Ter, Y. T.; Ernst, D. A.; Farfan-Pira, K. J.; Westerman, E. L.

Reproductive isolation is a central driver of speciation and can be reinforced by both innate mate preferences and mate preference learning. Pigmentation genes are strong candidates for pleiotropic effects on these processes because they shape visual traits used in mate choice and may also influence neural function, yet their role in learning is poorly understood. Here, we test whether the pigmentation gene yellow affects innate mate preference and aversive mate preference learning in female Bicyclus anynana, and whether these effects are associated with changes in brain dopamine levels. Using CRISPR-Cas9, we knocked out the yellow gene, developed a mutant line, and tested the mate preferences and mate preference learning ability of mutant females compared to wild-type (WT) females. We find that loss of yellow does not alter assortative mating based on pigmentation or disrupt innate visual or olfactory preferences. In contrast, loss of yellow does influence learning ability, as mutant females failed to modify mate preference following aversive premating experience, indicating an impairment in aversive learning. Despite yellow's role in the melanin biosynthesis pathway, brain dopamine levels remain unchanged in mutant females relative to WT females. These findings identify yellow as a pleiotropic gene influencing both pigmentation and aversive mate preference learning, providing evidence that pigmentation genes can shape behavioral processes important for reproductive isolation and speciation.