Identifying the end user requirements for a novel, field-based environmental DNA (eDNA) forensic collection method

Environmental DNA (eDNA) refers to the DNA derived from species and found in environmental samples such as water, air and soil. eDNA has been used in biodiversity and conservation studies but not routinely implemented in forensic investigations. Currently physical surveys are implemented at freshwater pearl mussel, Margaritifera margaritifera, crime scenes which are costly, invasive, reliant on practitioner experience and susceptible to false negative results. The aim of this study was to identify key end-user requirements for a novel eDNA sampling method collected through stakeholder discussion and questionnaire. Stakeholder discussions identified the following key criteria for method development: the end-user group, ease of collection, risk to end-user, speed of collection, cost of collection, and data accuracy. These criteria were used to develop three possible eDNA collection approaches: basic (2L water bottles for bankside collection), intermediate (Whirl-Pak® bags for bankside collection), and advanced (onsite Sterivex™ filtration equipment to use on samples collected by entering the watercourse). To compare the three possible methods in respect to their ease and risk, a questionnaire was disseminated to government officers, police officers, postgraduate students and university staff with expertise in either wildlife crime and/or eDNA approaches (N = 63). The time taken to collect samples together with the associated costs of equipment and analysis time were also calculated. Finally, impact of collection method on data accuracy was assessed through the development of a qPCR assay to measure the sensitivity, specificity and concentrations across two different DNA fragment sizes from hatchery reared populations of M. margaritifera. End-user questionnaire data reveals a preference for the basic and intermediate methods in respect to their ease of use and identified risks. Furthermore, these sampling strategies were quicker and cheaper to use although the advanced method could offer logistical benefits in terms of longer preservation times and reduced laboratory processing time. In respect to impact on data accuracy, the advanced method also showed poor detection rates of eDNA, a surprising result given the popularity of the method in conservation studies. Together, these data show the benefits of a co-developed and usable method for police and conservation first responders to use at wildlife crime investigations concerning M. margaritifera habitat destruction. Further research to develop a robust qPCR assay and assess the usability of the basic and intermediate collection methods on wild populations is recommended to determine the overall effectiveness of the described methods.