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. candidate 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:

Sonar fish counts on the Chignik River

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Myra Scholze steering ADFG’s skiff in Chignik, Alaska. Photo courtesy of Myra Scholze.

Field work is often seen as the glamorous part of science, where researchers get to experience the outdoors and be close to the subjects that they study. The sad reality is though that most scientists spend their time analyzing and processing data on computer screens at office desks. For Myra Scholze, a Fish and Wildlife Technician, for the Alaska Department of Fish and Game (ADFG), this is no unfamiliar territory.

Scholze began working for the ADFG seven years ago in the sport fisheries division in Kodiak. Two years ago, she began doing research for ADFG near Chignik, Alaska on the Alaskan Peninsula. The community of Chignik is primarily a fishing village that relies on the commercial and subsistence fisheries there.

Scholze’s work with ADFG helps manage those fisheries to maintain their sustainability. Her work is to count the salmon that swim up river between May and September.

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Myra Scholze on the Chignik River. Photo courtesy of Myra Scholze.

“Counting the fish is what tells fish and game when to open and close commercial fisheries,” Scholze said. “For each day of the month, in June and July, there’s escapement goals you’re supposed to meet that indicate that you’re going to meet your total number of fish that’s needed to maintain a sustainable run. We count the fish up and meet those goals then the manager at Chignik decides when and what areas to open and for how long.”

Specifically, Scholze is funded through a grant that is comparing and trying to find the correlation between fish counts made on a weir or on a sonar. The two fish counting methods generate a massive amount of data that must be processed.

Weir measurements are made by forcing salmon through a bottleneck in the river, the weir itself, and recording video of the salmon as they pass by. Researchers then go back and count how many individual salmon pass the camera lens.

Sonar doesn’t record video in a traditional sense, but rather records how sound moves through water. Sonar data is collected on both banks of the river and then a researcher must sit and watch back each of the videos and count how many fish blips they see on screen.

“We have two sonars and every ten minutes they create a file that looks kind of like a fish finder on a boat. That’s what you’re counting,” said Scholze. “Every bank creates 144 files per day, we have a sonar on each bank of the river, so we are creating 288 files a day. Over a month you’re creating about 10,000 files and that’s why we have such a back log and why I count files.”

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Myra Scholze adjusts the sonar in Chignik. Photo courtesy of Myra Scholze.

The massive amount of data and the nearly real-time nature (the videos can be sped up slightly when only a few fish are moving by) of watching back the files and counting fish makes for long work hours. Scholze has spent months outside of Chignik in the Kodiak ADFG office, in addition to long evenings at the bunkhouse in Chignik, just counting back fish on videos, so finding a correlation between weir and sonar counts may take years to come. The preliminary conclusions about correlation can’t even be made yet.

“They’ve looked at it [the correlation], but we don’t have enough done from 2016 yet.” Scholze said.

The work may be grueling to some, but to Scholze she loves being able to collect the data that helps inform management decisions for Alaskan fisheries. She intends to continue working for ADFG in Chignik for as long as they have files for her to count. She’s currently in Dutch Harbor, Alaska working for ADFG as a Fish and Wildlife Technician for the crab fishery there. Myra will return to Kodiak in the spring to restart her sonar counts before heading back to the field in Chignik as a Fishery Biologist.

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Myra Scholze collecting samples in Alitak, Alaska for a job she held with ADFG before working for ADFG in Chignik, Alaska. Photo courtesy of Myra Scholze.


Stickleback – The super fish

Darting through Cheney Lake in Anchorage, Alaska are thousands of small fish, about three inches in length, with three spiny projections that jut off the top of their bodies, pricking anything that dares touch them. The color of their scales varying in color depending on the season, sex, or population from which they descend. They gleam shiny silver, blue, or a dull brown, sometimes with a greenish hue. They’re named threespine stickleback, and they’ve become a powerhouse organism for study.  Found in nearly all Alaskan lakes and across most of the northern hemisphere, scientists have taken keen interest in these fish for the practical uses they hold for studying evolution and conducting research.

Threespine stickleback from Cheney Lake in Anchorage, Alaska during their reproductive stage of life. The fish with blue eyes is a reproductive male.

        At the University of Alaska Anchorage, Kat Milligan-Myhre, heads a laboratory of undergraduates, graduates, lab techs, and post docs who are all using threespine stickleback as a model organism for a variety of projects on host gut microbe interactions. The lab is able to study how the microbes within the gut of threespine stickleback, the host, affect a variety of things like development, physiology, behavior, and more. Milligan-Myhre developed a procedure that allows the lab to fertilize eggs of the fish and then make them free of all microbes. They can then add back in select microbes or none at all to study how the microbes are actually affecting the fish.

A 7-day-old transparent juvenile threespine stickleback. Milligan-Myhre has developed a protocol to rear threespine stickleback free of microbes until 14 days after the eggs they’ve hatched from have been fertilized.

        “Stickleback have a number of really cool qualities. One is that they are transparent so we can actually watch fluorescent microbes move around in the gut of a live stickleback,” said Milligan-Myhre, “We can make large amounts of genetically similar eggs from a single cross or a couple of crosses… with fish you can get 100 to up to 200, if you’re lucky, of genetically related fish. That allows us to have a lot of power so we can do some really good statistical analysis on these changes that we’re seeing when we treat these animals.”

Kelly Ireland and Kat Milligan-Myhre set traps for threespine stickleback in Cheney Lake in Anchorage, Alaska in May of 2017. The lab uses minnow traps that have a funnel and hole on either end of the trap that threespine stickleback then swim into and get trapped.

        They are studying a variety of populations from varying lakes across Alaska, but by far their most frequented lake of interest is Cheney Lake. The lake had threespine stickleback introduced to it in 2009 from a parental population found in Rabbit Slough, Alaska, by Frank Von Hippel, a former professor at UAA, who like Milligan-Myhre used them as a model organism. Von Hippel’s lab was interested primarily in the evolution of the fish, however.

Ryan Lucas, Emily Lescak, and Kelly Ireland of Kat Milligan-Myhre’s lab pull traps from Y Lake of the Talkeetna Lakes chain in Talkeetna, Alaska. The lab then does in field gut dissections to assess gut microbe composition within the threespine stickleback.

        “What really sets stickleback apart from zebrafish, which are the traditional go to fish model, is that we can take stickleback that have evolved in different environments and we can relate the environments in which they evolved to their physiological and genetic variation,” said Emily Lescak, former doctoral student of Von Hippel’s, currently working as a post-doctoral fellow in Milligan-Myhre’s lab, “Basically we can understand what selection pressures in the environment cause a fish to evolve in certain ways, so we can understand what sort of ecological pressures there are on fish populations.”

Threespine stickleback fish from Rabbit Slough, near Wasilla, Alaska. The Rabbit Slough population is anadromous meaning they’re born in freshwater, then travel to oceanic environments for most of their life, and then return to freshwater to mate.


       Incidentally there’s already evidence that the threespine stickleback Von Hippel introduced into Cheney Lake are already undergoing evolution from their anadromous (meaning the fish, like salmon, are born in freshwater, travel to the ocean, and then come back to the freshwater to mate) ancestral form, to freshwater forms. The threespine stickleback in Cheney Lake were introduced in 2009 after the Alaska Department of Fish and Game applied a Rotenone treatment in October, 2008, to the lake. Rotenone was used to eliminate northern pike that were introduced illegally. The Rotenone treatment wiped out all fish populations in the lake and allowed Fish and Game to restock Cheney Lake with rainbow trout, and Von Hippel to introduce threespine stickleback from a known population, Rabbit Slough. Milligan-Myhre’s lab has been collecting data on Cheney Lake and threespine stickleback from the lake monthly to assess the changes of the threespine stickleback population over time.


        “We can follow evolution in real time. That’s exciting,” said Milligan-Myhre.

        The lab is collaborating with a lab at Stony Brook University in New York to look at genetic differences as the population evolves. Milligan-Myhre’s lab hopes to also take a look at how as the population changes over time into their freshwater form the microbiota and threespine stickleback’s immune response to microbes also change.

Rachael Kramp an undergraduate student of Kat Milligan-Myhre’s lab, works with microbes from the guts of threespine stickleback from Cheney Lake in the anaerobic chamber of Milligan-Myhre’s lab at the University of Alaska Anchorage.

        The tools these fish offer are nearly limitless from using them as a model for biomedical research, as they have similar physiology to humans, to studying evolution, these fish also make great models for studying ecotoxicology, as well as, host microbe interactions, just to touch on a few of their benefits. The threespine stickleback came to be a model organism in the 1900s with the work of Nobel Prize laureates, Niko Tinbergen, Konrad Lorenz, and Karl von Frisch, because of the ease to which they could be manipulated in the lab, now in 2017 the threespine stickleback shows no signs of slowing down as being the model organism of many scientist’s dreams. In 2018, hundreds of researchers will even gather together for the 9th International Conference on Stickleback Behavior and Evolution in Kyoto, Japan. These prickly little fish may not seem like much to the majority of people, but to many scientists they are the crux of their entire careers.


Written by Kelly Ireland. Kelly Ireland is an undergraduate student doing research in Kat Milligan-Myhre’s lab.