A Noninvasive Tool to Assess the Distribution of Pacific Lamprey (Entosphenus tridentatus) in the Columbia River Basin

The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish once abundant throughout coastal basins of western North America that has suffered dramatic declines in the last century due primarily to human activities. Here, we describe the development of an environmental DNA (eDNA) assay to detect Pacific lamprey in the Columbia River basin. The eDNA assay successfully amplified tissue derived DNA of Pacific lamprey collected from 12 locations throughout the Columbia River basin. The assay amplifies DNA from other Entosphenus species found outside of the Columbia River basin, but is species-specific within this basin. As a result, the assay presented here may be useful for detecting Entosphenus spp. in geographic range beyond the Columbia River Basin. The assay did not amplify tissue or synthetically derived DNA of 14 commonly sympatric non-target species, including lampreys of the genus Lampetra, which are morphologically similar to Pacific lamprey in the freshwater larval stage.


Introduction
The Pacific lamprey (Entosphenus tridentatus) is an anadromous fish that was once abundant along the Pacific Coast of North America from Alaska to Mexico [1]. However, human activities and infrastructure have reduced habitat and restricted fish passage leading to dramatic declines of Pacific lamprey populations throughout Washington, Oregon, Idaho, and California [2]. The Pacific lamprey, along with the western brook lamprey (Lampetra richardsoni) and western river lamprey (L. ayresii), was petitioned in 2003 for protection under the U. S. Endangered Species Act, but the petition was denied due to insufficient data on species distribution and population structure of these fish [3]. Techniques that can rapidly and reliably detect species at low densities and accurately distinguish between species (particularly in early life stages) are necessary to further inform the status of all lamprey in this context. To assist in this effort, we developed an eDNA assay for detecting Pacific lamprey in the Columbia River basin where state, tribal, and federal efforts for species recovery are underway. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Methods and Results
To develop an eDNA assay specific to Pacific lamprey, we first sequenced DNA of the mitochondrial cytochrome c oxidase I (COI) of Pacific lamprey and unidentified freshwater lamprey. Tissue samples were obtained from previous studies conducted by the Columbia River Inter-Tribal Fish Commission, Nez Perce Tribe and the U.S. Forest Service. Freshwater lamprey species included in this study were obtained from various locations in the Willamette River basin, OR. DNA was extracted from tissue samples using the Qiagen DNEasy 1 Blood and Tissue Kit following manufacturer's protocol. We developed sequencing primers by aligning whole mitochondrial genome sequences of the least brook lamprey (Lampetra aepyptera), the American brook lamprey (Lethenteron appendix), and the European river lamprey (Lampetra fluviatilis; GenBank accession numbers: KP742974, KM267719, and Y18683 respectively) in MEGA 6 [4], and manually screening the COI region for conserved sequences among species. Within these conserved areas, we developed three sets of forward and reverse primers (Table 1), each amplifying a 534-600 base-segment of COI. PCR products for sequencing were obtained by amplifying DNA in 40.2 μl reaction volumes containing 1 mM of each primer, 2.5 mM MgCl 2 , 4 μl of 10X PCR buffer, 0.2 mM of each deoxynucleotide triphosphate (dNTP), 0.2 μl BSA, 0.2 μl 50X TITANIUM™ Taq DNA Polymerase (Clontech Laboratories), and the remaining volume with PCR-grade water. Cycling conditions consisted of 95˚C/12 min, [95˚C/1 min, 55˚C/1 min, 72˚C/1.5 min] × 35 cycles, with final extension at 72˚C/5 min. PCR products were cleaned with Exo-SAP-IT (Affymetrix) and DNA sequence data were obtained using the Big Dye kit and the 3700 DNA Analyzer (ABI; High Throughput Genomics Unit, Seattle, WA). The sequence data were analyzed and concatenated into a 1 421 base fragment using Sequencher 5.3 (Gene Codes Corporation, Ann Arbor, MI USA http://www.genecodes. com) and were uploaded to GenBank (accession numbers: KX389871-KX389877; KX443679-KX443687).
Lampetra individuals were presumed to be either L. ayresii or L. richardsoni based on the location of capture. However, we were unable to identify these individuals to the species level due to the lack of resolution in the taxonomy of Lampetra across western North America [5].
In addition to the sequence data obtained above, we obtained sequences of the COI region from GenBank for three additional Pacific lamprey individuals as well as 19 non-target species that are closely related or commonly co-occur (Table 2). We used the DECIPHER package [6] in R v. 3.0.1 [7] to obtain primers specific to Pacific lamprey. We then aligned sequences in MEGA 6, and visually identified a species-specific region to create a hydrolysis probe with a MGB quencher (Table 1). Primers were manufactured by IDT and purified using standard desalting methods. The probe was obtained from Life Technologies and was purified using HPLC. We assessed the melting temperatures of the primers (forward: 59.0˚C; reverse: 59.5˚C) and probe (70.0˚C) in Primer Express 3.0.1 (Life Technologies) and screened for secondary structures using IDT OligoAnalyzer (https://www.idtdna.com/calc/analyzer). We extracted DNA from tissue of 42 Pacific lamprey from 12 different locations in the Columbia River basin and 13 non-target species using methods outlined above (Table 3). DNA concentrations for tissues were measured using a Qubit 2.0 fluorometer (ThermoFisher Scientific) and ranged from 12.8-72 ng/μl. We were unable to obtain tissue from Pacific brook lamprey (Lampetra pacifica) for screening against the Pacific lamprey assay. To overcome this limitation, we used published sequence data (GenBank accessions KY072805-KY72808) to identify a DNA fragment matching a 449 basepair region of the COI gene encompassing the region of the Pacific lamprey assay. We then obtained a synthetic DNA fragment from IDT in the form of a plasmid with a PUVI restriction digest sequence inserted at the end of the 449 basepair sequence. The plasmid was linearized using PVUI restriction enzyme (New England Biosystems) following manufacturer's protocol and purified using a PureLink™ PCR Micro Kit (Invitrogen). We estimated the concentration of the linearized plasmid DNA using a Qubit 2.0 fluorometer (ThermoFisher Scientific) and diluted the DNA to 0.04 ng/ul in TE. We screened each non-target tissue and the synthetic L. pacifica DNA against our assay in a single 15-μl reaction using a StepOne Plus Real-time PCR Instrument (Life Technologies). Each reaction contained 7.5 μl Environmental Master Mix 2.0 (Life Technologies), 900 nM forward primer, 900 nM reverse primer, 250 nM probe, 4 μl DNA template (diluted 1:100 from extract), and 2.75 μl deionized water. Thermocycler conditions included 95˚C for 10 minutes followed by 45 cycles of denaturation at 95˚C for 15s and annealing and extension at 60˚C for 1 min. The assay amplified all Pacific lamprey samples (cycle threshold, C t , ranging from 19.1-21.9 Table 2. Species and source for DNA sequences used for in silico assay development. Source refers to GenBank accession number or location of collection for specimens analyzed in this study. Probe mismatches refers to the number of base-pair differences between the eDNA assay probe and the COI sequence for a particular species. Pacific brook lamprey (Lampetra pacifica) sequence data were obtained after primer and probe development.   River basin, OR), Vancouver lamprey (E. macrostomus; Lake Cowichan on Vancouver Island, British Columbia), and Northern California brook lamprey (E. folletti; portions of the Klamath River basin in CA). We optimized primer concentrations following methods outlined in Wilcox et al. [8] for final concentrations of 600 and 900 nM for the forward and reverse primer respectively. Using optimized assay concentrations and the cycling conditions above, we tested assay sensitivity by screening against a six-level standard curve dilution series (6 250, 1 250, 250, 50, 10, and 2 copies per 4 μl) created from target PCR product. We ran six replicates of each dilution resulting in an amplification efficiency of 97.2% (r 2 = 0.979), and observed amplification in all six reactions of 10 copies per 4 μl and five of six reactions at 2 copies per 4 μl, with an average Ct = 38.4 across the five positive reactions.
Finally, we screened the assay against eDNA samples collected from eight western U.S. sites for which the presence of Pacific lamprey was known from previous electrofishing surveys (Table 4). Environmental DNA was collected from 5-l water samples following methods described in Carim et al. [9] and extracted using Qiagen DNEasy 1 Blood and Tissue Kit following a modified protocol [10]. Using optimized assay concentrations, we analyzed these environmental samples in triplicate using the PCR recipe and cycling conditions above, but replacing 1.8 μl of water with an internal positive control consisting of water with 1.5 μl Table 4. Collection and species assemblage information for eDNA samples used to test the Pacific lamprey qPCR assay. Community assemblage information (fish species present) was obtained via a combination of eDNA analysis and personal communication with local tribal, state and federal biologists. TaqMan 10 X Exo IPC Mix and 0.3 μl TaqMan 50 X IPC DNA (ThermoFisher) to test for the presence of PCR inhibitors. To test for contamination in the qPCR setup, all eDNA samples were run alongside a negative control. As expected, the assay detected Pacific lamprey eDNA in all samples collected where this species was known to be present, but not in samples collected where this species was believed to be absent. We observed no DNA amplification in qPCR negative controls and there was no evidence of PCR inhibition in any of our eDNA samples.

Conclusions
This paper outlines the development of an eDNA assay that reliably detects Pacific lamprey in the Columbia River basin, and accurately distinguishes this species from other native lamprey (Lampetra spp.). As a result, this tool provides a sensitive and noninvasive sampling approach for determining the distribution of Pacific lamprey when individuals are present in low abundance, when physical sampling of individuals may be difficult or disruptive, and when accurate species level identification from morphological traits may be unreliable (but see [11] for tissue based genetic identification methods). As a result, this eDNA assay will be a valuable tool for management efforts focused on the assessment and monitoring of Pacific lamprey in the Columbia River basin. Conversely, this assay may not accurately separate other Entosphenus species that occur outside the Columbia River basin. As a result, this assay could serve as general assay for detection of Entosphenus spp. (such as E. lethophagus) where they may be sympatric. This assay may also serve to detect individual Entosphenus species (such as those native to the Klamath River basin) in areas where only one Entosphenus species is present. Nevertheless, we emphasize that this assay was developed for use in the Columbia River basin, and that validation of assay performance for other target and non-target species should be conducted prior to its use outside the Columbia River basin.