An extremely sensitive nested PCR-RFLP mitochondrial marker for detection and identification of salmonids in eDNA from water samples

Abstract

BackgroundSalmonids are native from the North Hemisphere but have been introduced for aquaculture and sport fishing in the South Hemisphere and inhabit most rivers and lakes in temperate and cold regions worldwide. Five species are included in the Global Invasive Species Database: rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar, brown trout Salmo trutta, brook trout Salvelinus fontinalis, and lake trout Salvelinus namaycush. In contrast, other salmonids are endangered in their native settings.MethodsHere we have developed a method to identify salmonid species directly from water samples, focusing on the Iberian Peninsula as a case study. We have designed nested Salmonidae-specific primers within the 16S rDNA region. From these primers and a PCR-RFLP procedure the target species can be unequivocally identified from DNA extracted from water samples.ResultsThe method was validated in aquarium experiments and in the field with water from watersheds with known salmonid populations. Finally, the method was applied to obtain a global view of the Salmonidae community in Nalón River (north coast of Spain).DiscussionThis new powerful, very sensitive (identifying the species down to 10 pg DNA/ml water) and economical tool can be applied for monitoring the presence of salmonids in a variety of situations, from checking upstream colonization after removal of river barriers to monitoring potential escapes from fish farms.

How can eDNA contribute in riverine macroinvertebrate assessment? A metabarcoding approach in the Nalón River (Asturias, Northern Spain)

“…Moreover, conventional sampling methods are invasive because the individuals are often removed from the river and killed for identification in the laboratory under a magnifying glass or the microscope (Clusa et al, 2017). Alternatively, the use of environmental DNA (eDNA), DNA that organisms expel or release in the environment (air, sediment, or water), could deal with these limitations of conventional assessments.…”

“…The filtration process followed the protocol described in Clusa et al (2017) to prevent contamination. The filtration process followed the protocol described in Clusa et al (2017) to prevent contamination.…”

“…Membrane Filter (Pall Corporation, Life Sciences) with 0.2 µm pore size and 47 mm diameter was used; one liter was filtered through one filter. The filtration process followed the protocol described in Clusa et al (2017) to prevent contamination. Briefly, water samples were filtered in a room separated from the molecular laboratory in which only water samples are handled.…”

Understanding and managing fish populations: keeping the toolbox fit for purpose

“…The development of effective PCR primers is central to the successful application of eDNA (Freeland, ; MacDonald & Sarre, ). As a result, a vast range of primer sets are available for fishes (Doi et al, ; Clusa et al, ). Metabarcoding primers, that simultaneously amplify eDNA from many fish species, have also been developed for monitoring entire fish communities (Miya et al, ; Valentini et al, ).…”

GoFish: A Streamlined Environmental DNA Presence/Absence Assay for Marine Vertebrates

“…Nested PCR is a highly sensitive and specific approach to identifying genetic variants [e.g., 45]. Nested PCR improves detection of earthworm eDNA from soil samples archived for more than 30 years [46] and enables highly sensitive eDNA assays for salmonid fishes and a fresh water mussel [47,48].…”