In the world of science, it's not always the largest or most charismatic creatures that make the biggest splash.
In a flurry of post-war productivity, a young Niko Tinbergen re-established his lab in Leiden and founded the journal Behaviour to serve the burgeoning field of ethology. Together with his senior assistant Jan van Iersel, he published a classic paper on "Displacement reactions in the three-spined stickleback" in the first issue of his new journal in 1948 1 . This small, unassuming fish began its modeling career among an enthusiastic core of Tinbergen students in the 1930s, and has since become a powerful model in the fields of behavioural ecology, evolutionary biology, developmental genetics, and ecotoxicology 1 .
Tinbergen's stickleback research exemplified his revolutionary framework for studying animal behavior—his famous "four questions" that demanded biological explanations for behavior at four complementary levels.
This tiny fish, with its distinctive spiny mohawk, has proven to be the perfect subject for addressing all four questions, helping transform ethology into a rigorous modern science 3 .
Tinbergen's 1963 seminal paper "On the aims and methods of ethology" proposed that a complete understanding of any behaviour requires answering four distinct but interconnected types of questions 2 6 . These questions operate at two different levels of explanation:
Take a longer perspective to explain why a behaviour evolved
Look into immediate mechanical reasons for behaviour
This framework structured the study of stickleback behaviour and continues to shape behavioural research today .
Male sticklebacks build elaborate nests and perform distinctive zigzag dances to attract females—behaviors that clearly function to increase reproductive success 1 .
Sticklebacks have repeatedly evolved similar traits when colonizing freshwater habitats from marine environments, allowing scientists to study evolutionary processes in real time 3 .
The red belly of the male stickleback during breeding season acts as a sign stimulus that triggers aggressive responses from other males, demonstrating how specific mechanisms elicit behavior 1 .
Young sticklebacks refine their predatory skills through experience, showing how behavior develops and changes throughout an individual's lifetime 1 .
In 2012, researchers at Stanford University and the Broad Institute announced a landmark achievement: they had analyzed the whole-genome sequence of 21 threespine sticklebacks from various geographic locations around the world 3 . This unprecedented view of the species' genetic diversity revealed astonishing insights into how new traits evolve.
The research team found that sticklebacks from different habitats—despite their different haunts—repeatedly developed the same traits through changes in similar regions of their genomes. They identified 147 regions that varied consistently between freshwater and marine sticklebacks 3 .
"What addresses a classic debate in evolutionary biology," said senior author David Kingsley. "How do new traits evolve in natural populations? Do they arise through mutations in the coding regions of genes, which alter the structure and function of encoded proteins? Or are new traits the result of modifications in the regulatory regions of genes, which control where and when already-established proteins are expressed?" 3
The stickleback genome provided a clear answer: about 80% of the changes involved regulatory regions of the genome rather than protein-coding regions themselves. This preference for regulatory changes makes evolutionary sense—altering where and when a gene is expressed allows refinement of traits without disrupting all the other functions a protein might have 3 .
of genomic changes in stickleback evolution involve regulatory regions
genomic regions vary consistently between marine and freshwater sticklebacks
| Chromosome Region | Key Genes | Adaptive Significance |
|---|---|---|
| Chromosome I | Atp1a1a (ion transport) | Osmoregulation in different salinity environments |
| Chromosome XI | KCNH4a (potassium channel) | Possibly gill function in freshwater |
| Chromosome XXI | Multiple morphological QTLs | Body shape, armor plate development |
One of the most ambitious stickleback experiments began unexpectedly when the Alaska Department of Fish & Game took measures to eradicate invasive northern pike from a lake system in the Kenai Peninsula in 2018 7 . The department treated eight lakes using rotenone, rendering them fishless and creating a rare opportunity for scientists to study how different ecotypes of sticklebacks respond to novel environments in nature 7 .
In 2019, researchers collected threespine sticklebacks from eight different source populations from lakes in the Mat-Su region and the Kenai Peninsula 7 .
The source populations represented two distinct ecotypes:
The researchers created an experimental design where each of the eight empty lakes received a mixture of either benthic or limnetic sticklebacks from different source populations.
In total, approximately 10,000 individual fish were collected, photographed, and fin-clipped before being released into their new habitat, allowing scientists to track genomic and morphological changes over time 7 .
| Experimental Component | Description | Scientific Purpose |
|---|---|---|
| Source Populations | 8 lakes with either limnetic or benthic ecotypes | Provide genetic variation for study |
| Experimental Lakes | 8 rotenone-treated lakes rendered fishless | Create novel environments for colonization |
| Monitoring Methods | Annual sampling, photography, genetic analysis | Track evolutionary changes in real time |
| Research Collaboration | 50+ scientists from 22 research groups | Comprehensive multi-disciplinary approach |
This project has grown into a large international collaboration involving over 50 scientists from 22 different research groups across 17 universities and institutions, making it one of the most comprehensive studies of eco-evolutionary dynamics in wild populations 7 .
Modern stickleback research employs a diverse array of scientific tools and techniques that have evolved dramatically from Tinbergen's early experiments with painted model fish 1 .
| Tool or Technique | Function | Application in Stickleback Research |
|---|---|---|
| Genome Sequencing | Determines complete DNA sequence | Identify genetic differences between ecotypes 3 |
| CRISPR/Cas9 | Precise genome editing | Test function of specific genes and regulatory regions 4 |
| piggyBac Transposon | Random gene insertion | Introduce transgenes into stickleback genome 4 |
| Geometric Morphometrics | Quantifies shape differences | Analyze body shape variation between populations |
| Stable Isotope Analysis | Trophic position determination | Study dietary differences between ecotypes |
| RAD Sequencing | Identifies genetic markers | Population genetics and phylogenetic studies |
The development of genetically-encoded tools to record and manipulate neurons in vivo has revolutionized neuroscience, and these approaches are now being extended to sticklebacks and other non-traditional model species 4 .
Like green fluorescent protein (GFP) and calcium indicators (GCaMP) that allow visualization of neuronal activity
Like channel rhodopsin that enable researchers to excite or inhibit specific neurons with light
Like GAL4-UAS that allow flexible use of reporters and effectors in specific tissues 4
The significance of stickleback research continues to grow as scientists address new questions. A 2025 study in Heredity directly tested the fitness effects of chromosomal inversions that differ between marine and freshwater sticklebacks across different salinity conditions 8 .
The researchers found that for most inversions, there were no strong deleterious effects and that fitness consequences weren't strongly influenced by salinity alone, suggesting more complex selective forces might be involved 8 .
This research exemplifies how sticklebacks continue to help scientists understand fundamental evolutionary processes. From Tinbergen's early behavioral observations to contemporary genomic analyses, the threespine stickleback has proven to be an invaluable model system 1 3 .
As one reviewer of "Tinbergen's Legacy in Behaviour" noted, the collection illustrates "the rapid development of this field from ethology towards up-to-date behavioural ecology and its manifold sub-areas" 1 .
Tinbergen begins studying stickleback behavior with his students
Tinbergen publishes "Displacement reactions in the three-spined stickleback" in the first issue of Behaviour 1
Tinbergen publishes "On the aims and methods of ethology" establishing the four questions framework 2 6
Whole-genome sequencing of 21 sticklebacks reveals evolutionary patterns 3
Alaska lake restocking experiment begins 7
Study on fitness effects of chromosomal inversions published in Heredity 8
The stickleback's extraordinary journey from subject of clever experiments with painted models to powerhouse of evolutionary genetics demonstrates how a well-chosen model organism can transform multiple scientific fields 1 . These tiny fish embody Tinbergen's legacy—their behaviors and adaptations continue to help scientists unravel the complex interplay between mechanism and adaptation, between individual development and evolutionary history.
As one scientist aptly put it, sticklebacks are essentially the "Darwin's finches of the sea"—their adaptation to novel environments is clearly visible and easily readable in the genome 3 . Their story reminds us that in science, as in nature, even the smallest creatures can teach us the biggest lessons.