Associative learning reconfigures colour-guided behaviour in Drosophila larvae

Colour-guided behaviour can be innate or learned, yet how a numerically simple nervous system assigns value to spectral cues remains unresolved. We developed an automated differential-conditioning assay in which third-instar Drosophila larvae were trained with blue and green light, one of which was paired with electric shock. Wild-type larvae subsequently avoided the shock-paired colour, whereas naive controls showed no strong blue-green preference. This learned avoidance persisted when the relative intensity cue was transferred to the alternative colour, indicating that behavioural choice followed spectral identity rather than simply tracking the dimmer stimulus. Disruption of either the blue-sensitive Rh5 or green-sensitive Rh6 photoreceptor pathway significantly weakened the training-induced behavioural shift relative to wild type, with Rh5 mutants showing only a small residual shift. Complementary extracellular recordings confirmed light-evoked activity in the larval visual system, with robust responses to ultraviolet and broadband white illumination but smaller and more variable responses to blue and green light. Together, these results show that larvae can discriminate blue from green, demonstrating elementary colour vision; that associative experience reconfigures their colour-guided behaviour; and that both photoreceptor pathways contribute to this learned response. This paradigm separates learned spectral preference from baseline light preference and provides a tractable framework for linking defined sensory inputs to the circuit mechanisms of visual memory.