Blowfly species of the family Calliphoridae can be used in forensic investigations to estimate the minimum post-mortem interval (PMI¬min). Lucilia caesar and Lucilia illustris (Diptera: Calliphoridae) are closely related and phenotypically similar, making reliable identification difficult, especially if specimens are in poor condition.  To identify potential markers to genetically distinguish these species five complete mitochondrial genomes were sequenced: three for L. caesar (KM657111- KM657113) and two mitochondrial genomes for L. illustris (KM657109, KM 657110).  The ND6 gene contained the most species-specific SNPs (1.71%), followed by the ND5 gene (1.68%) and then the COI gene (1.56%), identifying ND6 and ND5 as valuable loci for differentiating L. Caesar and L. illustris specimens

Ahmadzai, Urszula Krzeminska

Goodwin, William H

Schoofs, Kathleen

Blowfly species of the family Calliphoridae can be used in forensic investigations to estimate the minimum post-mortem interval (PMImin). Lucilia caesar and Lucilia illustris (Diptera: Calliphoridae) are closely related and phenotypically similar, making reliable identification difficult. To identify potential genetic markers to distinguish these species, five complete mitochondrial genomes were sequenced: three for L. caesar (KM657111-KM657113) and two for L. illustris (KM657109, KM657110). The ND6 gene contained the most species-specific SNPs (1.71%), followed by the ND5 gene (1.68%) and the COI gene (1.56%), identifying ND6 and ND5 as valuable loci for differentiating L. caesar and L. illustris specimens

Krzeminska Ahmadzai, Urszula

Goodwin, William

Ghent University Academic Bibliography

Analysis of the complete mitochondrial genomes of two forensically important blowfly species : Lucilia caesar and Lucilia illustris

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Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=tmdn20Mitochondrial DNA Part BResourcesISSN: (Print) 2380-2359 (Online) Journal homepage: http://www.tandfonline.com/loi/tmdn20Analysis of the complete mitochondrial genomesof two forensically important blowfly species:Lucilia caesar and Lucilia illustrisKathleen R. Schoofs, Urszula Krzeminska Ahmadzai & William GoodwinTo cite this article: Kathleen R. Schoofs, Urszula Krzeminska Ahmadzai & William Goodwin(2018) Analysis of the complete mitochondrial genomes of two forensically important blowflyspecies: Lucilia￿caesar and Lucilia￿illustris, Mitochondrial DNA Part B, 3:2, 1114-1116, DOI:10.1080/23802359.2018.1457991To link to this article:  https://doi.org/10.1080/23802359.2018.1457991© 2018 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup.Published online: 09 Oct 2018.Submit your article to this journal Article views: 24View Crossmark dataMITO COMMUNICATIONAnalysis of the complete mitochondrial genomes of two forensically importantblowfly species: Lucilia caesar and Lucilia illustrisKathleen R. Schoofs, Urszula Krzeminska Ahmadzai and William GoodwinSchool of Forensic and Applied Sciences, University of Central Lancashire, Preston, UKABSTRACTBlowfly species of the family Calliphoridae can be used in forensic investigations to estimate the min-imum post-mortem interval (PMImin). Lucilia caesar and Lucilia illustris (Diptera: Calliphoridae) are closelyrelated and phenotypically similar, making reliable identification difficult. To identify potential geneticmarkers to distinguish these species, five complete mitochondrial genomes were sequenced: three forL. caesar (KM657111–KM657113) and two for L. illustris (KM657109, KM657110). The ND6 gene con-tained the most species-specific SNPs (1.71%), followed by the ND5 gene (1.68%) and the COI gene(1.56%), identifying ND6 and ND5 as valuable loci for differentiating L. caesar and L. illustris specimens.ARTICLE HISTORYReceived 26 February 2018Accepted 18 March 2018KEYWORDSForensic entomology; post-mortem interval; speciesidentification; Lucilia caesar;Lucilia illustrisSpecies from the family Calliphoridae are typically the first tocolonize a corpse (Rodriguez and Bass 1983) and their devel-opment can be used to determine the time since oviposition(Amendt et al. 2007), and hence an estimation of the PMImin,which can aid legal investigations (Easton and Smith 1970;Erzincllioglu 1983; Kulshrestha and Chandra 1987). The twoclosely related greenbottles Lucilia caesar and Lucilia illustrisare both members of the family Calliphoridae and are com-monly found in the UK. These species are very similar andmorphological identification is a challenging task even forexperienced entomologists (Stevens and Wall 1996; Wallmanand Donnellan 2001).mtDNA-based identification has been explored in previousstudies, most frequently looking at COI, to distinguish species,including L. caesar and L. illustris (Malgorn and Coquoz 1999;Wells and Sperling 2000; Wallman 2001; Harvey et al. 2003;Sonet et al. 2012). The COII gene, the 16S ribosomal geneand the cyt b gene also showed insufficient interspecificvariation between L. caesar and L. illustris (Sonet et al. 2012).The samples were all collected from a site in Burnley UK(53.7661140 N, 2.2366190 W), which is at an altitude of268 m. DNA was extracted using the DNeasyVR Blood andTissue Kit (Qiagen, Hilden, Germany). The genomes of fivespecimens were amplified with 21 custom-made primer pairsand Sanger sequenced (KM657109.1, KM657110.1,KM657111.1, KM657112.1, KM657113.1). DNA extracts arestored at the University of Central Lancashire, UK.Genes were identified using ORF Finder (Wheeler et al.2002), tRNAscan-SE (v 1.21) (Lowe and Eddy 1996), andalignment with other complete Lucilia mitochondrial genomes(L. sericata AJ422212, L. porphyrina JX913758, L. cuprinaJX913744) (Stevens et al. 2008; Nelson et al. 2012).Newly sequenced mitochondrial genomes of both speciesconsisted of 13 protein-coding genes, 22 tRNA genes and tworRNA genes: 23 are located on the heavy strand (tRNA-Ile,tRNA-Met, ND2, tRNA-Trp, COI, tRNA-Leu(UUA), COII, tRNA-Lys,tRNA-Asp, ATP8, ATP6, COIII, tRNA-Gly, ND3, tRNA-Ala, tRNA-Arg,tRNA-Asn, tRNA-Ser(AGC), tRNA-Glu, tRNA-Thr, ND6, cyt b, andtRNA-Ser(UCA)). The other 14 genes are located on the lightstrand. This genome organization is consistent with otherLucilia species AJ422212, JX913758, and JX913744 (Stevenset al. 2008; Nelson et al. 2012).Start and stop codons were also similar to other DipteraElodia flavipalpis (Zhao et al. 2013) and Chrysomya chloropyga(Junqueira et al. 2004): the most used start codon was ATG(6), but ATT (4) and ATA (2) were also present; the COI startcodon is TCG (serine), which is not a typical start codon, buthas been observed in other Diptera (including L. cuprina andL. sericata) – the ATTTAA sequence adjacent to the startcodon is involved in translation initiation (Junqueira et al.2004). Incomplete termination codons (T) were observed forCOII, ND4, and ND5; it is thought that the termination codonis completed by polyadenylation (Lessinger et al. 2000).Two extra copies of tRNA-Leu and tRNA-Ser were found,which is typical for most Diptera. Some Diptera contain moreduplicate copies of tRNAs, but this was not the case for L.caesar and L. illustris (Lessinger et al. 2000; Harvey et al. 2003;Junqueira et al. 2004; Duarte et al. 2008). Anti-codons of thetRNAs were identified and showed to be consistent withother Diptera (L. sericata AJ422212, L. porphyrina, JX913758, L.cuprina JX913744). Based on tRNAscan-SE results and RNAfold(Lorenz et al. 2011), all identified tRNAs successfully foldedCONTACT William Goodwin whgoodwin@uclan.ac.uk School of Forensic and Applied Sciences, University of Central Lancashire, Preston, UK 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.MITOCHONDRIAL DNA PART B: RESOURCES, 2018VOL. 3, NO. 2, 1114–1116https://doi.org/10.1080/23802359.2018.1457991into typical cloverleaf structures, except for tRNA-Ser(AGC),which missed the D stem. However, this has been observedin other species as well, including Diptera (Junqueiraet al. 2004).The length of the ribosomal RNAs, 1327 bp for the 16Ssubunit and 787 bp for the 12S subunit was the same asother Lucilia species (L. sericata AJ422212, L. porphyrinaJX913758, and L. cuprina JX913744). The control regions of L.caesar and L. illustris were 1122 bp and 1121 bp, respectively;this is shorter than L. sericata (1125 bp) and L. cuprina (1136),but larger than L. porphyrina (1047 bp).Besides finding possible new genetic markers, the com-plete mitochondrial genomes provide insight into the evolu-tion of Calliphoridae. To infer phylogenetic relationshipsamong Calliphoridae, we performed Bayesian phylogeneticanalysis based on complete mitochondrial genome sequences(Figure 1). PartitionFinder (Lanfear et al. 2012) was run priorto phylogenetic analysis to find the most supported data par-titioning and associated substitution models based on theBayesian Information Criterion (BIC). Fourteen partitions wereidentified for the alignment of the complete mitochondrialgenomes of analysed Calliphoridae. All tested Lucilia speciesformed a monophyletic group. Phylogenetic analysis con-firmed L. illustris and L. caesar as sister species. There weretwo distinct clades among tested Calliphoridae species, oneincluded Lucilia spp., Calliphora spp., Hemipyrellia sp.,Aldrichina sp. while the other clade included Chrysomya spp.,Protophormia sp., and Phormia sp. A polytomy was present inthe final tree, represented by Cochliomyia sp.Most studies for genetic identification of L. caesar and L.illustris have been based on the COI gene. Other mitochon-drial genes that have been used are the COII gene, the 16Sribosomal gene and the cyt b gene, but these show insuffi-cient interspecific variation between these two Calliphoridaeindicating that additional genetic markers need to be devel-oped (Sonet et al. 2012). In this study, the ND6 and ND5genes contained the most species specific SNPs (1.71% and1.68%, respectively), followed by the COI gene (1.56%).Although these results are based on only few mitochondrialgenomes, the results are consistent with the study of Nelsonet al. (2012), where the ND6 gene showed the highestinterspecific genetic variation within Calliphoridae (andFigure 1. The Calliphoridae (NC_019636, NC_026668, NC_009733, NC_019637, KM657109-KM657113, NC_019573, NC_029486, NC_019638, NC_002660, NC_019635,NC_019634, NC_002697, NC_025338, NC_031381, NC_028412, NC_019633, NC_019632, NC_019631, NC_028411, NC_019639, NC_029215, NC_026996) phylogeneticsummary tree inferred using the Bayesian approach implemented in MrBayes 3.2 (Ronquist et al. 2012) with Sarcophagidae: Sarcophaga crassipalpis (NC_026667)and Ravinia pernix (NC_026196) as an outgroup (not shown). Mitogenomes sequenced in this study are highlighted. All nodes are well-supported (PP> 0.9). Fourindependent runs were performed for 10 million generations and sampled every 1000 generations. Convergence of replicates was verified in TRACER v. 1.6(Rambaut et al. 2014) by ensuring unimodality of posterior distributions and effective sample size (ESS >200) for all parameters. Trees were summarized using the50% majority rule method, after discarding the first 25% of the sample as burnin, and visualized using FIGTREE 1.4.2 (Rambaut 2012).MITOCHONDRIAL DNA PART B: RESOURCES 1115Chrysomyinae), when compared to other mitochondrialgenes. Further work, including population studies, is neededto evaluate the value of ND6 and NR5 as markers for differen-tiating between L. caesar and L. illustris in forensic casework.Disclosure statementThe authors report no conflict of interest and are responsible for the con-tent and writing of this article. The University of Central Lancashire’sEthics Committee approved all experimental procedures.ReferencesAmendt J, Campobasso CP, Gaudry E, Reiter C, Leblanc HN, Hall M. 2007.Best practice in forensic entomology-standards and guidelines. Int JLegal Med. 121:90–104.Duarte GT, De Azeredo-Espin AML, Junqueira ACM. 2008. 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Analysis of the complete mitochondrial genomes of two forensically important blowfly species: Lucilia caeasar and Lucilia illustris

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Analysis of the complete mitochondrial genomes of two forensically important blowfly species: Lucilia caeasar and Lucilia illustris

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