Photoperiodic and clock regulation of the vitamin A pathway in the brain mediates seasonal responsiveness in the monarch butterfly.

Seasonal adaptation to changes in light:dark regimes (i.e., photoperiod) allows organisms living at temperate latitudes to anticipate environmental changes. In nearly all animals studied so far, the circadian system has been implicated in measurement and response to the photoperiod. In insects, genetic evidence further supports the involvement of several clock genes in photoperiodic responses. Yet, the key molecular pathways linking clock genes or the circadian clock to insect photoperiodic responses remain largely unknown. Here, we show that inactivating the clock in the North American monarch butterfly using loss-of-function mutants for the circadian activators CLOCK and BMAL1 and the circadian repressor CRYPTOCHROME 2 abolishes photoperiodic responses in reproductive output. Transcriptomic approaches in the brain of monarchs raised in long and short photoperiods, summer monarchs, and fall migrants revealed a molecular signature of seasonal-specific rhythmic gene expression that included several genes belonging to the vitamin A pathway. We found that the rhythmic expression of these genes was abolished in clock-deficient mutants, suggesting that the vitamin A pathway operates downstream of the circadian clock. Importantly, we showed that a CRISPR/Cas9-mediated loss-of-function mutation in the gene encoding the pathway's rate-limiting enzyme, , abolished photoperiod responsiveness independently of visual function in the compound eye and without affecting circadian rhythms. Together, these results provide genetic evidence that the clock-controlled vitamin A pathway mediates photoperiod responsiveness in an insect. Given previously reported seasonal changes associated with this pathway in the mammalian brain, our findings suggest an evolutionarily conserved function of vitamin A in animal photoperiodism.