Environmental DNA for freshwater fish monitoring: insights for conservation within a protected area

Ethics statement

This project and the sampling carried out in protected spaces was authorized by the entity legally entitled to do so in Spain: the Government of the Asturias Principality, with the permit reference 101/16. The authors adhered to the European Code of Conduct for Research Integrity (Drenth, 2011).

Study area-upper Nalón Basin

The Upper Nalón Basin is located in the central part of the region of Asturias (Bay of Biscay, Spain). As part of the UNESCO (United Nations Educational, Scientific and Cultural Organization) Biosphere Reserve and Natural Park of Redes, it has a high faunal diversity (García-Ramos et al., 2006). In the streams (river headwaters) brown trout (Salmo trutta) are the only native fish species, because two consecutive impassable dams (Fig. 1) impede the arrival of native migratory European eel (Anguilla anguilla) and Atlantic salmon (Salmo salar), which occur downstream (Juanes et al., 2011). There is a rainbow trout fish farm located in one of the headwater streams in a zone denominated Veneros (Fig. 1), but escapes have not been reported and rainbow trout individuals were not recorded within the studied streams so far.

Field methods and macroinvertebrates sampling

Sampling took place in November 2015. Targeted sampling areas were typical juvenile habitat: shallow, well oxygenated and with moderate current velocity. At the sampling time of the year, in the particular river zones sampled trout juveniles around one-year-old are expected to be by far the most abundant life stage (Alonso, Gortázar & García de Jalón, 2010). Two sampling points were chosen along the upstream Nalón River (points n1, n2) and six on one of its head tributaries, Caleao river (c1 to c6; Fig. 1). The two streams are connected at the tail end of Tanes reservoir (Fig. 1). In each sampling point, the habitat was characterized based on the official protocol of the Spanish Ministry of Environment, Feeding and Agriculture (Alba et al., 2005), to check if it was appropriate for salmonids. A 20-meter transect was analysed per site. A profile of the substrate was drawn, characterized as percentage of blocks, boulders, gravel, sand and silt. The dominant vegetation type covering the river (river canopy) was identified as well as the shade percentage and continuity. Three measures of depth and width were taken within the 20-meter transect at each sampled site. From these data, the total amount of juvenile salmonid (‘rearing’) habitat units were estimated for each site based on the model described by Juanes et al. (2011). All profile variables were considered to assign a percentage of juvenile’s habitat to each sampling point. Then, it was standardized to 100 m² (estimated rearing units or ERU) ( Table S1).

Physical-chemical water properties were recorded with a Horiba U-50 multimeter at three different points in each sampled site. To minimize the potential for biased data (i.e.: after a storm) abiotic parameters were replicated three times with a seven-day interval between each reading and the average of these three measurements was presented. Measured parameters included water temperature (°C), pH and TDS (Total Dissolved Solids) (Table 1).

In addition to abiotic measures, macroinvertebrates communities were studied to calculate water quality indices. Individuals were sampled following the kick-net sampling methodology described in the protocol employed (Alba et al., 2005). Collected specimens were identified down to Family level using an identification key (Oscoz, Galicia & Miranda, 2011), and family presence-absence was recorded. Ecological quality ratio (EQR) and the ecological state of each point were calculated based on macroinvertebrates family’s composition, giving a punctuation to each family based on their tolerance to contamination (Alba et al., 2005) (Table 1).

One extra point was selected in Nalón River to collect only water samples (n3). Taken upstream n2 (where a rainbow trout fish farm is located, see Fig. 1), this water sample (n3) was collected 20 m upstream fish farm discharges as a control to discard the possibility that rainbow trout DNA comes only from fish farm runoff.

Water sample collection, filtration and eDNA extraction

Water samples of 1.5 L each were collected in sterile plastic bottles from the sampling points. Water samples were vacuum-filtered using a Supor^®-200 Membrane Filter (Pall Corporation, Life Sciences, Ann Arbor, MI, USA) with 0.2 µm pore size. The filtration room was free of external sources of contamination as it was separated from the molecular laboratory. The filtration system was cleaned up with 10% commercial chlorine based-bleach between samples to avoid contamination between sampling points. 1.5 L of Milli-Q water that was previously transported with the rest of the water samples from the field, was filtered at last following the same steps, and was considered as an extra sample to monitor possible field or filtering contaminations. Finally, filters were placed into 15mL tubes using sterile forceps and stored at −20 °C until DNA extraction.

DNA was extracted from filters with PowerWater^® DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA) and preserved at −20 °C until further processing. The DNA extractions were conducted under sterile conditions inside a laminar flow PCR-cabinet, following the manufacturer’s instructions. A negative control was added at this step to monitor contaminations during the extraction process.

Quantitative PCR procedures

Quantitative PCR from eDNA using specific primers has been validated for brown (Gustavson et al., 2015) and rainbow trout (Wilcox et al., 2015) (Table 2). Details about qPCR protocols are included in this section, as recommended by Bustin et al. (2009):

Two specific TaqMan assays were selected as molecular markers: for brown trout, a 61 base pairs (bp) fragment of the mitochondrial cytochrome oxidase I gene (COI: Cytochrome Oxidase Subunit I) (Gustavson et al., 2015); and a 102 bp fragment of the NADH gene for rainbow trout (Wilcox et al., 2015). In silico analysis were done using the Primer Blast application included in the NCBI webpage (Ye et al., 2012) to check the specificity of the markers. No coincidences were found with related nor cohabiting species.

Pre-PCR analyses of eDNA samples were carried out in a room separated from the molecular laboratory where there is no DNA nor tissue samples, inside a flow PCR-cabinet. Negative controls from the field, filtration and extraction processes were included in PCR runs as well as a PCR negative control.

The qPCR (quantitative Polymerase Chain Reaction) runs were performed using 7,900 HT Fast Real-Time PCR System (Life Technologies, Inc., Applied Biosystems, Carlsbad, CA, USA). Amplification reaction mixtures for brown trout included: 10 µl of TaqMan Environmental Master Mix 2.0 (Life Technologies, Carlsbad, CA, USA), 0.4 µl of each Primers (final concentration of 0.2 µM), and 0.4 µl hydrolysis probe (final concentration of 0.2 µM), and DNA template (6 µl of eDNA extracted from water samples, or from 43 ng of tissue DNA), up to a final 20 µl volume. Amplification reaction mixtures for rainbow trout included: 10 µl of TaqMan Environmental Master Mix 2.0 (Life Technologies, Carlsbad, CA, USA), 0.6 µl of Forward primer (final concentration of 0.3 µM), 1.2 µl of Reverse Primer (final concentration of 0.6 µM) and 0.5 µl of hydrolysis probe (final concentration of 0.25 µM) and DNA template (6 µl and 43 ng of eDNA and tissue DNA respectively), also up to a final 20 µl reaction volume.

As positive controls, DNA from tissue samples of each species were extracted with E.Z.N.A.^® Tissue DNA Kit (Omega, Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions. The two molecular markers were tested first on control DNAs. A mixture of control DNAs from rainbow and brown trout at known concentrations were PCR amplified with the two specific markers to check for possible co-amplification or interference between them (Fig. S1). On each qPCR run, a positive control from tissue extractions of the targeted species was also added to monitor PCR inhibition.

PCR amplicons were generated with the two primer sets from tissue DNA in a total volume of 20 µl, including Green GoTaq^® Buffer 1X, MgCl₂, 0.25 mM dNTPS, 0.25 µM of each primer, 4 µl of template DNA and 0.65U of DNA Taq polymerase (Madison, WI, USA). PCR conditions were 95 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, and a final step of elongation at 72 °C for 10 min.

The PCR amplicons obtained were quantified by fluorimetry using Qubit^® dsDNA BR Assay Kit (Thermofisher Scientific, Carlsbad, CA, USA). The amount of DNA was transformed into molecules per µl, calculated from the known base composition of the amplicon sequence. A standard curve was constructed including a serial dilution (from 2.34 × 10⁹ to 2.34 ×10³ molecules/µL for rainbow trout and from 6.3 ×10⁹ to 6.3 × 10³ molecules/µL for brown trout) and used as reference for DNA molecules quantification in water samples. A dilution series from tissue DNA of each species was done to determine the lowest copy number of target DNA per litre of water detectable from each assay.

All the analyses of tissue and amplicon samples were conducted separately from environmental samples, keeping them away from any source of contamination.

To confirm the correct target species detection, amplicons from some environmental samples were sequenced. Sequencing was carried out in the Sequencing Unit of University of Oviedo’s Scientific-Technical services.

Species-specific PCR-RFLP

The nested PCR-RFLP method was developed as described in Clusa et al. (2017) for detecting the two targeted species (brown and rainbow trout). Briefly: a first PCR was carried out to amplify a 567 bp fragment of the 16S rRNA gene, using as forward the 16S-new-F primer designed in the cited research (Table 3), and the reverse 16S-Br universal primer from Palumbi et al. (2002). A nested PCR amplification was then performed with the pair of Salmonidae-specific primers described in Zaiko et al. (2015) (Table 3). The nested PCR product was digested with TaaI and Tru1I FastDigest enzymes (Thermo Fisher Scientific Inc., Waltham, MA, USA) that produce diagnostic band patterns in agarose gel allowing to identify the two targeted species. With TaaI enzyme, brown trout DNA gives two bands of 205 and 272 bp, and with Tru1I, rainbow trout DNA gives a band of 66 bp.

Habitat quality measures

The physical-chemical characteristics of the sampling points were within the optimum range for Salmonid juveniles (Raleigh et al., 1984; Raleigh, Zuckerman & Nelson, 1986). The numbers of juvenile salmonid habitat units (ERU) ranged between 17 in c4 and 86.17 in n2 (Table 1).

The ecological state measured from macroinvertebrate communities (EQR) was good at the points from the upper zone of Caleao River and in Nalon River (c6, n1 and n2), and moderate in the rest of the points. Detailed results of macroinvertebrate families along sampling stations are summarized in Table S2.

qPCR assays

Assays of control DNAs provided the same quantification cycle values in the samples with single species DNA as well as in the mixture DNA samples (17 cycles for rainbow trout marker and 15 cycles for brown trout marker; see Figs. S1A and S1B respectively). Thus, there were no interferences caused by the presence of DNA from the other species (Fig. S1).

Standard curves for brown and rainbow trout fitted the equations y =  − 3.819x + 45.141, R² = 0.999 and y =  − 3.3128x + 39.2113, R² = 0.999 respectively. The lowest detectable number of copies was 7 ×10² and 2.6 ×10³ molecules per liter for brown and rainbow trout assays respectively. From eDNA, negative controls were clean as expected i.e., no evidence of amplification. As positive amplification was obtained from all eDNA extraction samples for brown trout assay (see below), PCR inhibition testing was not performed.

Values of quantification obtained from river water samples varied from 94.34 × 10² to 11.26 × 10² trout DNA molecules (Table 1). Brown trout eDNA was found from all nine sampling sites. Rainbow trout eDNA occurred from the three points located in Nalón River (Table 1), one 20 m upstream (Veneros) and two downstream from the rainbow trout fish farm located in that river. The amount of rainbow trout eDNA molecules was considerably higher than that of brown trout in the Nalón River sampling points (n1 to n3), especially in n1 where the highest amount (94342.76 eDNA molecules) was found. This point is located in the lowest zone of the sampling area (Fig. 1).

PCR-RFLP

The restriction patterns within the fragment amplified provided specific bands for brown trout (205 and 172 bp) after digestion with TaaI enzyme in all sampling points except n1 and n2 (Fig. 2A), where only the specific band for rainbow trout (66 bp) was found after digestion with Tru1I enzyme (Fig. 2B). Rainbow trout typical RFLP pattern was also found in n3 after Tru1I digestion (Fig. 2C).

Conclusion and implications for conservation

From our results, and supporting Gustavson et al. (2015), qPCR is more effective for species detection when the eDNA abundance is low or if the number of eDNA molecules of two targeted species are very different. The use of qPCR for species detection in those cases would be recommended.

This is an example of the potential of eDNA for investigating the distribution of native and exotic fish in running waters. In this case, it served for surveying trout species inside a protected area, where sampling using conventional methodology (i.e., electrofishing or netting) should be disregarded as much as possible so as to not disturb vulnerable communities from upstream mountain landscapes. Therefore, we would recommend this strategy for routine monitoring and early detection of exotic species within natural reserves. Although the results support the occurrence of undeclared exotic rainbow trout, they should be confirmed by independent observations and conventional standard monitoring, to sample individuals and to check if rainbow trout presence is sporadic or on the contrary it reproduces in the river. We consider that a stricter control of the fish farm would be recommended, and efficient containment measures of farmed fish should be taken to prevent escapes.