No Tissue Left Behind – Massive Collaborative Ecology and Evolution Project on the Kenai Peninsula

Invasive Northern Pike (Esox lucius) are a nuisance to endemic fish populations and can quickly take over waterways, but removal of Northern Pike presents a unique research opportunity. Rotenone, the chemical used to remove Northern Pike, also kills other fish, giving researchers the chance to reintroduce endemic species and do large scale ecology and evolution experiments in the wild. For example, nine lakes on the Kenai Peninsula that were treated with rotenone are now home to the “greatest ecology and evolution experiment” of all time, as project researchers like to say.

The project is looking at the ecology and evolution of reintroduced Threespine Stickleback (Gasterosteus aculeatus). The reintroduced Threespine Stickleback derive from donor populations of both benthic (bottom dwellers) and limnetic (dwellers of the light penetrating zone) morphs of the species from four lakes on the Kenai Peninsula (Tern, Watson, Spirit, and Wik) and four lakes in the Matanuska Susitna Valley (South Rolly, Finger, Long, and Walby). A mix of both benthic and limnetic ecomorphs were introduced into each recipient lake, allowing researchers to look at how the fish evolve over time and if the two ecomorphs converge, among other ecological and evolutionary questions.

“This is a rare opportunity to test predictions on how evolution proceeds in real time,” said Jesse Weber, an assistant professor at the University of Wisconsin-Madison and one of many professors working on the project. “Most similar experiments look at short lived microorganisms, like bacteria, yeast, or small arthropods. In this case, we have a fish with abundant ecological, genetic, and evolutionary data. We can test how ancestry and local environmental conditions interact and constrain or promote evolution across many different traits.”

Natalie Steinel checks a Threespine Stickleback for fibrosis before removing the head, kidney, and spleen from the fish. Photo by James Evans, University of Alaska Anchorage Advancement.

The rotenone treatments occurred in October 2018, and Threespine Stickleback were reintroduced in May and June of 2019. Two teams of researchers divided and conquered the workload of reintroductions. The first team was responsible for trapping Threespine Stickleback from donor populations and transporting thousands of fish down to the Kenai Peninsula for reintroduction into recipient lakes. The second team was responsible sampled the donor populations prior to donor fish being collected to establish a baseline on the donor populations themselves. The team sampled all aspects of the fish, measuring and weighing the fish before recording the numbers and types of external and internal parasites and the levels of fibrosis. Then the spleens, head, kidneys, liver, stomach, and intestine were removed before the fish had a filet of skin and muscle taken, and a fin clipped for genetic analysis. Finally, the remaining carcass was preserved in formalin. The team returned in 2020 and 2021 to collect the same samples from the recipient lakes to look at how the fish are changing over time.

The parasite load and fibrosis scores will help both Weber’s lab and Daniel Bolnick, a University of Connecticut professor, study the immune system of the fish. “My group is working on changes in the parasite community in each lake, how they get different parasites (diet changes in the different lakes), and how this drives evolution of their immune system,” said Bolnick. “My lab is particularly focused on the evolution of an immune defense involving fibrosis, the buildup of scar tissue from inflammation. Fibrosis also happens in people and contributes to about 40% of deaths in the U.S. (including contributing to heart disease and cancer). So, not only can we learn about fish adaptation, but in doing so we hope to better understand the genetics and function of an immune pathology also found in humans.”

Weber on the other hand is more interested in the genetics and gene expression behind immune response and predicts, “fish from different lakes will quickly converge on the same immune profiles when placed in similar environments…genetics doesn’t have a strong influence on short-term responses, but the long-term extent of immune convergence/divergence will be governed by: a) how much genetic variation any given population possesses for a specific immune trait; and b) which traits are most likely to allow fish to survive and reproduce across different lakes. I hope that there will be a disconnect between how fish respond in the short term (i.e., plastic changes) versus how they evolve in the long-term.”

In addition to the work that Weber and Bolnick are doing, assistant professor Natalie Steinel at the University of Massachusetts Lowell is also investigating the immune system. Steinel is using spleen samples to make histological sections and study the development of immune cells in the fish.

The parasite load, fibrosis scores, and immune cells also can impact other aspects of the fish and will lend more information to the other researchers on the project, including Dr. Kathryn Milligan-Myhre, assistant professor at the University of Connecticut, who is investigating the composition of the microbes in the gut, the gut microbiota. Milligan-Myhre is interested in how host genetic background and environment drive the gut microbiota composition. “I predict that the change in the microbiota is driven by the diet,” said Milligan-Myhre, “and as the populations come together that the diet will drive the microbiota more than the host genetic background.”

Outside of Weber, Bolnick, Steinel, and Milligan-Myhre’s work, numerous other researchers are examining other various questions about the reintroductions including Andrew Hendry, Alison Derry, Milan Malinsky, Kiyoko Gotanda, Alison Bell, Blake Matthews, Katie Peichel, Rowan Barrett, and Matt Walsh. Hendry acts as the project lead, Derry studies zooplankton and copepods, Malinsky – evolution and recombination rate and roles in adaptation, Gotanda and Bell – behavioral adaptation and evolution, Matthews – ecosystem dynamics, Peichel and Barrett – genome evolution, and Walsh evolution of Daphnia in response to stickleback introductions.

Daniel Bolnick displays a reproductive male Threespine Stickleback. Reproductive males usually have a characteristic red chin, blue eyes, and often bluish body pigmentation during the summer breeding season. Photo by Andrew Hendry.

“This is a new experience for many of us to work on such a large team. Logistics, communication, and openness are paramount, so this is a great exercise for many of us,” said Bolnick. “Physicists often form huge consortiums of research groups to do a single particle accelerator experiment that takes years of engineering and planning and analysis to do one experiment. Biologists, in contrast, are more often lone wolf researchers – a single lab group working on a unique problem. Or even competitors, racing each other for a solution. We are taking a more physicist mindset to biology, forming a large collaborative network that will draw lessons and inspiration from this experiment for decades to come. This requires careful coordination, so we don’t overly draw on landowner’s goodwill, and openness with each other about what our plans are (to minimize overuse of fish) and sharing data. Often sharing our data adds additional insights because the different things we study are interconnected.”

Weber also agrees with Bolnick about the collaboration involved in a project of this scale. “A project this size would be impossible without a huge group of collaborators, each providing diverse skills, perspectives, and resources such as supplies and funds. We were very lucky to be able to gather an extraordinarily good group of international labs,” said Weber. “Although the first few years of the project landed during difficult times, including heat waves, fires, and pandemics, the long-term hope is that all our lab groups, including undergraduate and graduate students, postdocs, and other professionals and community members, will be able to convene in Alaska each year to bond and learn about the amazing attributes of the fish and lakes in this region.”

The recipient lakes from this project have also been studied heavily by other researchers outside of the “greatest ecology and evolution experiment,” Patrick Tomco, assistant professor at the University of Alaska Anchorage, and his M.S. student Jordan Couture, looked at the rotenone degradation rates in the lakes as no previous work had examined how quickly the compound degrades in Alaska. It was previously thought that the colder temperatures might slow degradation. “The majority of the rotenone degraded rapidly over the first 14 days. It was completely gone, less than one part per billion, at 60 days,” said Tomco who mentioned that rotenolone, a less toxic transformation product of rotenone, was detected over 250 days.

While Tomco and Couture examined the degradation of the rotenone, Brandon Briggs, assistant professor at the University of Alaska Anchorage, and his M.S. student Jake Bozzini took samples of the lakes to determine how rotenone treatments impacted the microbial communities in the water. Bozzini found that the rotenone did not significantly alter the microbial community structure or function in any of the lakes. However, certain microbial genes were detected that may aid in the degradation of rotenone.

The 2019 stickleback sampling team. Back row: Natalie Steinel, Rachael Kramp, Elsa Diffo, Christopher Peterson, Trey Sasser, Jesse Weber, Kelly Ireland, and Kathryn Milligan-Myhre. Front row: Daniel Bolnick, Ana, and Roscoe. Photo by Andrew Myhre.

Overall, the research done on the rotenone treated lakes has been a large collaborative effort spanning across research labs, agencies, universities, and even countries. All of this research would also not be possible without the generous landowners that have provided access to the lakes via their properties, as well as their useful firsthand observations of what’s happening at the lakes day to day. It is sure that many exciting research papers will emerge from the project and many of the researchers are also hopeful that this will bolster support for effective and safe removal of Northern Pike using rotenone.

Kelly Ireland, author, has been a part of the stickleback reintroduction project since 2019. Ireland is a University of Alaska Fairbanks Ph.D. student in Brandon Briggs lab at the University of Alaska Anchorage and has been actively involved in field gear preparation, coordination, and sample processing for this project.

This story was written for the Alaska Chapter of the American Fisheries Society’s Fall 2021 newsletter Oncorhynchus 41(4). A copy of how the story appeared is below:

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