The aim of the present work was to investigate the mechanisms of circadian rhythms in the crayfish Procambarus clarkii during ontogeny, by studying a well described but controversial rhythm in adult crayfish: the motor activity rhythm. The temporal development of the clock pacemaker and of the clock entrainment mechanisms, and the spectral features of the circadian photoreceptors involved in this entrainment were studied. To investigate the first problem, the characteristics of the free running motor activity rhythm under controlled dark conditions were investigated, at different developmental stages of Procambarus clarkii after hatching. To determine the evolution of the rhythm entrainment mechanism, two batches of crayfish of similar age were submitted to either one of two different experimental conditions: 1) young animals were individually submitted to complete light-dark cycles (LD) 12:12 for ten or more days followed by a post-entrainment period of constant darkness; or 2) young animals were individually submitted to three trials with different skeleton photoperiod (SP) imitating LD 8:16, LD 12:12 and LD 20:4. Finally, three groups of juvenile instars showing evident circadian rhythms were used to determine the identity as well as the spectral characteristic of photoreceptors conveying light to the clock: 1) intact control animals, 2) animals lacking bilateral retina and lamina ganglionaris and 3) animals electrolytically-injured in different structures of the brain protocerebrum. All animals were monitored individually and qualitatively and quantitatively analysed. Results indicate that the pacemaker system responsible for locomotor activity rhythm may be present from the moment of hatching, but the coupling strength of this system might change along development. All studied ages showed a circadian rhythm able to entrain to the LD cycles by means of parametric and non-parametric mechanisms. Furthermore, results indicate that motor activity entrainment in crayfish is mediated by extra-retinal photoreceptors of different spectral sensitivity. Unpublished results from our laboratory suggest that the brain protocerebrum may be a locus of pacemaker oscillators.
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