Daily and circadian expression of cryptochrome during the ontogeny of crayfish

Cryptochromes (CRY) are proteins with a dual role in the circadian function of different animals, participating in phototransduction and light signaling to the clock and as a transcriptional repressor that provides negative feedback in the clock feedback loop. Here we characterize functional expression of CRY as a marker of the functionality of the circadian pacemaker of crayfish (Procambarus clarkii) throughout post-embryonic development. Using different experimental light protocols and by means of immunofluorescence and biochemical methods, we report that, as in the adult, in young crayfish from the first embryonic stage CRY is present in cells adjacent to the eyestalk hemiellipsoidal body and the anterior margin of the brain protocerebrum. In the brain, CRY cycles after 72 h darkness, entraining to LD cycles. Meanwhile, as in the adult eye, in juveniles CRY is driven by light, showing an arrhythmic pattern in DD and cycling under LD. These results, as well as the completely different period length found in the brain circadian oscillations of 2nd post-embryonic stage and juvenile animals, suggest important changes in the properties of the crayfish pacemaker through the development. Therefore these data support a previous idea about the functionality of the circadian system from hatching.