DNA degradation and genetic analysis of empty puparia: Genetic identification limits in forensic entomology
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 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], [6], [7], [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.
Section snippets
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 <5 years old (young puparia);
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group B: 7 puparia 5–20 years old (old puparia);
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group C: 3 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
Results and discussion
Genotyping succeeded only in 43 specimens (68.2%) out of 63 puparia (Table 1, Table 2). Table 1 shows the genotyping results for all young puparia of group A (<5 years old). Table 2 shows the genotyping results for old and very old puparia coming from groups B (5–20 years old) and C (>20 years old). MtDNA haplotypes matched eight Diptera species: 13 samples were Calliphora vicina 1 Calliphora vomitoria, 19 Lucilia sericata, 1 Chrysomya megacephala, 1 Phormia regina, 1 Sarcophaga crassipalpis, 6
Conclusion
The results demonstrate that DNA-based methods can be reliable for identification of Dipteran species using either entire, eclosed puparia or fragments of puparia. This is important because fly puparia have a very fragile structure and they are often recovered in fragments from a crime scene [14]. These results also raise the possibility of using the empty puparium or its fragments for genetic analysis, rather than the valuable adult specimen. When a crime-scene specimen is reared to the adult
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