Adenylate Cyclase 5 (Adcy5) Deficiency Impairs Pigment Granule Dispersion in Melanophores and Erythrophores in Nile Tilapia.

Adenylate cyclase 5 (Adcy5) generates cyclic adenosine monophosphate (cAMP) downstream of G protein-coupled receptor signaling, yet its role in vertebrate pigmentation remains incompletely understood. Here, we generated a CRISPR/Cas9-mediated adcy5 knockout line in Nile tilapia (Oreochromis niloticus) to investigate its function in chromatophore biology. Loss of adcy5 resulted in a pronounced disruption of body coloration, characterized by the absence of vertical black bars and a global reduction in pigmentation. Despite this, chromatophore number was largely unaffected, indicating that Adcy5 is not required for pigment cell specification but is essential for functional pigmentation. At the cellular level, pigment granules in melanophores and erythrophores failed to undergo dispersion and instead remained constitutively aggregated, revealing a primary defect in intracellular pigment granule transport. Consistently, adcy5 mutants exhibited reduced expression of key melanogenesis-associated genes, including mitfa, tyrb, tyrp1a, and tyrp1b, accompanied by decreased melanin content across multiple tissues. Pharmacological activation of cAMP signaling partially rescued the pigment dispersion defect, whereas stimulation of upstream α-MSH signaling produced only limited effects, placing Adcy5 upstream of intracellular cAMP production within the pigment regulatory cascade. Importantly, we further demonstrate that Adcy5 is required for erythrophore pigment granule dispersion, extending its functional role beyond melanophore biology. Together, these findings identify Adcy5 as a conserved regulator integrating cAMP-dependent pigment synthesis and granule transport across multiple chromatophore types in teleost fish.