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The estimation of the minimum time since death is one of the main applications of forensic entomology. This can be done by calculating the age of the immature stage of necrophagous flies developing on the corpse, which is confined to approximately 2–4 weeks, depending on temperature and species of the first colonizing wave of flies. Adding the age of the adult flies developed on the dead body could extend this time frame up to several weeks when the body is in a building or closed premise. However, the techniques for accurately estimating the age of adult flies are still in their beginning stages or not sufficiently validated. Here we review the current state of the art of analysing the aging of flies by evaluating the ovarian development, the amount of pteridine in the eyes, the degree of wing damage, the modification of their cuticular hydrocarbon patterns, and the increasing number of growth layers in the cuticula. New approaches, including the use of age specific molecular profiles based on the levels of gene and protein expression and the application of near infrared spectroscopy, are introduced, and the forensic relevance of these methods is discussed.
Determining the age of juvenile blow flies is one of the key tasks of forensic entomology when providing evidence for the minimum post mortem interval. While the age determination of blow fly larvae is well established using morphological parameters, the current study focuses on molecular methods for estimating the age of blow flies during the metamorphosis in the pupal stage, which lasts about half the total juvenile development. It has already been demonstrated in several studies that the intraspecific variance in expression of so far used genes in blow flies is often too high to assign a certain expression level to a distinct age, leading to an inaccurate prediction. To overcome this problem, we previously identified new markers, which show a very sharp age dependent expression course during pupal development of the forensically-important blow fly Calliphora vicina Robineau–Desvoidy 1830 (Diptera: Calliphoridae) by analyzing massive parallel sequencing (MPS) generated transcriptome data. We initially designed and validated two quantitative polymerase chain reaction (qPCR) assays for each of 15 defined pupal ages representing a daily progress during the total pupal development if grown at 17 °C. We also investigated whether the performance of these assays is affected by the ambient temperature, when rearing pupae of C. vicina at three different constant temperatures—namely 17 °C, 20 °C and 25 °C. A temperature dependency of the performance could not be observed, except for one marker. Hence, for each of the defined development landmarks, we can present gene expression profiles of one to two markers defining the mentioned progress in development.