ePIKE - Monitoring of pike with eDNA

ePIKE is divided into three complementary work packages. The research team consists of Anti Vasemägi, Martin Ogonowski, Göran Sundblad, Patrik Bohman, Josefin Sundin, and Erik Karlsson at the Department of Aquatic Resources, SLU.

Work package 1 – Evaluation and development of molecular methods

eDNA-methods have recently been applied successfully to detect pike in the environment using qPCR. The PCR-method is able to amplify DNA-molecules which allows very small amounts of DNA to be detected in the water. However, questions remain regarding which extraction methods to use and what the limit of quantification is. Therefore, we will test the efficiency of different filter types and optimize extraction methods to develop suitable lab protocols for qPCR. 

Work package 2 – Determination of an eDNA-biomass relationship

The amount of eDNA is expected to differ between different life stages of pike. It is therefore important to determine how the eDNA-signal varies in relation to fish size. In this work package we will investigate how the eDNA-biomass relationship differs between juvenile and adult pike under semi-natural conditions. It is also important to determine when it is most favourable to sample eDNA. Therefore we will study how the eDNA-signal fluctuates over the season as temperature and water quality change. 

Work package 3 – Field-based evaluation of the eDNA methodology

Our overarching aim is to evaluate the applicability of eDNA for monitoring of pike populations in the wild. Based on the results obtained from work packages 1 and 2, we will develop a series of protocols and sampling strategies appropriate for this type of monitoring. In conjunction, we will also compare the eDNA-based methods to other sampling methods aimed at specific life stages of pike. By these comparative studies we will be able to systematically evaluate the appropriateness of eDNA as a monitoring method for pike.  

Laboratory experiment with juvenile pike

The objective of this experiment was to:

1. Obtain eDNA-samples from pike to identify suitable primers (mtDNA, COI and 5S rDNA ITS)
2. Estimate DNA shedding and degradation rates
3. Determine the DNA-biomass relationship under controlled and nearly optimal conditions

Juvenile pike where caught in collaboration with the Swedish Angling Association “Sportfiskarna” in a nearby wetland in June 2019 and transported to aquaria at the institute of freshwater research, SLU, Drottningholm. After an acclimation period, we conducted a small scale experiment where different densities of juvenile pike (0, 1, 3, 9 individuals, 3 replicates each) were incubated in aquaria containing 15L filtered lake water. Water temperature and other environmental variables were monitored continuously. eDNA samples were collected by filtration through a 1.2 µm nitrocellulose filter at seven time points (before the addition of pike, during the incubation and after removal of pike).

Preliminary results from the experiment

Juvenile pike collected from the wild were successfully kept under laboratory conditions (in accordance with ethical permits). Individual isolation in Whitman-Vibert-boxes proved to be an effective method to prevent injuries and cannibalism when several individuals were kept together. The preliminary studies have provided valuable information and practical experience in stress-mitigation and survival of juvenile pike under laboratory conditions. Work analysing eDNA samples from the experiment and design of new 5S rDNA primer pairs is ongoing in collaboration with the Estonian University of Life Sciences in Tartu. .

Literature review

During 2019 we have conducted a comprehensive literature review which will be useful in the development of laboratory protocols and experimental designs.

Fieldwork

We conducted a comprehensive collection of eDNA samples from the Stockholm Archipelago during spring. We collected water samples for eDNA analysis from 22 shallow bays during the pike spawning period (March-May). The collection of eDNA was synchronized with the hook-and-line fish survey within the ReFisk project. This will enable the comparison of different sampling methods. All eDNA samples are frozen and will be analysed during 2021. 

Laboratory experiment

During 2020, our team was reinforced by a new Master’s student – Ofír Svensson. At the experimental facility in Drottningholm we conducted a series of outdoor mesocosm experiments where pike of different sizes, ranging a few kg to approximately 10 kg were incubated in natural lake water to determine the DNA-biomass relationship in adult pike. We also studied the effect of pike densities on eDNA shedding and different combinations of filtration setups in order to optimize the sampling and DNA recovery. 

During autumn, we have moved into the lab where we are testing different DNA extraction methods with the aim to find a cost efficient, reliable and effective protocol which will be used to analyze the accumulated samples from mescosm experiments and the field. The winter months will therefore be primarily dedicated to analysis of collected samples, but also to planning!

During 2021 we will conduct yet another field study where we aim to study how the eDNA-signal fluctuates seasonally. More info to come!

Fieldwork

On March 17th we installed a fish counter in the wetland Hemmesta sjöäng east of Stockholm. The counter is mounted at the entrance of the wetland where it will count, measure length and film all fishes that moves in and out of the wetland. Link to the counter

During spring, as pike spawn we will besides monitoring the counter also take water samples for eDNA analyses continuously over the entire spring season. By doing so we can investigate if the amount of eDNA in the outflow from the wetland can be used to successfully describe the patterns of the spawning migration. Sampling is done in accordance with techniques we have developed in earlier parts of the project.

The study at Hemmesta is done in collaboration with the Swedish Angler Association and Värmdö county, whom also involve school students to manage the wetland within the project Skolbäcken. We are very happy about these collaborations and the synergistic effects at the local level. We are also grateful to the volunteers that have contributed in different ways. The fish counter comes from TiVA and the management of the website is operated by Fiskevårdsteknik.

The large fieldworks within ePIKE are now completed, but we are still very active. During 2022 we have spent a lot of time in the lab, working on the development of a qPCR-assay targeting a multi-copy nuclear genetic marker instead of the more conventional mitochondrial markers. We hope that this approach will allow for equal, or even higher sensitivity making it possible to quantify considerably lower eDNA concentrations and provide a viable alternative to mitochondrial markers. Moreover, in combination with a mitochondrial marker we also anticipate to be able to detect active spawning events by studying the change in ratio between nuclear and mitochondrial gene copy numbers.

Results during 2022

We are very happy and proud to have the first ePIKE-publication coming out. Based on results from our earlier experiments it shows that the methods we have developed can quantify pike biomass, at least under controlled laboratory conditions (Karlsson et al. 2022).

We have also published a preprint of the fieldwork from 2020, where we collaborated with the ReFisk-project and compared pike abundance in spawning areas during spring (Ogonowski et al. 2022). Rod-fishing (i.e. angling) and eDNA showed a positive correlation, indicating that both methods can separate low-high pike abundance also in the field. However, we found that water temperature was very important for this relationship. Since there was more fish spawning with increasing temperature, thus releasing more DNA in to the water and increasing pike movements within the bays, we found an increased eDNA signal with increasing temperature. That rod-fishing and eDNA give slightly different results is not surprising since eDNA has the potential to sample a larger part of the population than angling, which is restricted to larger fish that are willing to strike the lure. The study also highlighted at what spatio-temporal scales variation in the eDNA signal was largest, which provides useful information for our continued work on developing a useful monitoring tool for Northern pike.

Ongoing 2023

The modified qPCR-assay using a nuclear genetic marker is currently being evaluated and optimized. Preliminary results indicate that the assay is, in all practical terms species specific and approximately 16 times more sensitive compared to the mitochondrial COI marker. The results of this study are expected to be published later this year.

The time-series data collected during 2021 from Hemmesta träsk combined with census data from the fish counter are currently being analysed with results expected later this year. We look forward to these results with excitement.