DNA barcoding allows identification of European Fanniidae (Diptera) of forensic interest
Introduction
Various arthropods have a close association with animal and human bodies. The aim of forensic entomology is to study the association of insects with cadavers and their biology for subsequent application in forensic practice [1]. Particularly, the use of entomological evidence allows for an accurate estimation of the minimum post-mortem interval (PMI), which often coincides with the period of insect activity [2]. Thus, accurate species identification is a prerequisite for any further analysis of the collected material [2], [3]. Recently, significant progress has been made in the field of identification of Diptera of medico-legal importance. High-quality and well-illustrated morphological keys facilitate identification of many forensically relevant species [4], [5], [6], [7]. However, precise identification of some taxa is still hampered by various factors: time-consuming rearing to the adult stage of some immature insects (eggs, larvae and pupae) [8]; high morphological similarity of closely related species [9]; occurrence of casual visitors that can be misidentified with regular elements of carrion fauna [10].
In case of any difficulties that hinder prompt and easy species determination, the contribution of experienced taxonomists to identify entomological material may often be required. This is also the case of the dipteran family Fanniidae. A long-standing belief that only a few species of Fanniidae are likely to constitute elements of the insect carrion community was challenged by recent studies [11], [12], [13]. In Europe, decomposing carrion and cadavers attract approximately 30 species of Fanniidae; this is in spite of the lower number of species that actually breed in these habitats [11], [12]. Fanniidae can be found either in forest habitats or synanthropic conditions [14], at the early, moist stages of cadaver decomposition [15] or advanced stages when larvae of other flies have finished development [12]. Fanniidae are known for their ability to exploit buried remains [16], [17] and those placed in indoor conditions [15], [18]. In the latter case, their presence may indicate neglect of the living person because fanniids are prone to oviposit on bodies before death if they are contaminated with faeces and urine [19]. Fanniidae despite their usefulness for medico-legal purposes [11], [12], [18], cannot be fully exploited in forensic investigations due to the issues of species identification. Male fanniids are relatively easy to identify because of their chaetotaxy details, body colour and dusting pattern, various leg armature modifications and details of the genitalia, which for many species comprise robust, species-specific characters [14]. However, the great majority of specimens collected from carrion and cadavers are females [20]. Contrary to males, females of many closely related species can only be discriminated based on very few vague characters [14]. On the other hand, females of some carrion-visiting species are still unknown [14]. Also existing keys for larval identification are confusing and do not allow for prompt and easy identification [14], [21].
Morphology-based taxonomy may be hindered by the aforementioned factors, yet these are not obstacles for a molecular identification approach [22]. DNA barcoding aims to provide taxonomic identification for examined specimens based on the fundamental assumption that each species has a unique barcode (a standardized, short DNA sequence), and this barcode indicates certain species [23]. The substantial advantage of a DNA-based identification approach is the possibility of identifying all life stages of a given species [24], [25]. An essential condition of extensive application of DNA barcoding is a reference library of gene sequences obtained from correctly, morphologically, identified specimens [22], [26]. This is a significant issue because mistakes in the initial stage of material identification will result in an erroneous assignment of gene sequence to species [26]. Up to 66% of insect species are expected to be infected with the intracellular symbiotic bacterium Wolbachia [27]. Wolbachia may disrupt the pattern of mitochondrial DNA (mtDNA) variation and impact identification accuracy by means of DNA barcoding [28]. Wolbachia may cause homogenization of mtDNA of different species (two species, one barcode) or increase intraspecific gene diversity (one species, two barcodes) [29]. Thus, screening of material for the presence of endosymbiotic bacteria should be considered a routine step in DNA barcoding studies. Originally about 650-bp long fragment of the 5′ end of the mitochondrial cytochrome c oxidase subunit I (COI) was proposed as a universal marker for metazoan DNA barcoding [23]. In comparison to this fragment, which has a greater than 97% species specificity, the 100-bp long 5′ end of the COI barcode region can already provide 90% success in metazoan identification [30]. So-called mini-barcodes can be used, for instance, in cases of old material identification, because shorter gene fragment amplification is not as much constrained by DNA degradation [30].
The usefulness of molecular taxonomy as a supplement/substitute for a morphology-based identification approach has also been validated for forensically relevant arthropods [22]. The most widely examined for DNA barcoding purposes is the COI marker [31], [32], [33], [34], [35]. However, with some exceptions [36], validation of COI mini-barcodes is still not considered to be a standard element of data analysis. Because COI did not allow for the discrimination of some taxa of medico-legal interest, several other markers were recently investigated: cyt b [37], ITS2 [37], [38], 16S [39], and EF1α [40]. Although significant progress has been observed in the application of DNA barcoding in the identification of dipterans of forensic interest, the majority of studies have mainly focused on insects that are most commonly used in forensic investigations, such as blowflies [33], [38] and flesh flies [36], [39]. Very few studies have focused on other, but still important, groups [31], [40]. To date, no comprehensive study has been undertaken to validate the usefulness of the DNA barcoding approach for taxonomic purposes of Fanniidae of medico-legal importance. Previous authors provided DNA sequences for a few species of forensic interest as an addition to studies of other forensically relevant dipteran families [32], [41], [42], [43]. Although numerous COI barcode region sequences can be found in the BOLD and GenBank depository databases, the great majority refer to common woodland species of no forensic importance.
The aim of this study was to assemble a reference library of the barcode region of COI for European Fanniidae of forensic interest. To validate application of molecular taxonomy methods we (1) studied intra- and interspecific gene diversity to test for the presence of a barcoding gap in newly obtained data and those retrieved from various depository databases, (2) studied specimen identification success for full-length and mini-barcode regions of COI for a few combinations of retrieved sequences, and (3) checked whether the obtained sequences form monophyletic clusters of species in phylogenetic analysis. Because maternally transmitted bacterial endosymbiotic parasites may affect the performance of DNA barcodes [28], we additionally screened all specimens used in this study for the presence of Wolbachia.
Section snippets
Sampling
We sampled the European Fanniidae of forensic interest, both those considered useful for medico-legal purposes and casual carrion visitors, from various regions in Asia, Europe and North America (Table S1, Appendix). The first set included species that are regularly reported to visit and breed in carrion and human cadavers: Fannia aequilineata Ringdahl, Fannia canicularis (Linnaeus), Fannia coracina (Loew), Fannia latipalpis (Stein), Fannia leucosticta (Meigen), Fannia manicata (Meigen), Fannia
Obtained sequences
We isolated genomic DNA for 90 specimens of Fanniidae representing 27 species (Table S1, Appendix). We successfully sequenced the full COI barcode region (658 bp) for 88 specimens. For two specimens, F. umbrosa and F. vesparia, we obtained shorter fragments that were 645-bp and 640-bp long, respectively. The universal primers Uni-MinibarF1 and Uni-MinibarR1 allowed for the successful amplification of a mini-barcode region in the examined species (data not shown). Additionally, we retrieved 2946
Discussion
Our study provides the most comprehensive reference library of COI barcode sequences for European Fanniidae of forensic interest. It is also the first attempt to investigate the usefulness of DNA barcoding for the identification of Fanniidae. Despite the large number of COI barcode sequences referring to Fanniidae in the BOLD and GenBank databases, the great majority of them are not useful for forensic entomologists as they refer to species of no medico-legal interest. Although we found some
Conclusions
Our analysis revealed that molecular taxonomy may be successfully applied for taxonomical purposes in European Fanniidae of forensic interest. The low intra- and relatively high interspecific diversity of COI barcode region allowed us to correctly identify species visiting and colonising animal and human bodies. Application of shorter fragments of the COI gene, mini-barcodes, allowed for specimen identification with an identification success comparable to that of using the full-length barcode
Acknowledgments
We would like to express our appreciation to Dr. Chong Chin Heo (Universiti Teknologi MARA, Malaysia), Ms. Nina Feddern (University Hospital Goethe University Frankfurt, Germany), Dr. Szymon Matuszewski (Adam Mickiewicz University, Poland), Dr. Thomas Pape (University of Copenhagen, Denmark), Dr. Adrian C. Pont (Oxford University Museum of Natural History, UK) and Mr. Nikita Vikhrev (Zoological Museum of Moscow University, Russia) for the aid in obtaining material of some species. We would also
References (66)
- et al.
An initial study of insect succession and carrion decomposition in various forest habitats of Central Europe
Forensic Sci. Int.
(2008) - et al.
Insect succession and carrion decomposition in selected forests of Central Europe. Part 2: composition and residency patterns of carrion fauna
Forensic Sci. Int.
(2010) - et al.
Long-term study of pig carrion entomofauna
Forensic Sci. Int.
(2015) - et al.
Entomofauna of a buried body: study of the exhumation of a human cadaver in Buenos Aires Argentina
Forensic Sci. Int.
(2014) - et al.
Neglect of the elderly: forensic entomology cases and considerations
Forensic Sci. Int.
(2004) - et al.
Child neglect and forensic entomology
Forensic Sci. Int.
(2001) - et al.
Identification of sarcosaprophagous Diptera species through DNA barcoding in wildlife forensics
Forensic Sci. Int.
(2013) - et al.
Molecular differentiation of Central European blowfly species (Diptera, Calliphoridae) using mitochondrial and nuclear genetic markers
Forensic Sci. Int.
(2014) - et al.
Molecular identification and phylogenetic analysis of the forensically important family Piophilidae (Diptera) from different European locations
Forensic Sci. Int.
(2016) - et al.
Potential use of DNA barcodes in regulatory science: identification of the U.S. Food and Drug Administration’s Dirty 22 contributors to the spread of foodborne pathogens
J. Food Prot.
(2013)