Detection of the Endangered Stone Crayfish Austropotamobius torrentium (Schrank, 1803) and Its Congeneric A. pallipes in Its Last Italian Biotope by eDNA Analysis

Chiara Manfrin,Victoria Bertucci-Maresca,Massimo Zanetti,Umberto Fattori,Piero G Giulianini,Alberto Pallavicini,David Stanković

doi:10.3390/d14030205

Chiara Manfrin, Victoria Bertucci-Maresca + Show 5 more

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https://doi.org/10.3390/d14030205

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Abstract

The stone crayfish, Austropotamobius torrentium, is a European freshwater crayfish. Although this species is relatively widespread throughout the continent, it is undergoing significant declines throughout its range. However, as the decline rates have not been quantified in detail, this species is classified as data deficient by the IUCN Red List of Threatened Species. The present study describes the development and validation of two species-specific assays based on hydrolysis probe chemistry for the detection of A. torrentium and A. pallipes environmental DNA (eDNA) in water samples collected in the Julian Alps of Italy (Friuli Venezia Giulia). The eDNA-based method was applied to 14 sites within the Danubian Slizza basin, known to be inhabited by A. torrentium, but with insufficient information on their distribution. In addition, one station in the Tagliamento River basin was sampled to test the performance of the A. pallipes probe. The presence of A. torrentium is confirmed at 6 out of 15 sites. At four of these sites, A. torrentium is detected for the first time. In contrast, the presence of A. torrentium was not detected at two sites already known to harbour the species. Finally, the presence of A. pallipes was confirmed in the station belonging to the Tagliamento basin. The methodology described, which allows the distinction between the two species, paves the way for the parallel detection of the stone crayfish and the white-clawed crayfish (A. pallipes) through eDNA analysis.

Highlights

The stone crayfish, Austropotamobius torrentium (Schrank, 1803), is a freshwater species native to Central and South-eastern Europe [1–3]
We present and evaluate the performance of two assays based on specific primers and hydrolysis probes targeting a variable region of 16S rRNA, that can discriminate between A. torrentium and A. pallipes complex
A. torrentium in the Friuli Venezia Giulia (FVG), the only Italian region where both species occur and for which the latest information dated back to 2015 [20]. The development of this methodology involved the following steps: (i) development of two species-specific assays based on probe hydrolysis allowing the detection of A. torrentium and A. pallipes environmental DNA (eDNA); (ii) testing the specificity of both assays on genomic DNA of the two target species and other crayfish present or potentially present in the FVG; (iii) testing the Limit of detections (LODs) of the method under laboratory conditions; and (iv) testing the performance of the assays in a pilot study on water samples collected in the wild

Summary

Introduction

The stone crayfish, Austropotamobius torrentium (Schrank, 1803), is a freshwater species native to Central and South-eastern Europe [1–3]. The species inhabits mainly small, cold-flowing waters at mid-to-high elevations, characterised by moderate gradient, high hydromorphological and bed heterogeneity, and abundant riparian vegetation [4]. Primarily due to channel modification (e.g., concrete revetment), agriculture, pollution, and exotic species that act as both competitors and parasitic disease vectors, threaten the survival of A. torrentium [5,6]. The risk of extinction is further increased by its slow life-cycle with low fecundity and high habitat specialization [2,4,7,8]. A. torrentium is listed as a “data deficient” (DD) species in the IUCN Red List of Threatened Species [13]. It is protected internationally by the Bern Convention

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