The crayfish Procambarus clarkii CRY shows daily and circadian variation

The circadian rhythms of crayfish are entrained by blue light, through putative extra retinal photoreceptors. We investigated the presence and daily variation of CRY, a protein photosensitive to blue light spectra and ubiquitous in animals and plants, in the putative pacemakers of Procambarus clarkii, namely the eyestalk and brain, at different times of the 24 h light:dark cycles. Using different experimental light protocols and by means of qualitative/quantitative immunofluorescence anatomical and biochemical methods, we identified CRY immunoreactivity in cells located in the medulla-terminalis-hemiellipsoidal complex (MT-HB) and the anterior margin of the median protocerebrum (PR). The immunoreaction varied with the time of day and the two neural structures showed a semi-mirror image. The results of the biochemical analysis matched these variations. Western blotting demonstrated statistically significant circadian rhythms in brain CRY abundance, but no daily circadian CRY abundance oscillations in the eyestalk. These immunocytochemical and biochemical results link a specific photoreceptor molecule to circadian rhythmicity. We propose that CRY may be linked to the photoreception of the clock and to the generation of circadian rhythmicity.