INVESTIGATING POSSIBLE IMPACTS OF <EM >PERIOD</EM> IN CIRCADIAN VS. CIRCALUNAR CLOCKS OF <EM>PLATYNEREIS DUMERILII</EM><EM>​</EM>

Biological clocks enable anticipation of environmental cycles to coordinate physiological functions and behaviour, giving a fitness advantage. While the circadian clock (~24h) has been extensively studied, the interaction between multiple biological clocks, such as the circadian and circalunar clocks (~29.5 days), is not well understood. In Platynereis dumerilii, circalunar rhythms persist when the circadian clock is inhibited, whereas circadian outputs show lunar-phase-dependent periodicity through a plastic circadian-circalunidian oscillator (PCC) varying between ~22h and ~25h. Conversely, in Clunio marinus the circasemilunar rhythm requires a synchronised circadian clock. To test whether circadian core components are required for the PCC in Platynereis dumerilii we investigated the period (per) gene, a transcriptional repressor of the circadian clock in many species, whose transcript in Platynereis is controlled by lunar phase.
We targeted the per locus via the CRISPR-Cas9 system and successfully generated two independent early frameshifts in the gene. Combining spawning assays and locomotor tracking, we assessed the impact on the circadian and circalunar clocks. Luciferase assays were used to verify the repressive capabilities of PER on a worm enhancer construct in tissue culture.
Homozygous mutants display reductions in overall activity and circadian rhythmicity, as well as delayed sexual maturation and growth, whereas the circalunar spawning pattern remains unchanged. Ongoing immunofluorescence aims to examine PER localization and dynamics underlying circadian regulation and its modulation by lunar phase.
Thus, per is required for robust circadian timing but likely dispensable for circalunar reproductive timing under the tested conditions, while its role in circadian-circalunidian plasticity is being determined.