Introduction

Medicolegal PMI estimation often involves examination of necrophagous insects developing on a corpse (reviewed in [1] and [2]). Blowflies (Diptera: Calliphoridae) are usually the first and most abundant colonizers and can therefore be used as indicators of the minimal time elapsed since death. However, several cases have been reported where small flies like the minute to medium sized scuttle flies (Phoridae) were present and even dominated the fauna of the carcass [3–10]. Although known to colonize a human corpse at a later time of decomposition, phorids may also be present at early stages of decay [7, 11, 12], especially when blowflies were unable to gain access [6, 11]. Unlike blowflies, they are able to move through the smallest openings or meters of soil, colonizing buried or concealed corpses and carcasses [3, 4, 13, 14]. Last but not least, some forensically important phorid species may also occur in cases of myiasis, the infestation of living humans or animals [6, 11]. In all these examples, they may provide decisive evidence in forensic investigations [15].

Over 3,000 of these typically humped-back species have been described so far and represent a diverse group of scavengers, herbivores, predators, and parasites. Buck [16] found over 40 necrophagous species in his study, about 50% belonging to the genus Megaselia. Surprisingly, only a minority has been reported to be attracted to decaying human corpses or carcasses of bigger vertebrates so far [6]; the majority infest invertebrate carrion like snails and insects [17–22]. However, as a precise identification based on morphological characteristics is hardly possible especially in the juvenile stages of these flies [15], our current knowledge about the abundance of scuttle flies on corpses may be biased [4, 6, 7, Amendt, unpublished data]. Species identification is also of major importance in forensic entomology because different species have different developmental rates at certain temperatures [7, 15, 23]. We believe that the difficult identification hampered the use of this important group in the past. Therefore, a tool which improves the identification of scuttle flies could be the starting point for research on forensically important phorid taxa, eventually leading to the routinely consideration of these flies in forensic entomology.

Within the past years, DNA-based techniques to identify forensically important fly species using either nuclear or mitochondrial DNA marker genes have been conducted [reviewed in 2]. Although there are still ambiguities about a universal marker for species identification [24], the mitochondrial COI gene is mostly used in forensic entomology and sufficient to identify many Diptera species [24]. Within the past years, COI became also popular for “DNA barcoding” and a 658-bp-long fragment has been suggested to use for standardized species identification [25]. Although still in their early steps, DNA barcoding approaches have been performed on a wide range of taxa so far [26–32] and it has also been applied on forensically important Chrysomya species (Diptera: Calliphoridae) [33].

The main purpose of this study is to characterize six forensically relevant scuttle fly taxa on the molecular level for the first time: Megaselia scalaris, Megaselia giraudii, Megaselia abdita, Megaselia rufipes, Conicera tibialis, and Puliciphora borinquenensis. The first five species have already been found on human corpses in the past [4–6, 8], only P. borinquenensis is still waiting to be detected in a forensic case.

We chose universal DNA barcoding primers [34] and gathered sequence data, which will be useful as reference standards for future determination of necrophagous phorid species. The need for such a reference database is subsequently described for three selected forensic cases, confirming the importance of Phoridae in forensic entomology.

Material and methods

Fly specimens

Adult flies of the necrophagous species M. scalaris (5), M. giraudii (5), M. abdita (8), M. rufipes (5), C. tibialis (5), and P. borinquenensis (6) were employed in the analysis. The majority of species originated from England. M. abdita and M. scalaris were from laboratory colonies in Cambridge and P. borinquenensis from a culture in Oxford. M. giraudii and M. rufipes were caught on bait in a Cambridge garden and C. tibialis was caught in Rüdesheim, Germany. All specimens were determined morphologically. At least five individuals of each species were used for molecular analysis. Numbers in parentheses indicate the number of individuals investigated.

DNA extraction

Genomic DNA from all specimens was extracted from the whole individual using a slightly modified phenol–chloroform extraction [35]. Subsequent ethanol precipitation a final elution in 50 μl distilled water was performed and extracts were stored at 4°C until PCR.

PCR amplification

Amplification of the COI barcoding region was performed using the primers LCO1490 (5′- GGTCAACAAATCATAAAGATATTGG-3′) and HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) [34]. Amplification was performed in a total reaction volume of 25 μl containing 1 unit/μl of Taq DNA polymerase, 2 mM of each dNTP, 8 mg/ml of BSA and 5 pmol of each primer. 5 μl of the DNA extracts were used as template.

All PCR amplifications were performed in a T3000 thermal cycler (Biometra). The thermal cycler program was the following: 1 min at 94°C followed by five cycles of 94°C for 1 min, 45°C for 1.5 min, and 72°C for 1.5 min followed by 35 cycles of 94°C for 1 min, 50°C for 1.5 min and 72°C for 1 min with a final extension step of 72°C for 8 min.

PCR products were detected by gel-electrophoresis in a 2.5% agarose gel, stained with ethidium bromide and visualized under UV light.

Sequencing analysis

PCR products were directly sequenced in both directions using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The protocol included a total reaction volume of 20 μl consisting 3 μl Big Dye, 2.5 μl 5× sequencing buffer, 5 pmol primer and 1 μl PCR product. Protocol for sequencing reaction was 28 cycles of 96°C for 10 s, 50°C for 5 s, and 55°C for 4 min.

Sequencing products were purified using gel-filtrated columns (Qiagen, DyeEx 2.0 Spin Kit) and run on an ABI3130 genetic analyzer (Applied Biosystems).

Sequence data for forward and reverse DNA strands were edited and aligned manually using the software Bioedit (Version 7.0.9).

Results and discussion

Identification

Current data present the first approach to use molecular tools to identify and distinguish between a variety of carrion-breeding scuttle flies (Phoridae). A total of 34 individuals were sequenced and aligned over 559 nucleotides of the COI barcoding fragment. A further two sequence data sets obtained from GenBank (http://www.ncbi.nlm.nih.gov/) served as outgroups for comparative purposes.

All new phorid sequences are deposited in GenBank under the following accession numbers: GU075399, GU075400, GU075401, GU075402, GU075403, GU075404, GU075405, GU075406, and GU075407.

Interspecific variation

Sequence analysis revealed a high interspecific nucleotide variation, which is sufficient for unambiguous identification and differentiation between the six species. Neighbor-joining analysis and distance matrix were performed in PAUP Version 3.1.1 [36] using default settings and are shown in Fig. 1 and Table 1, respectively.

Fig. 1
figure 1

Neighbor-joining tree. Relationships between the investigated scuttle fly species are presented based on the examined COI fragment. C. vicina and D. melanogaster were set as outgroups

Full size image
Table 1 Pairwise sequence differences between investigated scuttle flies and two outgroup species for the analyzed COI region (upper right panel: nucleotide divergence in %, lower left: absolute nucleotide differences)
Full size table

We found 147 variable positions and levels of interspecific nucleotide divergence ranged from 7.9% (M. giraudii to M. rufipes) to 18.6% (P. borinquenensis to M. rufipes). Within the genus Megaselia, genetic distances are relatively small with the lowest difference between M. giraudii and M. rufipes (7.9%) and the highest genus specific sequence divergence between M. scalaris/M. giraudii and M. scalaris/M. rufipes (11.4%; Table 1). All these values allow a clear differentiation of the species.

Comparing scuttle fly COI sequences to those of another representative carcass associated blowfly by phylogenetic analysis using PAUP 3.1.1, Calliphora vicina (accession number: AJ417702) and also to the fruit fly Drosophila melanogaster (accession number: AJ400907), we were able to detect a clear separation (Fig. 1), confirming that the three phorid genera belong to three different tribes, with Conicera being in a different subfamily. M. scalaris has been transported around the world by man, but was almost certainly Nearctic/Neotropic in origin. M. abdita, M. giraudii, and M. rufipes are also tramp species. All three are showing a holarctic but predominantly palaearctic distribution, while M. rufipes has been the most widely transported by man.

Intraspecific variation

Almost all specimens of one species showed identical nucleotide sequence except slight differences within the sequences of M. rufipes. Only two individuals shared the same haplotype (M. rufipes 1 and 2, Fig. 1 and Table 1) while the remaining three specimens differed to each other in one to two base pairs. However, intraspecific variation did not exceed 1% which allows species association in general [37, 38]. Current analysis demonstrates that intraspecific variation may affect RFLP approaches which are commonly used as cheap and fast alternatives to sequence-based species identification; point mutations may alter restriction sites and thus lead to misidentifications if not properly interpreted [39, 40].

Case 1

On July 11th, the body of a 53-year-old man was found on a mattress in his apartment. The corpse was in an advanced stage of decay and mummification. There were no signs of a third party fault. Insect fauna was dominated just by maggots and pupae of an unknown phorid species, which was later identified as M. abdita. Partial mummification of the body and the absence of blowflies indicated that death occurred in the cold season, when blowflies are not active. Later investigations revealed a possible time of death in late October of the previous year.

Case 2

On October 4th, the heavily decomposed body of a newborn baby was discovered in the storage room of a charitable foundation. The cadaver was wrapped in a towel and in a jacket and placed in a plastic bag. This bag was deposited in a closed cupboard. The corpse was strongly infested by phorid flies of all life stages, which were identified via DNA analysis as C. tibialis and M. scalaris. No other insect taxa were found. While M. scalaris needs about 3 weeks to reach the pupal stage at 15°C (the ambient temperature in the basement) [41], there is a lack of reliable developmental data for C. tibialis. However, Bourel et al. [13] noted C. tibialis as an indicator for a minimum post-mortem interval of about 2 months. Later investigations revealed a probable time of death of approximately 4 months prior to discovery.

Case 3

On December 6th, a 65-year-old woman was found dead in her apartment. The cause of death was unknown and the partner of the woman had disappeared. As it was certain, due to witness evidence, that he was still in the apartment on November 25th, investigators wanted to know if the deceased was still alive at that time. Entomological samples showed L3 larvae of the blowfly C. vicina and several scuttle fly pupae. DNA analysis identified them as M. scalaris. While C. vicina needs about 5 days at the recorded temperature of 20°C to reach the measured size and stage of development, M. scalaris needs between 10–11 days at that temperature to reach the pupal stage [41]. This indicated a minimum post-mortem interval which reconciled with the last activity of the now disappeared partner in the apartment, who was suspected of being involved in the death of the woman.

Conclusion

Phoridae regularly infest human cadavers [5, 6, 10, 12, 15] and can serve as a very powerful evidence in the estimation of the post-mortem interval. They especially colonize buried or concealed corpses even at an early stage of decomposition and their occurrence might be helpful in death investigations when access for blowflies is restricted. Despite that advantage, their use in forensic entomology is still uncommon. One of the main reasons for this is the difficult identification of the specimens found on a corpse. Here, DNA sequence analysis can lead to unambiguous species determination. A proper species identification will also improve our knowledge of the biology and development of the scuttle flies, as one of the basic requirements in developmental studies in the laboratory is the guarantee of having an uncorrupted colony of a known species.