Assessing the Efficiency of Environmental DNA and Metabarcoding for Conservation of the Barred Grass Snakes (Natrix helvetica)

Abstract

The rapidly developing technology of DNA analysis has gained much significance in biology, offering non-invasive methods to study the conservation ecology of elusive, cryptic, and endangered species. In the last decade, environmental DNA survey (eDNA) and metabarcoding methods have been increasingly used to study aquatic, semi-aquatic, marine, and terrestrial organisms. Moreover, most investigations into environmental DNA and metabarcoding have focused on amphibians and fish while limited to reptiles. This thesis explores the efficiency of environmental DNA (eDNA) for detecting the presence of barred grass snakes (Natrix helvetica) and metabarcoding for analysing diet habits, highlighting these methods as practical tools for conserving reptile species. Environmental DNA (eDNA) is genetic material, such as urine, faeces, saliva, slough skin, and gametes of target species, that is released into the environment. This genetic material is then extracted from environmental samples such as water, soil, sediment, and air. We developed and validated a novel species-specific qPCR assay to detect N. helvetica from two environmental samples (water from the ponds and roller swabs of entire artificial cover objects (ACO)). The results proved the assay's efficiency, specifically and sensitivity in detecting N.helvetica at low concentrations of eDNA. At the same time, water eDNA samples yielded reliable detections compared to roller samples. However, we suggest that using an ACO that is flat in shape with a non-porous surface could enhance eDNA recovery and improve detection rates. To further understand the dietary habits of N. helvetica, the metabarcoding approach, in which different universal primers were used to detect multiple species of prey, was performed using faecal samples collected from two distinct populations. Our results found amphibians as the primary dietary component, supplemented by small mammals, birds, and insects, with dietary variations observed across age, sex, and populations. We recommend that future monitoring and conservation strategies incorporate eDNA and metabarcoding, as these tools offer precise, cost-effective alternatives to traditional surveys for reptiles, contributing to biodiversity protection and habitat management.