Molecular identification and phylogenetic analysis of the forensically important family Piophilidae (Diptera) from different European locations.

Species identification plays an important role in forensic entomology and is mandatory for an accurate calculation of the minimum post-mortem interval. Many important Diptera and Coleoptera taxa of the cadaver community can already be identified by common barcoding approaches, i.e., by sequencing a 658bp region in the mitochondrial cytochrome c oxidase subunit I (coI) gene. Nevertheless, there is still a lack of reference barcodes for species, in particular, that can be found on cadavers at later decomposition stages. Flies of the family Piophilidae illustrate this gap of knowledge perfectly. Due to the fact that a reliable morphological identification key for the immature stages of this flies is still missing and the immature stages of many piophilids cannot be assigned to a certain species, there is need for additional tools to identify forensically relevant taxa. We collected adult piophilid specimens at 10 locations in five European countries: Spain (n=3 locations), Germany (n=3), Portugal (n=2), Poland (n=1) and Switzerland (n=1). Apart from the coI barcoding region, we additionally analyzed a 398bp long region of the nuclear elongation factor 1 alpha (ef1a) and subsequently established the molecular identifier for nine piophilid species. In addition, we present the molecular phylogeny of the examined taxa.

Species composition of forensically important blow flies (Diptera: Calliphoridae) and flesh flies (Diptera: Sarcophagidae) through space and time.

Weekly monitoring of forensically important flight-active blow flies (Diptera: Calliphoridae) and flesh flies (Diptera: Sarcophagidae) was performed using small baited traps. Sampling took place in two rural, one suburban and two urban habitats in and around Frankfurt (Main), Germany, lasting two years and eight months. Highest values for species richness and Chao-Shen entropy estimator for Shannon's index in both families were found at the urban sites, peaking during summer. Space-time interaction was tested and found to be significant, demonstrating the value of a statistical approach recently developed for community surveys in ecology. K-means partitioning and analysis of indicator species gave significant temporal and habitat associations of particular taxa. Calliphora vicina was an indicator species for lower temperatures without being associated with a particular habitat. Lucilia sericata was an indicator for urban sites, whereas Lucilia ampullacea and Lucilia caesar were indicators for rural sites, supplemented by the less frequent species Calliphora vomitoria. Sarcophagidae were observed during a clearly shorter period of year. Sarcophaga subvicina+Sarcophaga variegata was found to be an indicator for urban habitats during summer as well as Sarcophaga albiceps for rural habitats. A significant association of Sarcophaga caerulescens to rural habitats as well as one of Sarcophaga similis to urban habitats was observed.

Diapause-specific gene expression in Calliphora vicina (Diptera: Calliphoridae)--a useful diagnostic tool for forensic entomology.

Estimating the post mortem interval (PMImin) by age determination of blow fly larvae has been well-established for moderate temperatures. Low-temperature developmental data is only available sparsely and usually does not take overwintering strategies into account. The blow fly Calliphora vicina hibernates by diapausing in the third larval stage extending the duration of this developmental stage up to several weeks or even months. As the diagnosis of the diapause status is not possible by morphological characteristics, PMImin estimations might be biased during the cold season if only based on age determination of third instar larvae of C. vicina. Molecular markers were searched for which allows one to identify diapause in larvae. Expression analysis of 19 genes was performed in diapausing and non-diapausing larvae. Three genes encoding for heat shock proteins (hsp23, hsp24 and hsp70) were found to be up-regulated distinctly in diapausing larvae and at 1 day in non-diapausing larvae. If several larvae are subjected to an analysis, a high variance in the expression level of the gene encoding for the anterior fat body protein is a further marker for diapause. The present study proves the potential use of gene expression analysis as a suitable diagnosis tool for diapause in C. vicina.

Lucilia silvarum Meigen, 1826 (Diptera: Calliphoridae)--a new species of interest for forensic entomology in Europe.

In Europe, the blowfly genus Lucilia is represented in Forensic Entomology mainly by the species L. ampullacea, L. caesar, L. illustris and L. sericata. In the US, Lucilia silvarum is rarely recorded as a carrion breeding species but usually as a more or less exclusive parasite of frogs and toads. We present three forensic cases from different European countries reporting, for the first time, L. silvarum on human bodies that were found close to lakes, wetlands, or riversides. To use this species for post-mortem interval estimations, thermal development data is needed. The first step is accurate identification by morphological and molecular means. Therefore, we analysed a 611 bp part of the mitochondrial COI region for 23 specimens of L. silvarum from 9 different geographical regions, all of which give the same haplotype. Differences within the haplotype varied by up to 0.2%. Comparison between the haplotype found and those published on GenBank showed up to 1.2% variance. Moreover, we present an updated key for the morphological identification of the third larval instars of European Lucilia spp. of forensic importance, adding not only L. silvarum, but also L. cuprina which was recorded in Europe for the first time about 20 years ago.

Fast and simple DNA extraction from saliva and sperm cells obtained from the skin or isolated from swabs.

The forensic scientist often has to cope with problematic samples from the crime scene due to their minute size and thus the low amount of extractable DNA. The retrieval of DNA from swabs taken from the surface of the skin, for example, in cases of strangulation, can be especially difficult. We systematically investigated swabs taken from the skin (to obtain a genetic profile from the victim and also from a possible offender) and from sperm cell containing swabs using two extraction kits: the Invisorb forensic and the Invisorb spin swab kit (both Invitek, Germany). DNA quality and quantity were tested on ethidium bromide containing agarose gels and in a highly sensitive duplex-PCR, which amplifies fragments specific for mitochondrial and nuclear DNA. Absolute quantification was done using real time PCR. Samples, which were positive in the duplex-PCR, were also employed to genetic fingerprinting using the Powerplex ES and the AmpFlSTRIdentifiler(TM) kits. Our study shows that the easy-to-use Invisorb spin swab kit is very suitable for DNA isolation from swabs taken from the skin and also from sperm cells. Retrieval of cells from the skin with swabs moistened in extraction buffer, not in distilled water, led to a significant higher DNA yield.