These methods, instrumentation and/or protocols apply to all data in this dataset:| Methods and protocols used in the collection of this data package |
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| Description: | Study site:
The focus of our study was Hidden Lake, Banff National Park, Canada, a high elevation mountain lake in the Canadian Rockies that received rotenone treatment during the summers of 2018 and 2019 to eradicate non-native brook trout (Salvelinus fontinalis) that were introduced and established in the 1970s, leading to the extirpation of a population of Westslope cutthroat trout (Oncorhynchus clarkii lewisi). The rotenone treatment was done accordingly to Montana State (USA) rotenone policy in the absence of a Canadian equivalent. This policy recommends two rotenone treatment for brook trout eradication because their spawning is not perfectly synchronous and because brook trout eggs in the gravel are not susceptible to rotenone (MFWP 2017). Moreover, several fish and traces of environmental DNA from brook trout were detected between the two rotenone treatments in summer 2018 and 2019.
The rotenone formulation applied to Hidden Lake (21-22 Aug 2018 and 13 Aug 2019) was Nusyn-Noxfish® and contained 2.5 % rotenone active ingredient. The theoretical rotenone concentration of Hidden Lake once it penetrated the thermocline by pumping was 30 ppb and 25 ppb in 2018 and 2019 respectively (Parks Canada 2020).
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| | Description: | Samples collection:
Samples collected for the evaluation of the efficacy of brook trout removal efforts from the Hidden Lake watershed were collected from Hidden Lake as well as from Corral Creek and tributary streams. Environmental DNA samples were collected from Hidden Lake, Hidden Creek, and Corral Creek following rotenone application at four time points: (i) five weeks prior to the first rotenone application, from Hidden Lake only on July 12 2018; (ii) approximately three weeks after the first application of rotenone, from Hidden Lake and Hidden Creek on 7 September 2018 and from Corral Creek on 9 September 2018; (iii) approximately 10 months after the first rotenone application, on 10 July 2019 on Corral Creek and Hidden Creek and 11 July 2019 on Hidden Lake; and (iv) one year after the final rotenone treatment, on 19 August 2020 in Hidden Lake, Hidden creek and Corral creek. Because of travel restrictions associated with the COVID19 pandemic for university researchers, eDNA samples from the fifth period (one year after 2nd rotenone treatment) were collected and analyzed by Aquality Environmental Consulting Ltd (Calgary, Alberta) using similar field sampling and laboratory analytical protocols (see methods below for further details).
Hidden Lake. Sample sites on Hidden Lake were equidistantly distributed throughout the lake, with four samples collected in both the littoral and pelagic zones of the lake for each sampling event, with two 700 ml Whirl-Pak™ bags per site and from a bleached inflatable kayak. The four littoral samples were collected approximately 1-3 m from the shore at a minimum depth of 30 cm but approximately 15 cm above the lakebed to avoid the collection of sediments, which can both inhibit downstream DNA applications and represent a significant source of potential DNA contamination (Turner et al. 2014). Four pelagic samples were collected in a transect along the longest axis of the lake from the surface waters of the euphotic zone at a depth of approximately 0.5 to 1.0 m. In total, this made 8 samples per time point.
Hidden Creek and Corral Creek. Sample sites on Hidden Creek and Corral Creek were distributed throughout the streams. Hidden Creek and Corral Creek have a number of small primary tributaries that could potentially be inhabited by brook trout; limited sampling time precluded collecting samples from all such tributaries. Emphasis was therefore placed on collecting eDNA samples 5-10 m downstream of the confluence of tributaries, on the assumption that a positive detection of eDNA in the main stem immediately downstream of primary tributaries could indicate the presence of brook trout in the tributary, particularly if upstream samples were negative, for a total of 13 sampling sites along Hidden Creek and Corral Creek. For these stream samples, the water was collected using two 700 ml Whirl-Pak™ bags per site at mid-stream locations without entering stream habitat while wearing sterile nitrile gloves.
eDNA sampling collected one year after the final rotenone application was focused on stream sites within the watershed where trace eDNA was found during the third eDNA sampling period. For this last sampling period, sample replicates were collected using a backpack eDNA sampler (Smith-Root, WA, USA) following manufacturer recommendations; sampling was conducted by Aquality.
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| | Description: | Filtering and DNA extraction:
eDNA samples from 2018 and 2019 were immediately filtered on the lakeshore or streamside on a chlorophyll filtering manifold (Wildco, Florida, USA). One liter of water from each site was filtered through a 0.7 µm glass fibre filter (GE Healthcare Life Sciences, Ontario, Canada) using a vacuum hand pump (Soil Moisture, California, USA). As a negative control, 500 mL of distilled water was filtered prior to filtering lake or stream samples for each day of field work. After filtration, filters were folded and stored in a sterile 2 mL microcentrifuge tube containing 700 µL of ATL buffer that was then labelled and individually sealed in a zippered plastic bag. Filters were immediately placed in a cooler bag containing two frozen gel packs, and transported to a -20 ℃ freezer right away, until they were driven to Montreal while stored on dry ice. In Montréal, filters were stored in a -80 ℃ freezer until further analyses.
At one year after the final rotenone treatment, water samples were also collected from an unnamed water source known to contain brook trout as a positive field control. A negative control sample composed of 1 L of Lake Louise municipality tap water was also filtered on site. eDNA samples were immediately chilled for the duration of transport (36 hours) back to the laboratory where they were frozen in a -20 ℃ freezer until extraction. Due to differences in the teams conducting the sampling, water samples from 2020 were pumped through self-preserving, single use, 1.2 µm polyethersulfone (PES) filter membrane apparatuses (Thomas et al. 2019).
DNA from each filter was extracted using Qiagen DNeasy Blood and TissueTM kit and QiashredderTM columns following a modified extraction protocol (see Appendix A in Yates et al. (2020) for details). Extracted DNA was eluted into 130 µL of AE buffer and stored in a clean -20 ℃ freezer solely dedicated to the storage of extracted eDNA product (i.e. no post-PCR products or tissue samples). An extraction blank of 700 µL of ATL buffer was included in all batches. Environmental DNA samples from 2020 were extracted from filters using the same Qiagen DneasyTM Blood and Tissue kit as for the other samples, but without the Qiashredder spin columns and following manufacturer’s protocol, with the exception that during lysis filters were beat with 1-mm silicacarbide beads for 10 min on high using a Bead Mill Homogenizer (Fisher Scientific).
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| | Description: | Quantitative PCR (qPCR)
Brook trout eDNA concentration was quantified using quantitative PCR (qPCR) with the TaqMan minor groove BRK2 assay published in Wilcox et al. (2013). Details on PCR reaction preparation, component concentrations, standard curve preparation, and cycling conditions are in the Appendix 1 of Yates et al. 2020. All samples were run in triplicate at a 20 µL reaction volume with 5 µL of template DNA on a Stratagene MX 3000P thermal cycler. All reactions were spiked with an internal positive control (IPC) to test for inhibition, which was defined as a 1 > Ct shift in the IPC relative to a reaction containing 5 µL of nuclease-free ultrapure water (Appendix 1 in Yates et al. 2020). Brook trout eDNA concentrations at each site were calculated by averaging replicate values, and final copy number data was presented as mean copies per reaction. Quantitative PCR for samples from 2020 were also performed using the protocol detailed by Yates et al. (2020), with the exception that IDT PrimeTime Gene expression was used as a master mix, a six-point standard curve ranging from 50,000 to 0.5 copies of the target gene was used, and samples were spiked with an internal positive control published in (Rudko et al. 2017) to assess sample inhibition.
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| | Description: | Contamination prevention:
All lake samples were collected from an inflatable kayak that was decontaminated 48 h prior to sampling by a complete soaking in a 2% household bleach solution for 15 minutes. The kayak paddle and life jacket used were similarly decontaminated. All samples were collected with Whirl-Pak™ bags (Uline, Ontario, Canada) while the individual collecting the sample wore sterile nitrile gloves. The vacuum hand pump (Soil Moisture, California, USA) used for filtration was wiped with a 30% household bleach solution and allowed to rest for ten minutes before rinsing with distilled water before the sampling day. The filtering manifold components were soaked in a 30% household bleach solution for a duration of 8 minutes and rinsed with distilled water between each sample to avoid cross-contamination of eDNA. Filters were handled with a pair of metal forceps that were soaked in a 30% household bleach solution for 8 minutes between sample filtrations before rinsing in distilled water.
For transport, manifolds were transported in a backpack cooler (Polar Bear Coolers, Georgia, USA) whose interior was wiped with a 30% bleach solution and allowed to rest for ten minutes before rinsing with distilled water. Manifold components were stored in sealed individual plastic zippered bags for transportation to Hidden Lake and Corral Creek. Writing utensils (pencils and markers) were also wiped with a 30% bleach solution and stored in zippered bags. The cooler bag used to keep eDNA samples cold and transportation was decontaminated by wiping with a 30% household bleach solution and allowed to rest for 10 minutes before rinsing with distilled water. This freezer contained two frozen gel packs that were decontaminated by soaking in 30% household bleach solution for ten minutes and rinsing with distilled water. Filters were immediately transported to Kootenay crossing, where they were stored in a zippered plastic bag in a -20 ℃ freezer that was previously decontaminated by wiping with a 30% household bleach solution.
To avoid cross-contamination between lake and stream sites, as well as time periods, extractions were conducted in batches corresponding to sampling period and lake or stream sites (i.e. only samples from the lake or only samples from streams from a single sampling period were extracted in a batch). Extracted DNA was eluted into 130 µL of AE buffer and stored in a clean -20 ℃ freezer solely dedicated to the storage of extracted eDNA product (i.e. no post-PCR products or tissue samples). All extractions were conducted in a room dedicated solely to the extraction of eDNA samples that is cleaned on a weekly basis with a 10% household bleach solution and is free from PCR products, animal tissues, or high-concentration DNA. All individuals entering and working in the room are required to wear dedicated and clean lab coats, dedicated footwear with shoe covers, hair nets, and nitrile gloves. Laboratory surfaces were soaked with a 20% household bleach solution for ten minutes before and after each extraction batch. PCR Clean Wipes™ (Thermo Scientific, Massachusetts, USA) were also used to decontaminate all lab surfaces and micropipettes before and after each extraction batch.
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| | Description: | Aquality methods:
Field
The 2020 set of eDNA samples were collected following the second rotenone application, with an emphasis on and additional stream site within the watershed. Two additional sites were added on a tributary stream flowing into Hidden Creek that was identified as potential spawning grounds and rearing habitat for juveniles. Triplicate 1 L sample replicates were collected per site using a backpack eDNA sampler (Smith-Root, WA, USA) following manufacturer recommendations. Clogged filters at some sites resulted in less than 1 L being collected on some replicates and greater than 1 L on others; however, 3 L total were filtered at all sites except for CC14, where only 1 L total of water was collected due to filter clogging. Samples were collected instream from pool habitats without entering the stream.
Filtering
Water was pumped through self-preserving polyethersulfone (PES) filter membrane apparatuses (Thomas et al. 2019). Samples in Hidden Lake were collected via an inflatable kayak. As a positive field control, water samples were also collected from an unnamed water course known to contain brook trout. A negative control sample composed of 1 L of Lake Louise tap water was also filtered on site. Environmental DNA samples were immediately chilled for the duration of transport (36 hours) back to the laboratory where they were immediately frozen in a -20 ℃ freezer until extraction.
DNA extractions
Environmental DNA was extracted from filters using a Qiagen DNeasyTM Blood and Tissue kit following manufacturer’s protocol, with the exception that during lysis filters were beat with 1-mm silicacarbide beads for 10 min on high using a vortex. Environmental DNA analysis was conducted within a laboratory workflow which included separate clean rooms, pre-amplification, post-amplification, and DNA extraction spaces to prevent sample cross-contamination.
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| | Description: | References:
Canada Parks. 2020. Summary report for the chemical removal of brook trout from Hidden Lake, Upper Corral Creek and Tributaries. Radium Hot-Springs (BC): Internal Report.
[MFWP] Montana Fish Wildlife and Parks: Fisheries Division. 2017. Montana rotenone policy. Approved by: Eileen Ryce, Fisheries Division Administrator. Issued Apr 18, 1996. Revised Apr 5, 2017.
Rudko SP, Ruecker NJ, Ashbolt NJ, Neumann NF, Hanington PC. 2017. Enterobius vermicularis as a novel surrogate for the presence of helminth ova in tertiary wastewater treatment plants. Appl Environ Microbiol. 83(11):1–13.
Thomas AC, Nguyen PL, Howard J, Goldberg CS. 2019. A self-preserving, partially biodegradable eDNA filter. Methods Ecol Evol. 10(8):1136–1141.
Wilcox TM, Mckelvey KS, Young MK, Jane SF, Lowe WH, Whiteley AR, Schwartz MK. 2013. Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity. PLoS One. 8(3):e59520
Yates MC, Glaser DM, Post JR, Cristescu ME, Fraser DJ, Derry AM. 2020. The relationship between eDNA particle concentration and organism abundance in nature is strengthened by allometric scaling. Mol Ecol. Early view:1–15.
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