A characterisation of biological rhythms in behaviour and holobiont-wide gene expression in the foraminifer Heterostegina depressa from laboratory culture

Algal symbiosis facilitates the success of Large Benthic Foraminifera (LBF) as major carbonate producers in the ocean. Throughout the evolution of these associations, microalgae and their foraminiferal hosts are exposed to periodic changes in external conditions (e.g. tidal and daily cycles), driven by astronomical cycles such as Earth’s rotation and orbital motions. Many other unicellular and multicellular organisms have evolved biological rhythms, with period lengths similar to those of environmental cycles, as adaptations to these periodic changes. These rhythms can either be exogenously driven as responses to environmental cycles or can emerge from endogenous molecular pacemakers. It has been shown that organisms with internal periods closely aligned with the environmental cycles gain significant advantages. However, climatic changes can lead to disruptions and desynchronization of biological rhythms with adverse effects on the fitness of organisms and ecosystem functions, making the characterisation of biological rhythms an important subject in LBF ecology. While biological rhythms in microalgal model systems, such as diatoms (e.g., Phaeodactylum tricornutum) and dinoflagellates (e.g., Symbiodiniaceae), have gained increasing attention, little is known about the persistence of rhythmic processes in associations with LBF. These foraminifers exhibit reticulopodial locomotion and photoprotective behaviour in response to diurnal changes in irradiance, which are widely regarded to be governed by their microalgal symbionts.

In this experimental study, we characterise the behavioural and holobiont-wide molecular rhythms of the diatom-bearing calcareous LBF Heterostegina depressa. Cultured cells were maintained under light-dark conditions (14:10, LD), at a constant temperature of 25°C. For the behavioural characterisation, locomotor activity was quantified using time-lapse imaging. Behavioural recordings with lengths ranging from 3 to 7 days were conducted to assess rhythmicity and determine dominant period lengths. Transcriptomic dynamics were assessed through bulk RNA sequencing, de novo transcriptome assembly, and subsequent differential gene expression analysis. Cells for the differential gene expression analysis were sampled every 4 hours over a 48-hour period. Rhythm analysis of the activity patterns derived from behavioural recordings revealed substantial inter-individual variability, with some individuals exhibiting recurring spikes in activity with a period length of 24 hours. Additionally, we identified a set of significantly rhythmic transcripts, cycling with a period length of 24 hours.

Our findings suggest that timepoints of observations in studies of LBF ecology need to account for temporal changes across a 24-hour period, even under constant temperature conditions. Beyond these findings, we present insights from locomotion behaviour and gene expression under constant dim light (LL) conditions, highlight enriched pathways, and discuss potential endogenously driven rhythms in transcript expression.