Original Article

Molecular Species Identification of Six Forensically Important Iranian Flesh Flies (Diptera)


Background: Flesh flies (Diptera: Sarcophagidae) are considered as myiasis agents and important evidences in forensic investigations. However, their use has been restricted because, at all larval stages and female adults, morphological species identification is difficult or very challenging. This study investigated to test utility of mitochondrial cytochrome oxidase subunit I (mt-COI) sequences for differentiation of six forensically important Iranian flesh flies namely, Sarcophaga crassipalpis, S. flagellifera, S. hirtipes, S. aegyptica, S. africa and S. argyrostoma.Methods: Male specimens were morphologically identified to species level and then the genomic DNA of the flies were extracted and subjected to polymerase chain reaction (PCR) against mt-COI gene. The PCR products were sequenced and the obtained sequences were analyzed for the species specific restriction fragment length polymorphisms (RFLPs).Results: Rate of genetic variation between species was 6–10% which was enough to find restriction enzymes (RE) that were able to produce species-specific RFLP profiles. Combinations of three REs: BsrFI, RsaI and HinfI, provided diagnostic bands for identification of the six Sarcophaga species.Conclusions: The results of this study showed that molecular markers such as RFLPs enhancing the use of evidence from flesh flies in forensic investigation. However, lack proper restriction sites in the COI region inhibited introduction of a single restriction enzyme for easy species identification. It is recommended to apply larger part of DNA such as combination of COI and COII genes to provide better RFLP markers for species identification of flesh flies.
1. Amendt J, Campobasso CP, Gaudry E, Reiter C, LeBlanc HN, Hall MJ (2007) Best practice in forensic entomology--stand¬ards and guidelines. Int J Legal Med. 121(2): 90–104.
2. Amendt J, Krettek R, Zehner R (2004) Fo-rensic entomology. Die Naturwissen-schaf¬ten. 91(2): 51–65.
3. Byrd JH, Castner JL (2001) Forensic En-tomology the Utility of Arthropods in Legal Investigations. CRC Press, New York.
4. Goff ML (1993) Estimation of postmortem interval using arthropod development and successional patterns. Forensic Sci Rev. 5(2): 81–94.
5. Kamal AS (1958) Comparative study of thirteen species of sarcosaprophagous Cal¬liphoridae and Sarcophagidae (Diptera) I. Bionomics. Ann Entomol Soc Am. 51 (3): 261–271.
6. Wells JD, Pape T, Sperling FA (2001) DNA-based identification and molecular systematics of forensically important Sarcophagidae (Diptera). J Forensic Sci. 46 (5): 1098–1102.
7. Pape T (1996) Catalogue of the Sarcopha-gidae of the World (Insecta: Diptera). Associated Publishers, Gainesville, Flor-ida.
8. Saigusa K, Takamiya M, Aoki Y (2005) Spe¬cies identification of the forensically im¬portant flies in Iwate prefecture, Japan based on mitochondrial cytochrome oxi¬dase gene subunit I (COI) sequences. Le¬gal Med-Tokyo. 7(3): 175–178.
9. Dahlem GA DW (1996) Revision of the ge-nus Boetcheria in America north of Mex¬ico (Diptera: Sarcophagidae). Insecta Mun¬di. 10: 77–103.
10. Povolný DVY (1997) The flesh flies of Central Europe. Spixiana Zeit Zool Suppl.
24: 1–263.
11. Wells JD, Stevens JR (2008) Application of DNA-based methods in forensic en-tomology. Annu Rev Entomol. 53: 103–120.
12. Zehner R, Amendt J, Schutt S, Sauer J, Krettek R, Povolny D (2004) Genetic identification of forensically important flesh flies (Diptera: Sarcophagidae). Int J Legal Med. 118(4): 245–247.
13. Sperling FA, Anderson GS, Hickey DA (1994) A DNA-based approach to the identification of insect species used for postmortem interval estimation. J Fo-rensic Sci. 39(2): 418–427.
14. Harvey AG, Bryant RA, Tarrier N (2003) Cognitive behaviour therapy for post-traumatic stress disorder. Clin Psychol Rev. 23(3): 501–522.
15. Meier R, Zhang G, Ali F (2008) The use of mean instead of smallest interspecific distances exaggerates the size of the “Barcoding Gap” and leads to misiden-tification. Syst Biol. 57(5): 809–813.
16. Whitworth TL, Dawson RD, Magalon H, Baudry E (2007) DNA barcoding can-not reliably identify species of the blow-fly genus Protocalliphora (Diptera: Cal-liphoridae). Proc Royal Soc. 274(1619): 1731–1739.
17. Oshaghi MA, Yaghoobi F, Abai MR (2006) Pattern of mitochondrial DNA variation between and within Anopheles stephensi (Diptera: Culicidae) biological forms sug¬gests extensive gene flow. Acta Trop. 99(2–3): 226–233.
18. Oshaghi MA, Shemshad K, Yaghobi-Er-shadi MR, Pedram M, Vatandoost H, Abai MR, Akbarzadeh K, Mohtarami F (2007) Genetic structure of the malaria vector Anoph¬eles superpictus in Iran using mitochondrial cytochrome oxidase (COI and COII) and morphologic mark-ers: a new species complex? Acta Trop. 101(3): 241–248.
19. Karimian F, Oshaghi MA, Sedaghat MM,
Waterhouse RM, Vatandoost H, Hanafi-Bojd AA, Ravasan NM, Chavshin AR (2014) Phylogenetic analysis of the Oriental-Palearctic-Afrotropical members of Anopheles (Culicidae: Diptera) based on nuclear rDNA and mitochondrial DNA characteristics. Jpn J Infect Dis. 67(5): 361–367.
20. Hashemi-Aghdam SS, Oshaghi MA (2015) A checklist of Iranian cockroaches (Blat¬todea) with description of Polyphaga sp as a new species in Iran. J Arthropod Borne Dis. 9(2): 161–175.
21. Hashemi-Aghdam SS, Rafie G, Akbari S, Oshaghi MA (2017) Utility of mtDNA-COI barcode region for phylogenetic re-lationship and diagnosis of five common pest cockroaches. J Arthropod Borne Dis. 11(2): 182–193.
22. Khanzadeh F, Khaghaninia S, Maleki-Rav¬asan N, Oshaghi MA, Adler PH (2020) Black flies (Diptera: Simuliidae) of the Aras River Basin: species composition and floral visitation. Acta Trop. 209: 105536.
22. Ratcliffe ST, Webb DW, Weinzievr RA, Robertson HM (2003) PCR-RFLP iden-tification of Diptera (Calliphoridae, Mus¬cidae and Sarcophagidae)--a generally applicable method. J Forensic Sci. 48 (4): 783–785.
23. Guo YD, Cai JF, Meng FM, Chang YF, Gu Y, Lan LM, Liang L, Wen JF (2012) Identification of forensically important flesh flies based on a shorter fragment of the cytochrome oxidase subunit I gene in China. Med Vet Entomol. 26(3): 307–313.
24. Guo Y, Zha L, Yan W, Li P, Cai J, Wu L (2014) Identification of forensically im-portant sarcophagid flies (Diptera: Sar-cophagidae) in China based on COI and period gene. Int J Legal Med. 128(1): 221–228.
25. Jordaens K, Sonet G, Richet R, Dupont E, Braet Y, Desmyter S (2013) Identifica-tion of forensically important Sarcophaga spe¬cies (Diptera: Sarcophagidae) us¬ing the mitochondrial COI gene. Int J Legal Med. 127(2): 491–504.
26. Meiklejohn KA, Wallman JF, Dowton M (2011) DNA-based identification of fo-ren¬sically important Australian Sarcoph-agidae (Diptera). Int J Legal Med. 125 (1): 27–32.
27. Kim YH, Shin SE, Ham CS, Kim SY, Ko KS, Jo TH (2014) Molecular identifica-tion of necrophagous muscidae and sar-cophagidae fly species collected in Ko-rea by mitochondrial cytochrome C oxi-dase subunit I nucleotide sequences. Sci World J. 2014: 275085.
28. Zhang C, Fu X, Xie K, Yan W, Guo Y (2015) MtDNA analysis for genetic iden¬tification of forensically important sar¬cophagid flies (Diptera: Sarcopha-gidae) in China. J Med Entomol. 52(6): 1225–1233.
29. Sharma M, Singh D, Sharma AK (2015) Mitochondrial DNA based identifica-tion of forensically important Indian flesh flies (Diptera: Sarcophagidae). Forensic Sci Int. 247: 1–6.
30. Talebzadeh F (2017) Insect fauna of hu-man cadavers in Tehran d istrict. [MSc Thesis]. School of Public Health, Tehran University of Medical Sciences, Iran.
31. Zumpt F (1965) Myiasis in Man and An-imals in the Old World, a textbook for physicians, veterinarians and zoologist. Butterwort, London.
32. McAlpine JF (1981) Morphology and ter-minology-adults. In: McAlpine JF, Pe-terson BV, Shewell GE, Teskey HJ, Vock¬eroth JR, Wood DM (Eds): Manual of Nearctic Diptera. Vol. 1. Research Branch, Agriculture Canada, Monograph No. 27, pp. 9–63.
33. Folmer O, Black M, Hoeh W, Lutz R, Vri-jenhoek R (1994) DNA primers for am-plification of mitochondrial cytochrome c oxidase subunit I from diverse meta-zoan invertebrates. Mol Mar Biol Bio-tech¬nol. 3(5): 294–299.
34. Larkin MA BG, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics. 23(21): 2947–2948.
35. Akbarzadeh K, Saghafipour A, Jesri N, Karami-Jooshin M, Arzamani K, Haz-ratian T, Kordshouli RS, Afshar AA (2018) Spatial distribution of necro-phagous flies of infraorder Muscomor-pha in Iran using geographical infor-mation system. J Med Entomol. 55(5): 1071–1085.
36. Crozier RH, Crozier YC (1993) The mito-chondrial genome of the honeybee Apis mellifera: complete sequence and genome organization. Genetics. 133(1): 97–117.
37. Hebert PD, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through DNA barcodes. Proc Biol Sci. 270(1512): 313–321.
38. Hebert PD, Ratnasingham S, deWaard JR (2003) Barcoding animal life: cyto-chrome c oxidase subunit 1 divergences among closely related species. Proc Biol Sci. 270(Suppl 1): S96–9.
39. Ball SL HP, Burian SK, Webb JM (2005) Biological identifications of mayflies (Ephemeroptera) using DNA barcodes. J North Am Benthol Soc. 24: 508–524.
40. Tan M, Alhajj R, Polat F (2010) Scalable approach for effective control of gene regulatory networks. Artif Intell Med. 48 (1): 51–59.
41. Jafari S, Oshaghi MA, Akbarzadeh K, Abai MR, Koosha M, Mohtarami F (2019) Iden¬tification of forensically important flesh flies using the cytochrome c oxi¬dase subunits I and II genes. J Med En¬tomol. 56(5): 1253–1259.
42. Bakhshi H, Oshaghi MA, Abai MR, Rassi Y, Akhavan AA, Mohebali M (2013) MtDNA CytB structure of Rhombomys opimus (Rodentia: Gerbellidae), the main reservoir of cutaneous leishmaniasis in the borderline of Iran-Turkmenistan. J Arthropod Borne Dis. 7(2): 173–184.
43. Oshaghi MA, Rafinejad J, Choubdar N, Piazak N, Vatandoost H, Telmadarraiy Z (2011) Discrimination of relapsing fe-ver Borrelia persica and Borrelia mi-crotti by diagnostic species-specific pri-mers and polymerase chain reaction-re-striction fragment length polymorphism. Vector Borne Zoonotic Dis. 11(3): 201–207.
44. Oshaghi MA, Ravasan NM, Hide M, Ja-vadian EA, Rassi Y, Sedaghat MM (2009) Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L. infantum/L. do¬novani discrimination. Exp Parasitol. 122 (1): 61–65.
45. Oshaghi MA, Rasolian M, Shirzadi MR, Mohtarami F, Doosti S (2010) First re-port on isolation of Leishmania tropica from sandflies of a classical urban cuta-neous leishmaniasis focus in southern Iran. Exp Parasitol. 126(4): 445–450.
46. Rassi Y, Oshaghi MA, Azani SM, Abai MR, Rafizadeh S, Mohebai M (2011) Molecular detection of Leishmania in-fection due to Leishmania major and Leishmania turanica in the vectors and reservoir host in Iran. Vector Borne Zo-onotic Dis. 11(2): 145–150.
47. Oshaghi MA, Chavshin AR, Vatandoost H (2006) Analysis of mosquito blood-meals using RFLP markers. Exp Parasi-tol. 114(4): 259–264.
48. Anjomruz M, Oshaghi MA, Sedaghat MM, Pourfatollah AA, Raeisi A, Vatandoost H (2014) ABO blood groups of residents and the ABO host choice of malaria vec¬tors in southern Iran. Exp Parasitol. 136: 63–67.
IssueVol 14 No 4 (2020) QRcode
SectionOriginal Article
DOI https://doi.org/10.18502/jad.v14i4.5279
Sarcophagidae; Forensic entomology; Flesh fly; mtDNA COI; PCR-RFLP

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Talebzadeh F, Oshaghi MA, Akbarzadeh K, Panahi-Moghadam S. Molecular Species Identification of Six Forensically Important Iranian Flesh Flies (Diptera). J Arthropod Borne Dis. 14(4):416–424.