Temperature-responsive spatial-temporal regulation underlies phenotypic plasticity of body pigmentation in Eastern honey bee

Abstract

Temperature-induced variation in body coloration is a widespread form of phenotypic plasticity among insects, often mediated by the differential expression of pigmentation-related genes. Despite extensive documentation of this phenomenon, the molecular mechanisms by which environmental cues modulate pigmentation pathways remain largely unresolved. In this study, we investigated seasonal darkness plasticity in Apis cerana, the Eastern honey bee exhibiting a characteristic “yellow-black” abdominal pattern that responds dynamically to ambient temperature. Individuals reared under lower temperature conditions developed a markedly darker phenotype compared to those maintained at higher temperatures, highlighting a temperature-dependent shift in cuticular pigmentation. Exposure to low temperature also resulted in thicker adult cuticles and a concomitant reduction in water loss rate. The co-occurrence of darkened pigmentation and cuticle thickening corresponds with the colder and drier conditions characteristic of winter in the natural habitat of A. cerana. Furthermore, the Hedgehog (Hh) pathway was found to be enriched during early development stages. Notably, its target gene optomotor-blind (omb), a key temperature-sensitive regulator, was upregulated under decreased temperature, establishing the spatial arrangement of the black stripe at the posterior end of each abdominal tergite. Moreover, transcriptional suppression of omb induced upregulation of the pigmentation gene ebony, whose intrinsic thermally responsive expression directly enhances phenotypic plasticity in epidermal pigmentation, serving as a regulatory amplifier for environment-dependent melanin patterning. At the Pb (black eyes with an unpigmented body) in the mid-pupal period, temperature significantly influences 20-hydroxyecdysone (20E) titers. Integrated analyses combining in vivo 20E injection and LC-MS-based hormone quantification revealed that elevated developmental temperature upregulates 20E titers during the temperature-sensitive window, thereby suppressing the expression of pigmentation-related genes. These findings reveal a two-phase mechanism in which spatial patterning and hormonal signaling are temporally decoupled: omb acts early to establish the pigmentation boundary, while 20E acts later to adjust pigment intensity in a temperature-dependent manner. This layered control system allows A. cerana to fine-tune both the position and extent of abdominal pigmentation, providing a flexible strategy for thermal adaptation.