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

Over the years, after the advent of minisatellite and microsatellite DNA typing, investigators generally use conventional sources like body fluids and cadaver as a source of DNA evidence for establishing identification. But in cases of extreme body decomposition or in the absence of the body, removed from the crime scene, these conventional methods fail to obtain the identity of the victim, paving way for the use of non-conventional sources as source of DNA evidence. DNA extraction from larval gut content of Megaselia scalaris larvae (non-conventional source), can be used for victim identification. While most of the work focus on PMI estimation, DNA analysis of gut contents of larvae found at a crime scene and the determination of their food source is a relatively new field and is still in its initial periods of development and till now is only documented on larvae of big size, mainly belonging to species in the family Calliphoridae. In this study DNA was successfully extracted from the gut content of M. scalaris (Diptera, Phoridae) larvae fed on Bos taurusmeat. The post feeding 3rd instar larvae were fixed using 5 different methods - hot water (>80 ̊C) for 30s on the larvae, only freezing the larvae (-20 ̊C), freezing (-20 ̊C) the larvae first and then placing them in EtOH, placing the larvae directly in EtOH (100%) and pouring hot water (>80 ̊C) for 30s on the larvae and then placing them in EtOH (100%).DNA extraction was performed using the Qiagen Investigator Kit. ANOVA test followed by a Post-Hoc analysis (Tukey HSD) revealed that there was statistical significant difference among all methods (p=0.000). Method 4 was seen to provide the maximum DNA yield (3.36 ±0.14ng/μl). After this, amplification was done using the ABI PCR System of the mitochondrial gene Cytochrome B and ribosomal gene 16s rRNA. The sequences obtained showed positive results in almost all samples as well as in the controls. After a positive amplification result with both PCR and quantification analysis in qPCR, a successful STR analysis revealed an exact match of the STR profiles of the sample of DNA extracted from the gut and DNA extracted from the Bos taurusmeat that was fed to the larvae. This proves that DNA can successfully be extracted and characterised from larval gut content irrespective of its size. The results obtained with Megaselia scalaris are particularly important since this species is found in both indoor and buried crime scenes. This study improves the ability to extract DNA from Dipteran larvae of forensic interest and it can also be used as an effective method to support forensic investigation.