Abstract. Circadian rhythms are prevalent on Earth and temporally organize behaviour and physiology of organisms to occur in species-specific ‘temporal niches’. However, species differ in how strictly individuals are controlled by their circadian clock, suggesting that it may offer a selective advantage for an individual to extend its temporal niche under certain circumstances, for example during stressful environmental conditions. A potential mechanism controlling temporal niche adherence involves the evolutionary capacitor and chaperon protein HSP90, known to assist the proper folding of important signalling molecules. If HSP90 becomes rate limiting (e.g., under environmental stress) hidden genetic variation will be expressed, producing novel and potentially beneficial phenotypes for the individual. While this role of HSP90 is well established for morphological traits, we show here that it extends to regulation of temporal behavioural patterns. We show that within a small subset of clock neurons in the fly brain, HSP83, the fly homologue of HSP90, mitigates inter-individual behavioural variability. We provide evidence for the requirement of HSP83 for efficient transcription of the gene encoding the circadian neuropeptide Pigment Dispersing Factor (PDF), and for correct PDF accumulation in central clock neurons. Strikingly, Hsp83 mutants affect synchronized oscillations of the clock protein PERIOD (PER) in subsets of circadian clock neurons in the same way as flies without PDF, further supporting a role of Hsp83 in regulating Pdf. Our findings therefore provide a mechanistic explanation for HSP83 function in regulation of behavioural variability, and offer an explanation for how to restrict temporal niche extension to stressful environmental conditions.

The repository contained the files and data required to understand the findings described in this manuscript. Each .zip files is named with the figure and/or table that they were used for. Notes: There are 4 extra runs to the Fig4 and Tab2. Those runs were not used for the HMM, actograms, histograms and sinchronisation index, but they were included in the phase analysis (Fig.4) and the quantification or % of rhythmic flies and period length in DD (Tab 2). .zip file description: Fig1_Fig3_Tab1_HMM: data formatted for HMM analysis. Data divided by experiments. Files names indicate respectively "run_DAMmonitor_experiment". Dead animals are excluded. Description_days file: details about light ON and light OFF. Reports files shows the HMM analysis workflow. Fig1_Tab1_data_Hsp83mut: DAM monitors and metadata.csv file to create actograms, histograms and to analyse sincronyzation index (SI). Fig2_Fig4_FigS2_phase: phase data obtained from flytoolbox (Matlab). Data divided by experiments. File names indicate respectively "Figure_experiment". Fig3_Tab1_data_CRISPR_KO: DAM monitors and metadata.csv file to create actograms, histograms and to analyse synchronisation index (SI). Fig6_luciferase: luciferase data divided by genotype. Tab2_rhythmicity_DD and TabS2_rhythmicity_LD: csv files from flytoolbox (Matlab) used to analyse % rhythmicity and period. Data divided by genotype. Files names indicate respectively "period3d_genotype_all". Dead animals are excluded. Scripts: Rstudio scripts used to plot and summarise the phase data and for the synchronisation index (SI). Actograms and histograms were plotted following the Rethomics Rstudio package.