Journal List > Korean J Leg Med > v.37(4) > 1004710

Park and Shin: Molecular Species Identification of Forensically Important Flies in Korea

Abstract

To estimate the postmortem interval (PMI) by using entomological evidence, species identification of forensically important flies is mandatory. However, the traditional species identification method, which relies on the key morphological features of adult flies, is not always available to investigators and has limitations to the immature samples. Because of these limitations, species identification using DNA sequences has long been an issue in the field of forensic entomology. In this review, I have briefly described the basic principles of molecular species identification and phylogenetic analysis and their applications in forensic entomology. I also recommend an experimental and statistical method to identify unknown fly samples obtained from the field.

REFERENCES

1. Gennard DE. Forensic entomology: an introduction. 2nd ed.Chichester: John Wiley & Sons;2012.
2. Sperling FA, Anderson GS, Hickey DA. A DNA-based approach to the identification of insect species used for postmortem interval estimation. J Forensic Sci. 1994; 39:418–27.
3. Singh B, Wells JD. Molecular systematics of the Calliphoridae (Diptera: Oestroidea): evidence from one mitochondrial and three nuclear genes. J Med Entomol. 2013; 50:15–23.
crossref
4. Zaidi F, Wei Sj, Shi M, et al. Utility of multi-gene loci for forensic species diagnosis of blowflies. J Insect Sci. 2011; 11:1–12.
crossref
5. Boehme P, Amendt J, Zehner R. The use of COI barcodes for molecular identification of forensically important fly species in Germany. Parasitol Res. 2012; 110:2325–32.
crossref
6. Cognato AI. Standard percent DNA sequence difference for insects does not predict species boundaries. J Econ Entomol. 2006; 99:1037–45.
crossref
7. Wells JD, Lunt N, Villet MH. Recent African derivation of Chrysomya putoria from C. chloropyga and mitochondrial DNA paraphyly of cytochrome oxidase subunit one in blowflies of forensic importance. Med Vet Entomol. 2004; 18:445–8.
crossref
8. Kumar S, Nei M, Dudley J, et al. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform. 2008; 9:299–306.
crossref
9. Kumar S, Tamura K, Nei M. MEGA: Molecular evolutionary genetics analysis software for microcomputers. Comput Appl Biosci. 1994; 10:189–91.
crossref
10. Tamura K, Dudley J, Nei M, et al. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007; 24:1596–9.
crossref
11. Graur D, Li W-H. Fundamentals of molecular evolution. 2nd ed.Sunderland: Sinauer Associates;2000.
12. Huelsenbeck JP, Ronquist F. MRBAYES: Bayesian infer-ence of phylogenetic trees. Bioinformatics. 2001; 17:754–5.
crossref
13. Caine ′ LM, Real FC, Salon ̃a-Bordas MI, et al. DNA typing of Diptera collected from human corpses in Portugal. Forensic Sci Int. 2009; 184:e21–3.
14. Chen WY, Hung TH, Shiao SF. Molecular identification of forensically important blow fly species (Diptera: Calliphoridae) in Taiwan. J Med Entomol. 2004; 41:47–57.
crossref
15. Debry RW, Timm AE, Dahlem GA, et al. mtDNA-based identification of Lucilia cuprina (Wiedemann) and Lucilia sericata (Meigen) (Diptera: Calliphoridae) in the continental United States. Forensic Sci Int. 2010; 202:102–9.
crossref
16. GilArriortua M, Salona Bordas MI, Caine ′ LM, et al. Cytochrome b as a useful tool for the identification of blowflies of forensic interest (Diptera, Calliphoridae). Forensic Sci Int. 2013; 228:132–6.
crossref
17. Guo YD, Cai JF, Li X, et al. Identification of the forensically important sarcophagid flies Boerttcherisca peregrina, Parasarcophaga albiceps and Parasarcophaga dux (Diptera: Sarcophagidae) based on COII gene in China. Trop Biomed. 2010; 27:451–60.
18. Harvey ML, Dadour IR, Gaudieri S. Mitochondrial DNA cytochrome oxidase I gene: potential for distinction between immature stages of some forensically important fly species (Diptera) in western Australia. Forensic Sci Int. 2003; 131:134–9.
crossref
19. Jordaens K, Sonet G, Richet R, et al. Identification of forensically important Sarcophaga species (Diptera: Sarcophagidae) using the mitochondrial COI gene. Int J Legal Med. 2013; 127:491–504.
crossref
20. Kavitha R, Nazni WA, Tan TC, et al. Molecular identification of blow flies recovered from human cadavers during crime scene investigations in Malaysia. Malays J Pathol. 2012; 34:127–32.
21. Meiklejohn KA, Wallman JF, Dowton M. DNA-based identification of forensically important Australian Sarcophagidae (Diptera). Int J Legal Med. 2011; 125:27–32.
crossref
22. Meiklejohn KA, Wallman JF, Dowton M. DNA barcoding identifies all immature life stages of a forensically important flesh fly (Diptera: Sarcophagidae). J Forensic Sci. 2013; 58:184–7.
crossref
23. Park SH, Zhang Y, Piao H, et al. Use of cytochrome c oxidase subunit i (COI) nucleotide sequences for identification of the Korean Luciliinae fly species (Diptera: Calliphoridae) in forensic investigations. J Korean Med Sci. 2009; 24:1058–63.
crossref
24. Park SH, Zhang Y, Piao H, et al. Sequences of the cytochrome C oxidase subunit I (COI) gene are suitable for species identification of Korean Calliphorinae flies of forensic importance (Diptera: Calliphoridae). J Forensic Sci. 2009; 54:1131–4.
crossref
25. Saigusa K, Matsumasa M, Yashima Y, et al. Practical applications of molecular biological species identification of forensically important flies. Leg Med (Tokyo). 2009; 11(Suppl 1):S344–7.
crossref
26. Saigusa K, Takamiya M, Aoki Y. Species identification of the forensically important flies in Iwate prefecture, Japan based on mitochondrial cytochrome oxidase gene subunit I (COI) sequences. Leg Med (Tokyo). 2005; 7:175–8.
crossref
27. Stevens J, Wall R. Species, sub-species and hybrid populations of the blowflies Lucilia cuprina and Lucilia sericata (Diptera: Calliphoridae). Proc Biol Sci. 1996; 263:1335–41.
28. Stevens JR, Wall R, Wells JD. Paraphyly in Hawaiian hybrid blowfly populations and the evolutionary history of anthropophilic species. Insect Mol Biol. 2002; 11:141–8.
crossref
29. Wallman JF, Donnellan SC. The utility of mitochondrial D-NA sequences for the identification of forensically important blowflies (Diptera: Calliphoridae) in southeastern Australia. Forensic Sci Int. 2001; 120:60–7.
crossref
30. Wells JD, Pape T, Sperling FA. DNA-based identification and molecular systematics of forensically important Sarcophagidae (Diptera). J Forensic Sci. 2001; 46:1098–102.
crossref
31. Wells JD, Sperling FA. A DNA-based approach to the identification of insect species used for postmortem interval estimation and partial sequencing of the cytochrome oxy-dase b subunit gene I: a tool for the identification of European species of blow flies for postmortem interval estimation. J Forensic Sci. 2000; 45:1358–9.
32. Wells JD, Wall R, Stevens JR. Phylogenetic analysis of forensically important Lucilia flies based on cytochrome oxidase I sequence: a cautionary tale for forensic species determination. Int J Legal Med. 2007; 121:229–33.
crossref
33. Sonet G, Jordaens K, Braet Y, et al. Why is the molecular identification of the forensically important blowfly species Lucilia caesar and L. illustris (family Calliphoridae) so problematic? Forensic Sci Int. 2012; 223:153–9.
34. Piao HG, Chung U, Shin SE, et al. DNA-based identification of necrophagous fly species using abdominal-B (Abd-B) homeobox sequence. Korean J Leg Med. 2012; 36:74–84.
35. Park SH, Park CH, Zhang Y, et al. Using the developmental gene bicoid to identify species of forensically important blowflies (Diptera: Calliphoridae). Biomed Res Int. 2013. DOI: doi: 10.1155/2013/538051. Epub 2013 Mar 18.
36. Zhang Y. Identification of carrion flies by ultrabithorax (Ubx) and abdominal-A (abd-A) homeobox. Seoul: Korea University;2007.
37. Park SH. Molecular identification of necrophagous fly species by sequence analysis of Dfd and Scr homeoboxes. Seoul: Korea University;2007.
38. Kim SY, Park SH, Piao H, et al. Vouchering of forensically important fly specimens by nondestructive DNA extraction. ISRN Entomology. 2013. Article ID 286182.
crossref

Fig. 1.
An example of a neighbor-joining phylogenetic tree, using COI sequences of modern human (Homo sapiens), Neanderthal man (Homo neanderthalensis), chimpanzee (Pan troglodytes), bonobo (Pan paniscus), gorilla (Gorilla gorilla), and orangutan (Pongo abelii), illustrates the key features of a phylogenetic tree such as nodes, branches, bootstrap numbers, and the scale.
kjlm-37-177f1.tif
Table 1.
List of Forensically Important Fly Species Commonly Collected in Korea
Family Subfamily Species Genbank
Fanniidae   Fannia prisca JX861413-7
Muscidae Azelinae Hydrotaea dentipes JX861420-JX861429
  Hydrotaea occulta JX861430
  Ophyra chalcogaster JX861452-JX861456
  Ophyra leucostoma JX861457-JX861459
  Ophyra nigra JX861460-JX861468
  Muscina angustifrons JX861436-JX861444
  Muscina stabulans JX861449-JX861451
  Muscina pascuorum JX861445-JX861448
Muscinae Musca domestica JX861431-JX861435
Phaoniinae Phaonia aurea JX861481&JX861482
Calliphoridae Calliphorinae Adrichina grahami EU880180-EU880182
  Calliphora lata EU880183-EU880187
  Calliphora vicina EU880188-EU880192
  Triceratopyga calliphoroides EU880176-EU880179
Chrysomyinae Chrysomya megacephala KF037969
  Chrysomya pinguis FJ195381
  Phormia regina JN257239&FJ360867
Luciliinae Hemipyrellia ligurriens EU880206&EU880207
  Lucilia ampullaceae EU925394
  Lucilia caesar EU880193-EU880196
  Lucilia illustris EU880197-EU880205
  Phaenicia sericata EU880208-EU880212
Sarcophagidae Sarcophaginae Sarcophaga haemorrhoidalis JX861406-JX861408
  Boettcherisca peregrina JX861409-JX861412
  Helicophagella melanura JX861418&JX861419
  Parasarcophaga albiceps JX861469-JX861473
  Parasarcophaga harpax JX861474&JX861475
  Parasarcophaga similis JX861476-JX861480

Because the COI sequences of Korean Chrysomyinae flies were not submitted to NCBI Genbank yet, the listed accession numbers are those of conspecifics of which sequences are consistent with Korean counterparts.

Table 2.
Universal Primer Sequences for COI
Name Sequence Binding site
F1 5 5′ -CCTTTAGAATTGCAGTCTAATGTCA-3′ tRNA-cysteine
F2 5 5′ -GGAGGATTTGGAAATTGATTAGTTCC-3′ ′220-245 on COI
F3 5 5′ -CTGCTACTTTATGAGCTTTAGG-3′ 1000-1022 on COI
R1 5 5′ -CCTAAATTTGCTCATGTTGACA-3′ 2-23 on COII
R2 5 5′ -CAAGTTGTGTAAGCATC-3′ 1327-1343 on COI
R3 5 5′ -CCAAAGAATCAAAATAAATGTTG-3′ 688-710 on COI

COI: Cytochrome c oxidase subunit I;

COII: Cytochrome c oxidase subunit II

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