DNA characterisation from gut content of larvae of Megaselia scalaris (Diptera, Phoridae) for forensic investigations

The role of DNA in crime scene investigation over the last couple of decades has been immense. DNA materials as evidence are routinely collected from conventional sources (body fluids) from a wide range of crime scenes. In the absence of conventional sources, DNA evidence can be obtained from non-conventional sources, like touch DNA and gut contents of Dipteran larvae found on or near the body. While most studies about insects and their larval stages obtained from crime scenes have been done for PMI estimation, the use of gut contents from Megaselia scalaris (Diptera, Phoridae) larvae for human identification has not been yet investigated. The larvae’s ability to crawl through tight spaces make them an important species for both indoor crime scenes and also in the cases of buried corpses. In the present study, a comprehensive framework has been developed to extract non-insect DNA from the gut contents of larvae of M. scalaris (Diptera, Phoridae), fed on Sus scrofa tissue, and use it for STR analysis, making a tool for human identification, aiding forensic investigations. The larvae were fixed using 5 different protocols: (a) suspending the larvae in hot water (>80°C); (b) larvae kept at -20°C; (c) larvae kept in EtOH (98%) and stored at -20°C; (d) larvae kept at -20°C for 4hrs and later kept in EtOH; (e) larvae first suspended in hot water (>80°C) and kept in EtOH (98%) -20°C. Despite the small size of the larvae (2.0 ± 0.5 mm) and low amount of gut content (0.2-0.5 mg), DNA extraction of the gut contents of larvae was undertaken successfully using the Qiagen® Investigator Extraction Kit. The extracted samples were quantified and the maximum quantification was obtained from the larvae fixed by freezing at -20°C, with an average of 3.67 ± 0.05 ng/μl per sample, followed by larvae fixed with EtOH at -20°C with 2.55 ± 0.06 ng/μl per sample. A positive PCR amplification result was obtained from the mitochondrial gene cytochrome b (149bp) and ribosomal gene 16s rRNA (138bp), which was confirmed by analysis through BlastN, showing a positive result of Sus scrofa DNA sequence. STR analysis of the samples was done using Multiplex PCR test kit with 11 autosomal markers and 1 gender specific marker for Sus scrofa. A complete STR profile was obtained from the samples (minimum 1 crop) with a match on all loci when compared to the control sample. The results obtained from this study are significant, since M. scalaris is an important fly of forensic interest with a cosmopolitan distribution, generally encountered by investigators in crime scenes. The results obtained also show that preservation of larvae with EtOH (-20°C) and only freezing (-20°C) help in proper DNA typing, which is helpful for investigators as it is a more practical and easy method for proper collection and preservation of the larvae.