Detection of Proteus Anguinus Using Environmental DNA in Croatia

Abstract

The olm (Proteus anguinus) is the only cave dwelling vertebrate in Europe, inhabiting the karst cave systems of the Dinaric Alpes. It is a troglomorphic species, having a hidden way of living. Since it inhabits the interiors of caves, it is hardly seen on the surface and we are lacking a lot of information about its exact distribution. Recent studies have shown that by sampling the environment, we can get information about the species living there (Ficetola et al. 2008, Thomsen et al. 2011). To gather further data about the olm, a new, non-invasive monitoring method is urgently needed. The aim of this study was to develop a method by which we can detect olm DNA from cave waters. In the first phase, we took 15 ml samples from aquariums of the Zoo of Zagreb and from Miljacka II cave in June 2012. In the second phase 10, 5 and 2 l filtered samples were taken from Rupećica cave system in September 2012. We developed specific primers to amplify short DNA sequences from the mitochondrial D-loop region (87 bp) to determine the presence of the species in different environments. The reliability of the results was tested by sequencing and the sequences were compared to the DNA extracted from olm tissue and obtained from the GenBank. With this method, first time applied eDNA technique on a cave dwelling organism, we were able to detect P. anguinus DNA from one sample from Rupećica (filtered), one from Miljacka (not filtered) and one from aquarium of the Zoo of Zagreb (not filtered). However, further improvement of the PCR reactions and the sequencing technique is needed.