Forensic Science International 195 (2010) 99–102
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DNA degradation and genetic analysis of empty puparia: Genetic identification limits in forensic entomology Morena Mazzanti a, Federica Alessandrini a, Adriano Tagliabracci a, Jeffrey D. Wells b, Carlo P. Campobasso c,* a
Section of Legal Medicine, Department of Neuroscience, Universita` Politecnica delle Marche, Via Conca 71, 60126 Ancona, Italy Department of Biology, University of West Virginia, Morgantown, WV 26506-6057, USA c Department of Health Sciences (SpeS), University of Molise, Via De Sanctis, snc, 86100 Campobasso, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 March 2009 Received in revised form 25 November 2009 Accepted 25 November 2009
Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Forensic entomology Species identification Empty puparia Mitochondrial DNA DNA degradation
1. Introduction Puparial cases are common remnants of necrophagous flies in crime investigations. They can represent the most developed carrion insect life stage collected, which makes them very useful for the estimation of the post-mortem interval (PMI). In fact, at the end of metamorphosis, an emerging adult fly leaves behind the hardened puparium and thin exuvial membranes (the puparial shell and the prepupal skin) formed from the last two larval cuticles. In order to use empty puparia effectively in forensic settings, it is crucial to identify the species of fly eclosed from each puparium. But at external examination, fly puparia are similar in general appearance (usually light brown to dark brown or black in colour), and the heavy sclerotization makes it difficult to see important features. Diagnostic larval characters may still be visible [1] however, even an accurate morphological analysis of pupae and
* Corresponding author. Tel.: +39 0874 404 776; fax: +39 0874 404 778. E-mail addresses:
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[email protected] (C.P. Campobasso). 0379-0738/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2009.11.022
puparia cannot discriminate among some closely related species. Sometimes length and width of puparial cases can be helpful to discriminate between sarcophagids and calliphorids according with their own diagnostic features (posterior spiracles, in particular) but it is not enough for species identification [2]. Morphological identification of immature Diptera often requires specialized taxonomic knowledge and methods [3,4], and only very few experts have such knowledge. For this reason several experts still prefer rearing the immature stages up to eclosion of the adult fly, and apply the identification keys only on adult specimens. But time-consuming rearing of larvae to adults for identification may delay a criminal investigation or cause significant problems when rearing fails. Based on the above disadvantages of the morphological identification process, a forensic entomological investigation can benefit from molecular genotyping methods. There are several reports on the use of DNA techniques for identification of forensically important flies carried out on immature insect stages and adult flies [5–8]. The present study deals with mtDNA analysis of empty puparia, left behind after adult emergence. The goal is to demonstrate that a reliable genetic identification of flies can be made also from complete empty puparia or their fragments.
M. Mazzanti et al. / Forensic Science International 195 (2010) 99–102
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Fig. 1. Sequencing primers and analyzed fragments size.
2. Materials and methods 63 empty puparia of Diptera mostly coming from real cases and field carcass experiments were analyzed. Puparia were divided into three groups according to their time period between eclosion and our analyses:
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group A: 53 puparia 20 years old (very old puparia).
For group A, each pupa was reared and stored in a singular tube closed with gauze until eclosion of the adult fly. The adult fly was then killed by freezing at 20 8C and the corresponding puparium was stored at room temperature without preservatives. All puparia were measured in length to discriminate between Diptera families. However, size can be to some extent dependant on food availability. Therefore, other diagnostic features were also used for predicting the family such as the analysis of posterior spiracles still available and better discriminator than length. Among the 53 puparia coming from group A only 11 puparia were >0.7 cm in length having a posterior spiracular pit of Sarcophagidae (Sphd) family while the other 42 puparia were 0.7 cm in length suggesting a sarcophagid individual (according with diagnostic features of its posterior spiracles) while the other 9 puparia were
