Community Research and Development Information Service - CORDIS


ALH Report Summary

Project ID: 639192
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - ALH (Alternative life histories: linking genes to phenotypes to demography)

Reporting period: 2015-05-01 to 2016-10-31

Summary of the context and overall objectives of the project

"Understanding how and why individuals develop strikingly different phenotypes, or follow alternative life history pathways, is a major goal in evolutionary biology. It is also a prerequisite for conserving important biodiversity within species and predicting the impacts of environmental change on wildlife populations. The aim of this ERC project is to examine alternative migratory and life history tactics in fish using brown trout (Salmo trutta) as a model system. This species exhibits ""facultative anadromy"", a phenomenon where some trout (known as anadromous individuals) migrate to sea for part of their lives, while others from the same population remain resident in freshwater and never go to sea. The two forms are known to interbreed on returning to shared freshwater spawning areas and anadromous parents are capable of producing non-anadromous offspring, and vice versa. Many important questions remain, however, regarding the relative importance of genetic versus environmental factors in shaping migratory phenotypes, the genetic architecture of facultative anadromy and associated traits such as age and size at first reproduction, and the speed with which tactic frequency can evolve or respond to ecological changes via phenotypic plasticity.

Our team are examining these and other questions using a combination of large-scale laboratory and field experiments, integrating several previously independent perspectives from evolutionary ecology, ecophysiology and genomics. Recent advances in molecular parentage assignment, quantitative genetics and genomics (next generation sequencing and bioinformatics) will allow novel insights into how complex phenotypes are moulded by the interaction between genes and environment. To provide additional mechanistic understanding of these processes, the balance between metabolic requirements during growth and available extrinsic resources will be investigated as the major physiological driver of migratory behaviour. Together these results will be used to develop a predictive model to explore the consequences of rapid environmental change for trout populations, accounting for both ecological and evolutionary processes.

The results of this project will generate general insights into facultative migration, a phenomenon not limited to trout (e.g. many fish, bird and insect species exhibit co-existing migratory and non-migratory forms). Migration is a crucial aspect of biological responses to climate change and better understanding of how (and the speed with which) populations can shift their migratory tactics will inform conservation and adaptive management strategies. Salmonid fishes are iconic and economically important species in European freshwaters and coastal seas, and this project strives to increase knowledge of their basic biology. Our goal is to create a predictive model informed by empirical data that can eventually be used to explore how facultatively anadromous salmonid populations might respond to human-induced changes in their environments.

More generally, humans may value certain phenotypes over others (e.g. so-called trophy specimens, such as the biggest fish in a population, or animals with the largest horns or antlers), but often our actions lead unintentionally to the demise of the very thing we value more. For example, fishing activities may preferentially target the larger sea-going trout in a population, effectively selecting against any genes involved in anadromy or large body size. Harvested fish populations may therefore evolve changes in migration behaviours, or in age and size at maturation, with potential consequences for their resilience in the face of environmental change, and/or knock-on impacts on other species and the wider ecosystem. By improving our understanding of eco-evolutionary processes in wild or semi-wild populations, the insights from this project may help to guard against such unintended consequences."

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

This project (code name ALH, for “alternative life histories”) has four interlinked goals:
1. To analyse how genes and environment interactively determine ALH tactics, using two large-scale experiments (one laboratory-based, the other field-based);
2. To relate ALH tactics to variation in how fish acquire and utilise energy;
3. To advance understanding of the genomic basis of ALH tactics in variable environments;
4. To explore how environmental change might lead to rapid life history shifts in facultatively migratory populations using an eco-genetic simulation model.

Here follows a brief summary of progress to-date on each goal:

Goal 1:
Two large-scale experiments were planned as part of Goal 1 (note that the data and samples generated by these experiments also feed into Goals 2-4). The first of these experiments is a lab-based study, where trout sourced from different populations in the West of Ireland are being reared in tanks in an indoor recirculating aquaculture system (RAS). Our primary objective in the first 18 months of this project has been to set up this RAS and to get this crucial experiment up-and-running. This required a considerable amount of work, as while there was some existing equipment already in place at the Host Institute, much of it was old and needed replacing, and we also decided to change the original experimental design slightly in order to increase the chances of success. Originally, the plan was to have two separate recirculation systems running simultaneously, each on a different temperature regime (because a major aspect of this experiment is to explore the effects of temperature on trout life history decisions and associated metabolic mechanisms). That original design had one major limitation, however, in that there would have been no replication – i.e. there would be one set of inter-linked tanks in the “normal temperature” treatment, and another set of inter-linked tanks in the “high temperature” treatment. While we would have strived to keep all other factors constant across these treatments, we could not have guaranteed this.

Thus we decided to instead build a single, large RAS that contains 18 tanks all connected to the same filtration systems. Once the water that drains from these tanks has passed through mechanical and biological filters, it then flows into two separate sumps. The temperature in each sump is controlled by separate conditioning units, and thus one can be set to “normal temperature” and the other to “high temperature”). The water is then pumped from both sumps around to the tanks, but each tank then has a “tap” that can be either set to “warm” (i.e. water from warm sump flows into tank) or “cold” (i.e. water from cold sump flows in). This gives us full tank-level control over water temperature and allows for replication across tanks of a given water temperature treatment. Moreover, the water from all tanks remixes as it drains out and goes through the same filtration processes, plus the light-levels and oxygen levels for each tank are carefully controlled and standardised. Hence the only thing that varies between tanks is water temperature and/or food supply (each tank has its own automatic feeder, and food is the second variable of interest that we are manipulating in this experiment), which is a more robust experimental design than the original plan.

The construction of this system required both the re-use of old components from the pre-existing systems, and the purchasing of new components, new tanks and automatic feeders and lights. This system is now fully up-and-running and the experiment is underway.

A PhD student (Ms Louise Archer) was taken on to work specifically on this lab-based fish rearing experiment, and a Research Assistant (Mr Stephen Hutton) was also hired to oversee the construction of the system and the day-to-day running of the equipment, fish husbandry and health, and data recording. After careful discussion with the PhD student, it was decided to modify slightly the original experimental design to further expand the scope of the experiment. The original plan was to explore the interacting effects of temperature (two levels: “normal” and “high”) and food quantity (two levels: “low” and “high”) on trout life histories, and this is still being done for a subset of the tanks in our RAS. In a companion experiment that is running in parallel in the same system, we are also exploring the effects of food timing by having four different treatments: (1) low food quantity delivered in the first year of life followed by low food in the second year (Low-Low); (2) low food in the first year followed by high food in the second (Low-High); (3) High-Low; and (4) High-High. This will allow us to better isolate when the “decision windows” for anadromy and/or early maturation occur in the juvenile phase of the trout life-history, i.e. whether their migratory and life-history phenotypes are more sensitive to food abundance in the first or second year of freshwater life.

For both of the above parallel experiments, each treatment level is replicated in two tanks. The fish also come from two different genetic backgrounds: one population (“Bunavela”) is derived from a lake-resident stock of brown trout from the Burrishoole system in the West of Ireland, which do not migrate naturally to sea. The other population (“Erriff”) is derived from a river system in the West of Ireland where the trout naturally exhibit facultative anadromy – i.e. a mix of migratory sea trout and freshwater-resident trout. Wild broodstock (i.e. reproductively active adults) for this experiment were collected from each wild river system in the winter of 2015 and experimental families were produced. Originally, the plan was to produce both pure and hybrid families, but in the end we could not produce hybrid families as our broodstock from the two populations were ready to spawn at different times, and hence creating hybrids was impossible. Thus we have “pure Bunavela” families and “pure Erriff” families, with paternal half-sib families within each group. The eggs from these families were kept separately in an incubation facility and then introduced to the tanks in our experimental RAS after hatching (after a transitionary period in temporary holding tanks, while the single large RAS was being constructed). After some initial losses due to disease issues (a common problem in such experiments) that were dealt with according to standard operating procedures, the trout are now growing rapidly and are in good health.

This experiment will continue for at least another year, more likely two, depending on how rapidly the fish adopt their final life history tactics (i.e. either assume a migratory, non-maturing phenotype, or a freshwater-maturing phenotype). The early indications are that a large fraction of the experimental fish may either smoltify (assume the migratory phenotype) or mature by the summer of 2017. The experiment will continue to yield important data over the course of the coming months and years, and will form a major part of the PhD thesis of Louise Archer. The ultimate aims from this experiment are to explore how environmental factors (food quantity, food timing and temperature) and genetic background (Bunavela versus Erriff populations) affect trout life histories, both independently and in interaction with each other. Because fish from each population (which can be distinguished by unique physical tags and DNA fingerprinting) are mixed in our tanks and exposed to different environmental treatments, this allows for both genetic and environmental effects and “G x E” effects to be characterised.

The second major experiment in this project involves a reciprocal transplant experiment in the wild, where the goal is also to explore the interaction between genes and environment in determining alternative life histories and migratory tactics, but in a fully natural environment instead of a controlled captive-rearing environment as per Experiment 1 above. The broodstock for this second experiment are being collected in Nov/Dec of 2016, and the planned crosses will be made just before Christmas. We have already started collecting these broodstock from our two study populations in the West of Ireland, and the goal is still to produce both pure families and hybrid families (by targeting groups of adult fish from each population due to spawn around the same date). All necessary facilities for housing the broodstock, incubating the eggs and conducting the subsequent fieldwork (as per the original project proposal) are already in place and we are confident that the experiment will be pulled-off successfully. We also have some back-up plans in place, should there be any unforeseen deviations from this plan.

For both of the above experiments, all necessary ethical permission and licences are in place and meticulous records of fish health and procedures carried out are being maintained.

The second PhD student for this ERC project has already been offered the position, and he (Mr Robert Wynne) will start the position in January 2017. This PhD student will focus on the genetic and genomic bases of facultative migration and alternative life histories.

Goal 2:
In order to relate ALH tactics to variation in how fish acquire and utilise energy, we are measuring a range of traits of interest on the fish in our laboratory experiment that is already underway. The mass and length of representative samples of fish from each tank are being measured at regular intervals, which will allow for growth trajectories to be calculated for each population in each experimental treatment. A sub-sample of fish in each tank have also been uniquely tagged using visible implant elastomer (VIE) tags, which allows us to characterise individual-level growth trajectories. We are also measuring at regular intervals the metabolic phenotypes of samples of fish from each tank, using an intermittent flow respirometry system. In the original proposal, the plan was to measure metabolic rates of fish using indirect techniques (e.g. using otolith measurements, and also using a mercury mass balance method to estimate individual consumption rates, and coupling this with growth measures to indirectly estimate metabolic rate), but in the end we decided (and were advised by colleagues) that it would be much better to directly measure oxygen uptake rates of individual fish, as a more accurate proxy for their metabolic rates. We therefore purchased respirometry chambers and associated components and software from Loligo® Systems (Viborg, Denmark) and these are now set up and collecting on-going data at regular time intervals. While this was an unforeseen expense at the time the proposal was written, the original plan was to use a chunk of the Consumables budget to undertake the mercury measurements necessary for indirectly estimating metabolic rates – so this money has effectively now been reallocated to measure the same thing in a more accurate and efficient way. By coupling metabolic measurements with growth measurements, this will allow us to characterise the energy budgets of individuals or groups of fish and relate them to their life history trajectories in the experiments.

These respirometry measurements will also be made on samples of wild-caught trout as part of Experiment 2, once our experimental families have hatched in the wild stream environments and are old enough to sample (likely beginning summer of 2017). We will also be undertaking measurements of fat content of our fish in both the lab-based experiment and the field experiment, which will further allow us to relate ALH tactics to patterns of energy uptake, storage and usage.

Goal 3:
A multi-pronged approach is being followed to interrogate the genomic basis of alternative life histories in variable environments. Firstly, the lab-based experiment will yield crucial phenotypic data on life histories and associated energetic traits in different food and temperature environments. Once this phenotypic information is available to us, the goal is then to tissue sample the same individual fish to obtain high-quality DNA, and then to genotype the individuals using a bespoke panel of single nucleotide polymorphism (SNP) markers distributed across the trout genome. A genome-wide association study (GWAS) will then be carried out to test for statistical associations between genotype and phenotype, which will hopefully permit us to hone in on specific chromosomal regions linked with anadromy and alternative life histories (or with phenotypic plasticity in these traits, given that we can measure fish from different genetic backgrounds in different experimental environments).

A similar GWAS approach will be adopted in the reciprocal transplant experiment to take place in the wild starting in 2017. There, we will be able to measure the phenotypes of fish from different genetic backgrounds (including hybrids between our Bunavela and Erriff populations) in two different wild river environments, affording a unique opportunity to relate genotype to phenotype in a fully-natural ecological setting. It will be intriguing to see whether the same chromosomal regions identified in the GWAS on the lab-experiment fish show up as being important in the wild-experiment fish.

In order to develop our bespoke panel of informative SNPs, we are undertaking an initial study using wild-caught adult fish from an important sea-trout producing river in the West of Ireland. We have already secured samples from a number of adult sea trout (fish that chose anadromy) and freshwater brown trout (fish that chose freshwater residency) and we are in the process of isolating and amplifying high-quality DNA from these fish. The samples will then be genotyped on a high-density SNP array (>100K SNPs) developed for Atlantic salmon – a sister species of brown trout – which should yield several tens of thousands of trout SNPs. We will then undertake a GWAS to identify a subset of the most-informative SNPs, i.e. the ones that correlate significantly with our alternative life histories (anadromy versus non-anadromy) of interest.

This should provide us with a valuable tool going forward, which can be used to study for example levels of adaptive divergence between wild populations of brown trout (e.g. across geographic or ecological gradients), and to identify chromosomal regions associated with migration/anadromy in our experimental trout populations. The ultimate goal is to eventually annotate species genes that are involved, but this will be very much dependent on how successful we are in these preliminary association-study stages.

In addition to these GWAS approaches, our goal is to also undertake some gene expression/transcriptomic work. We are already collecting the necessary tissue samples (from a range of potentially relevant fish organs such as the brain, liver, kidneys, etc.) from adult trout of different life history phenotypes, and we hope to do the same for juvenile trout in our experiments at strategic time-points.

The second PhD student (Mr Robert Wynne) will undertake much of the work in Goal 3, together with a bio-informatics post-doc that we plan to hire on in 2017 or 2018, who will also provide important training to the PhD students.

Goal 4:
The final goal of this project is to integrate all the data and results from the previous goals and couple these with existing data from the literature and also long-term demographic studies of trout in our West of Ireland populations of interest, in order to develop a predictive model. This model will capture key evolutionary and ecological processes and be used to explore how different scenarios of environmental change might impact trout life histories and population dynamics, for example related to
climate change or changes in marine survival rates associated with fish farming activities or fishing pressures. This aspect of the project will necessarily take place towards the end of the project (years 4 and 5) and we will hire on a post-doc with the necessary quantitative skills to undertake much of the work, with inputs from the PI and the rest of the team. Our hope is that this modelling framework will provide a useful tool for fisheries managers and conservation biologists to assess potential threats and the scope for adaptive responses of trout and other species in the face of a range of environmental threats/changes.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The main progress made so far on this ERC project has been:

* To set up a large-scale, challenging laboratory (aquaculture) experiment. Here we are exploring how food quantity and temperature interactively affect life history decisions, which has never before been tested in the context of facultative anadromy in salmonids. Moreover, we have fish from different genetic backgrounds in this experiment, allowing us to tease apart the relative roles of genes versus environment (i.e. G x E interactions). G x E is a fundamental topic in modern evolutionary biology that remains poorly understood, particularly in non-model organisms, and hence we aspire to break new ground here.

* To plan and get the ball rolling on a parallel, field-based experiment that involves reciprocally transplanting trout from different genetic backgrounds (including population-hybrids) between two different, wild river systems. Reciprocal transplant experiments are the gold standard for deciphering the extent to which phenotypic differences among populations (in this case related to migratory, life history and associated energetic traits) have a genetic basis versus being shaped by phenotypic plasticity. Again, G x E interactions can also be explored in this experiment, but this time in fully wild environments, which is very rare in studies of non-model vertebrate organisms. Additionally, this reciprocal transplant experiment doubles as a local adaptation experiment – and again, such experiments are few-and-far-between in fishes, or vertebrates in general, owing to the logistical difficulties involved. We expect the results to therefore generate one or more high-impact publications.

* To initiate a genome-wide association study, where the aim is to identify chromosomal regions associated with anadromy and migration. While recent work on steelhead/rainbow trout in North America has made substantial advances in this area, studies on brown trout in Europe have lagged far behind. The “holy grail” of this aspect of the project is identity actual genes involved in alternative life histories, which would represent a major scientific advance of both fundamental and applied relevance (e.g. for the aquaculture, fish farming and fishing industries).

* By drawing attention to fundamentally important biological questions in an iconic species of major economic importance in Europe, this ALH project aspires to enhance the wider societal impacts of evolutionary ecology research and ultimately to generate results that will be of practical utility to fisheries managers and conservation biologists.

* An important element of the ALH project is also the training of the next generation of scientists, through taking on two PhD students and two post-doctoral researchers who all aspire to publish high-impact papers as part of the project – a crucial aspect of getting ahead in an increasingly competitive European research environment.

* Together with Irish and Scottish colleagues, the PI on this project has produced a thorough review that is very relevant to the subject matter of the ALH project, which will be published as a chapter in a new book on sea trout biology and management, which will be published in 2017:
Andrew Ferguson, Thomas E. Reed, Philip McGinnity and Paulo Prodöhl (In Press). Anadromy in brown trout (Salmo trutta): A review of the relative roles of genes and environmental factors and the implications for management and conservation. In: Sea Trout: Management and Science (Editors: Graeme Harris), Troubador Publishing Ltd.
This review presents the current state of knowledge in this particular topic, which lies at the heart of the ALH project, and will be an extremely useful resource for academics, agency scientists and fisheries managers.
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