfcI restriction enzyme sites. This is important as variation at these sites might lead to false species identication. 5. Conclusions This work has therefore indicated that the use of the COI region followed by a SfcI restriction enzyme digest provides a simple and relatively fast method of accurately distinguishing between two of the main UK Calliphora blowy species. This region of the COI gene also provides potential variation between other forensically important blowy species. Acknowledgements Helene Le Blanc from The University of Derby for providing some samples. Steven Tann-Ailward for help with sample collection. References
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