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Abstract Coloration is a multifaceted trait that serves various functions, including predator defense, thermoregulation, and immune response, among others. We investigated pupal color variation in Chlosyne lacinia pupae, focusing on identifying the cue that influences variation in melanization. Through laboratory experiments, we demonstrated that pupae reared on black backgrounds exhibited significantly higher melanization compared to those on white backgrounds. Additionally, black pupae experienced longer developmental periods, suggesting a trade-off between defense and developmental time. Our findings support crypsis as a likely evolutionary driver for increased melanization in response to substrate color. We discuss potential implications for predator avoidance, immune response, and developmental costs associated with melanization. This study provides insights into the adaptive significance of pupal melanization in response to environmental cues, shedding light on the complex interplay between life history traits in butterflies.