Quantifying Threespine Stickleback Gasterosteus aculeatus Coloration for Population Analysis: Method Development and Validation

Fish coloration plays an important role in reproduction and camouflage, yet capturing color variation in field conditions remains challenging. We present a standardized, semi-automated protocol for measuring body coloration in the popular model fish threespine stickleback (Gasterosteus aculeatus). Individuals are photographed in a controlled light box within minutes of capture, and color is sampled from eight anatomically defined standard sites in human-perception–based CIELAB space. Analyses combine univariate color metrics, multivariate statistics, and the ΔE* perceptual difference index to detect subtle shifts in hue and brightness. Validation on laboratory-reared and wild-caught pre-spawning fish shows the method reliably distinguishes males and females well before full breeding colors develop. Although it currently omits ultraviolet signals and fine-scale patterning, the approach scales efficiently to large sample sizes and varying lighting conditions, making it well suited for population-level surveys of camouflage dynamics, sexual dimorphism, and environmental influences on coloration in sticklebacks and other fish species.