Forensic Science International 229 (2013) e26e29

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Forensic Science International

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Case report

Human DNA extraction from empty puparia

Daniela Marchetti a, Elisa Arena a,b,*, Ilaria Boschi a, Stefano Vanin c
a

Institute of Legal Medicine, Catholic University, Medical School, Rome, Italy INPS, National Institute of Social Security, Rome, Italy c Department of Chemical & Biological Sciences, School of Applied Science, University of Hudderseld, Queensgate, Hudderseld HD1 3DH, UK
b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 September 2012 Received in revised form 18 March 2013 Accepted 23 March 2013 Available online 23 April 2013 Keywords: Forensic genetics Forensic entomology DNA characterization Diptera Lucilia sericata

Empty puparia, as well as larvae at different developmental stages, are potentially useful in the identication of a victim where a corpse has been removed from the scene of a forensic investigation. To evaluate the relevance and the reliability of this substrate to be used as forensic evidence, the authors report for the rst time the extraction and typing of human DNA from empty puparia using STR analysis, in two actual cases where the bodies of the victims were still present thereby enabling validation of the typing. 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction At the end of y metamorphosis, an emerging adult y leaves behind the hardened puparium and thin exuvial membranes (the puparial shell and the prepupal skin). Puparia represent the outer skin of the last developmental immature stage of the holometabolous carrion breeding ies. The heavy sclerotization of the outer surface varies from light brown to dark brown or black in color and, with a few exceptions between closely related species, does not allow for discrimination between species. In Post Mortem Interval (PMI) estimation, a correct species identication is crucial [1,2]. For this reason, puparia have been recently identied using mt-DNA analysis [3]. To our knowledge, there are no published reports that demonstrate the extraction and complete characterization of human DNA from puparia. Theoretically, obtaining this DNA is of forensic importance for corpse identication if puparia have been found in cases where a corpse is missing or it has been removed from a crime scene after a complete or partial decomposition [4]. A review of the literature shows that human DNA has been successfully recovered from the alimentary canal of 3rd instar larvae [5,6] as well as from the gut contents of beetle larvae, lice, ticks and mosquitoes [79].

Host tissue ingested by y larvae is usually eliminated from the alimentary canal in the post feeding larval stage and during metamorphosis. It is possible that host tissue is not completely lost but some is trapped among the cuticular spines and folds of the pupa. According to Replogle et al. [10], who recovered host DNA from lice excrement, it is also possible that host material can be extracted in the meconium and eliminated at the end of metamorphosis. A noncellular peritrophic matrix is formed around the food bolus in the midgut of many arthropod species [11] and separates the midgut epithelial cells from ingested food [12]. The authors hypothesize that human DNA can be present inside empty puparia, in the meconial peritrophic membrane, as well as in other larval structures (e.g., the cephalopharyngeal skeleton), and that human DNA can be found on the external side of the empty puparia that are in direct contact with the feeding substratum. Our study focuses on the possibility to isolate and identify human DNA from puparia using STR analysis. 2. Materials and methods 2.1. Sampling and preservation Empty puparia were obtained after adults had emerged from whole puparia collected from two cases that were investigated at the Forensic Institute of the University Sacro Cuore of Rome, in August 2011. One case involved a male whose identity was unknown, found hanging in the countryside near Rome in summer. The corpse was

* Corresponding author at: Institute of Legal Medicine, Catholic University, Largo F. Vito, 1 00168 Rome, Italy. Tel.: +39 06 35507031; fax: +39 06 35507033. E-mail address: elisarena@libero.it (E. Arena). 0379-0738/$ see front matter 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.forsciint.2013.03.043

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in an advanced state of decomposition. Cause of death was dened by autopsy as asphyxia. A genetic identication of the body was required. The second case involved a 50-years-old female, found naked in a natural park near Rome. She had been missing for about 7 days prior to her discovery. The corpse showed signs of putrefaction. The autopsy and the toxicological examination revealed death due to acute drug intoxication. A genetic investigation for sexual abuse was required. The genetic results showed only the victims prole. The bodies were colonized by insects, mainly by y larvae, at different developmental stages. Larvae at different stages were taken from each corpse, killed in boiling water and preserved in 80% ethanol as suggested by Amendt et al. [13]. In addition, two pupae were collected from each corpse and they were placed into separate containers that were one-third lled with sand and covered with a mesh lid. The containers were placed into a cabinet maintained at a constant temperature of 24 (1)8C. After ve days, adult ies emerged from the hardened puparia. All of the adult ies were killed and placed in a 70% ethanol solution. Puparia were maintained in the cabinet for seven days before being stored at 20 8C for two months and then processed for DNA extraction. The puparia from case 1 were labeled A and B and those from case 2, C and D. Cleaning puparial surfaces. The external surface of each puparium was carefully cleaned using a swab moistened with sterile distilled water. A second dry swab was used to remove traces of water and external contamination. Puparial cases were dissected using a sterile blade, cut in small pieces, placed in 1.5 ml Eppendorf tubes and immediately processed. 2.2. Isolation and extraction of DNA In case 1, DNA was extracted using two different methods. DNA from sample A was extracted using the Chelex1-100 (Bio-Rad) procedure whereas DNA from sample B was extracted using a PrepFiler1 Kit (Applied Biosystem). In case 2, DNA from both puparia was extracted using the PrepFiler1 Kit. Chelex extraction was performed according to the protocol reported by Walsh et al. (1): 500 ml of 5% Chelex 100 resin in sterile H2O was added to sample and incubated at 56 8C for 60 min. The sample was then boiled for 810 min, and the resin was separated from DNA by centrifugation. The PrepFiler1 Kit (Applied Biosystem) was used according to Users Manual protocol [14] with some few volumes modication as routinely adopted in our laboratory to improve DNA extraction from epithelial cells on thick substrate (data unpublished): 400 ml of Lysis buffer and 3 ml of 1.0 M dithiotreitol (DTT) were added to samples in 1.5 ml tubes. The lysis mixture was incubated at 70 8C for 120 min, and the solution was vortexed 45 times. Cheratinic substrate was removed by using PrepFiler1 lter columns after 2 min of centrifugation. Then, 180 ml of isopropanol and 20 ml of magnetic particles were added to the lysate mixture to bind genomic DNA. Then DNA was washed following the Users Manual protocol. A total of 30 ml of elution buffer was added to the resin to release the DNA from the magnetic particles, and the tubes were vortexed at maximum speed for 10 s and incubated at 70 8C for 8 min. Finally, the tubes were placed on the magnetic stand for 50 . DNA solution was then recovered by pipetting and stored at 20 8C. A total of 35 ml of DNA solution was obtained from each puparium. DNA samples were quantied by real-time PCR using a Quantiler1 Human kit (Applied Biosystems). PCR was performed using a 7.500 Real-Time system and a standard protocol. Each sample was amplied in duplicate to obtain an average quantication.

All samples were amplied using AmpFlSTR NGMTM SElect amplication kit (Applied Biosystems). For each reaction unit, a positive and a negative control were used. PCR reactions were performed in 25 ml of nal volume, using 0.61.5 ng of DNA, following the producers recommendation without any modication of the number of cycles. 3. Results Larvae and ies emerged from the 4 pupae were identied using the morphological characters reported in the identication keys published by Rognes [15] and Szpila [16] as belonging to the species Lucilia sericata (Diptera, Calliphoridae), a common summer species [17]. Case 1. The Chelex1-100 (Bio-Rad) method used failed to extract DNA from sample A. The PrepFiler1 method used for puparium B extracted 35 ml of approximately 0.10.2 ng/ml of good quality DNA. The STR analysis showed a full prole matching that of the victim. Case 2. The DNA proles was extracted from puparia C and D using the PrepFiler1 method. The STR proles obtained from these two puparia matched each other and the victims prole (Fig. 1). In this case, the quantity of DNA recovered from the puparia was higher compared with the amount obtained from sample B (case 1), yielding approximately 810 ng of good quality DNA from each sample. 4. Conclusion To our knowledge, neither human DNA nor any host DNA has ever been extracted from puparia, but puparia have been used in the genetic identication of y species using mt-DNA [3]. Carvalho et al. [18] obtained sheep DNA from the digestive canal of a twoday-old pupa. However, the authors did not obtain any results from older pupae, possibly due to DNA degradation into fragments smaller than 87 bp or due to the low sensitivity of the method used. Both methods used to extract DNA are commonly/routinely used in forensic investigations although the PrepFiler1 technique is currently preferred for its sensitivity [19,20]. In both cases reported in this paper, the latter technique worked better and allowed us to obtain a good quality (Fig. 1), fully ampliable human DNA from puparia maintained for seven days at 24 8C after eclosion. Puparia, as well as the digestive canal contents from y larvae and pupae, as previously analyzed [17], may be used to identify human corpses; in very rare cases, a puparium is the only item remaining behind that could be used to identify the missing body. This interesting opportunity for forensic investigation has some limitations. Infact, although STR typing was successful and a complete STR prole was obtained for all specimens, currently, it is not clear where human DNA is located in the puparium. Due to the rigorous cleaning of the external surfaces of the empty puparium that efciently removed adhered human DNA, we can suggest that the origin of victims DNA can be from the internal surfaces or within the cuticular material. Further studies are required to better understand how environmental conditions (humidity, temperature and UV radiation) as well as the age of the puparia and the species from which they came could inuence the DNA extractability and quality. In conclusion, this study demonstrated the possibility to extract and to type human DNA from puparia and has a practical implication because empty carrion y puparia are notable for their durability compared to other entomological evidence.

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Fig. 1. STR prole obtained from the puparium C.

Acknowledgment Authors like to thank Prof Bryan Turner for his useful comments on the manuscript and for the English revision.

References
[1] C.P. Campobasso, F. Introna, The forensic entomologist in the context of the forensic pathologists role, Forensic Sci. Int. 120 (2001) 132139. [2] J.H. Byrd, J.L. Castner, Insects of forensic importance, in: J.H. Byrd, J.L. Castner (Eds.), Forensic Entomology. The Utility of Arthropods in Legal Investigations, CRC press, Boca Raton, FL, USA, 2010, pp. 116122. [3] M. Mazzanti, F. Alessandrini, A. Tagliabracci, J.D. Wells, C.P. Campobasso, DNA degradation and genetic analysis of empty puparia: genetic identication limits in forensic entomology, Forensic Sci. Int. 195 (2010) 99102.

[4] J.D. Wells, K.R. Stevens, Application of DNA-based methods in forensic entomology, Annu. Rev. Entomol. 53 (2008) 103120. [5] C.P. Campobasso, J.G. Linville, F. Introna, Forensic genetic analysis of insect gut contents, Am. J. Forensic Med. Pathol. 26 (2005) 161165. [6] X.J. Li, J. Cai, Y.D. Guo, F. Xiong, et al., Mitochondrial DNA and STR analyses for human DNA from maggots crop contents: a forensic entomology case from central-southern China, Trop. Biomed. 28 (2011) 333338. [7] W.R. Mukabana, W. Takken, G.F. Killeen, W.A. Hawley, B.G. Knols, Extent of digestion affects the success of amplifying human DNA from blood meals of Anopheles gambiae (Diptera: Culicidae), Bull. Entomol. Res. 92 (2002) 233239. [8] K.Y. Mumcuoglu, N. Gallili, A. Reshef, P. Brauner, H. Grant, Use of human lice in forensic entomology, J. Med. Entomol. 41 (2004) 803806. [9] J. Kreife, S. Kempfer, Isolation and characterization of human DNA from mosquitoes (Culicidae), Int. J. Legal. Med. 112 (1999) 380382. [10] J. Replogle, W.D. Lord, B. Budowle, T.L. Meinking, D. Taplin, Identication of host DNA by amplied fragment length polymorphism analysis: preliminary analysis of human crab louse (Anoplura: Pediculidae) excreta, J. Med. Entomol. 31 (1994) 686690.

D. Marchetti et al. / Forensic Science International 229 (2013) e26e29 [11] A.C. Moncayo, K. Lerdthusnee, R. Leon, R.M. Robich, W.S. Romoser, Meconialperitrophic matrix structure, formation, and meconial degeneration in mosquito pupae/pharate adults: histological and ultrastructural aspects, J. Med. Entomol. 42 (2005) 939944. [12] M.J. Lehane, Peritrophic matrix structure and function, Ann. Rev. Entomol. 42 (1997) 525550. [13] J. Amendt, C.P. Campobasso, E. Gaudry, et al., Best practice in forensic entomology standards and guidelines, Int. J. Legal Med. 121 (2006) 90104. [14] PrepFilerTM, Forensic DNA extraction Kit Users Guide, Publication Part Number 4390932 Rev. B, Revision date November 2008, Applied Biosystem. [15] K. Rognes, Blowies (Diptera, Calliphoridae) of Fennoscandia and Denmark, in: E.J. Brill (Ed.), Fauna Entomologica Scandinava, vol. 24, Scannavian Science Press Ltd, Leiden, Netherlands, 1991. [16] K. Szpila, Key for the identication of third instars of European blowies (Diptera: Calliphoridae) of forensic importance, in: J. Amendt, M.L. Goff, M. Grassberger, C.P.

e29

[17]

[18]

[19]

[20]

Campobasso (Eds.), Current Concept in Forensic Entomology, Springer, Dordrecht, Heidelberg, London, New York, 2010, pp. 4356. S. Vanin, P. Tasinato, G. Ducolin, et al., Use of Lucilia species (Diptera: Calliphoridae) for forensic investigations in Southern Europe, Forensic Sci. Int. 177 (2008) 3741F. F. Carvalho, I.R. Dadour, D.M. Groth, M.L. Harvey, Isolation and detection of ingested DNA from the immature stages of Calliphora dubia (Diptera: Calliphoridae), Forensic Sci. Med. Path. 1 (2005) 261263. P.S. Walsh, D.A. Metzger, R. Higuchi, Celex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material, Biotecniques 10 (1991) 506513. M.G. Brevnov, H.S. Pawar, J. Mundt, L.M. Calandro, M.R. Furtado, J.G. Shewale, Developmental validation of the PrepFiler Forensic DNA Extraction Kit for extraction of genomic DNA from biological samples, J. Forensic Sci. 54 (2009) 599607.