Expression of clock genes tracks daily and tidal time in brains of intertidal crustaceans Eurydice pulchra and Parhyale hawaiensis

Intertidal organisms, such as the crustaceans Eurydice pulchra and Parhyale hawaiensis , express daily and tidal rhythms of physiology and behaviour to adapt to their temporally complex environments. Although the molecular-genetic basis of the circadian clocks driving daily rhythms in terrestrial animals is well understood, the nature and mechanism of the circatidal clocks driving tidal rhythms remain a mystery. Using in situ hybridisation, we identified discrete clusters of ∼60 putative “clock” cells co-expressing canonical circadian clock genes with comparable distributions across the protocerebrum of E. pulchra and P. hawaiensis brains. In tidally rhythmic, field-collected E. pulchra sampled under a light:dark (LD) cycle, the expression of period ( per ) and cryptochrome 2 ( cry2 ) exhibited daily rhythms in particular cell groups (e.g., medioposterior cells) whereas timeless ( tim ) showed 12-hour rhythms in others (e.g., medial cells). In tidally rhythmic laboratory-reared P. hawaiensis , previously entrained to 12.4-hour cycles of agitation under LD and sampled under continuous darkness, several cell groups (e.g., medioposterior cells) exhibited circadian expression of per and cry2 . In contrast, dorsal-lateral cells in the protocerebrum exhibited robust ∼12-hour, i.e., circatidal, rhythms of per and cry2 , phased to the prior tidal agitation but not the prior LD. In P. hawaiensis exhibiting daily behaviour under LD without tidal agitation, robust daily rhythms of per and cry2 expression were evident in medioposterior and other cells whereas expression in dorsal-lateral cells was not rhythmic, underlining their intrinsic tidal periodicity. These results implicate canonical circadian mechanisms in circatidal time-keeping and reveal conserved brain networks as potential neural substrates for the generation of interactive daily and tidal rhythms appropriate to intertidal habitats.