Biological rhythms in the deep-sea hydrothermal mussel Bathymodiolus azoricus

Abstract

Biological rhythms are a fundamental property of life. The deep ocean covers 66% of our planet surface and is one of the largest biomes. The deep sea has long been considered as an arrhythmic environment because sunlight is totally absent below 1,000 m depth. In the present study, we have sequenced the temporal transcriptomes of a deep-sea species, the ecosystem-structuring vent mussel Bathymodiolus azoricus. We reveal that tidal cycles predominate in the transcriptome and physiology of mussels fixed directly at hydrothermal vents at 1,688 m depth at the Mid-Atlantic Ridge, whereas daily cycles prevail in mussels sampled after laboratory acclimation. We identify B. azoricus canonical circadian clock genes, and show that oscillations observed in deep-sea mussels could be either a direct response to environmental stimulus, or be driven endogenously by one or more biological clocks. This work generates in situ insights into temporal organisation in a deep-sea organism.

Fig. 1. Sampling strategy to allow deep-sea chronobiology studies.

a The work was carried out on Bathymodiolus azoricus mussels from the Mid-Atlantic Ridge (MAR). The in situ study was performed on the Eiffel Tower edifice, at the Lucky Strike vent field (1688 m depth). For the laboratory experiment, mussels were sampled at the Menez Gwen vent field (834 m depth). Sources: Esri, GEBCO,NOAA, National Geographic, DeLorme, HERE, Geonames.org. b A typical work session of the Remotely Operated Vehicle (ROV) Victor6000 in the deep sea, using artificial white light. © JY Collet - Bienvenue Productions - Ifremer. c The sampling strategy was designed to sample and readily stabilise mussel tissues under red light in the deep sea. MOMARSAT 2017 cruise. © Ifremer.

Fig. 2. Bathymodiolus azoricus valve opening was rhythmic on the Mid-Atlantic Ridge.

a The ecological observation module TEMPO records 2-min video sequences four times a day, under white artificial light, on the Eiffel Tower vent edifice, Lucky Strike vent field (1688 m depth). b Proportion of B. azoricus mussels (n = 31) that were open at 0, 6, 12, and 18 h UTC between 27 July and 31 August 2014. c Whittaker–Robinson periodogram analysis of mussels’ valve behaviour. Black dots indicate significant periods.

Fig. 3. Bathymodiolus azoricus gill transcriptome was cyclic at the hydrothermal vent field (1688 m depth).

Normalisation performed with down-sampling and DESeq2. a Heatmap of median-normalised expression patterns of all 2502 rhythmic transcripts detected in the interval 10.4–14.4 h. b Euler diagrams detailing the number of rhythmic transcripts detected in the interval 10.4–14.4 h and specifically with a period of 12.4 h using both RAIN (dark and light brown disks) and ABSR (dark and light blue disks) methods. c Seawater temperature (brown continuous line) and pressure (blue dotted line) profiles recorded on the Lucky Strike vent field at the time of sampling. d Heatmap of median-normalised expression patterns of all 869 rhythmic transcripts detected in the interval 20–24.8 h. e Euler diagrams showing the number of rhythmic transcripts detected in the interval 20–24.8 h and specifically with a period of 24.8 h using both RAIN (dark and light green disks) and ABSR (dark and light orange disks) methods. f GO terms associated to all the rhythmic transcripts detected in the range 10.4–14.4 h and g, in the range 20–24.8 h. All heatmaps are single-plotted and represent five individuals per time point. Rhythmic transcripts are ordered by phase. Heatmap colours: median-normalised expression levels greater than 1.5-fold are shown as gold yellow; expression levels less than 0.5-fold are shown as light blue. Heatmap heights and disk areas are proportional to the number of transcripts.

Fig. 4. Simplified Maximum-likelihood phylogenies of the canonical circadian clock genes.

a CLOCK and BMAL/CYCLE proteins. b PERIOD proteins. c TIMELESS and TIMEOUT proteins. d CRYPTOCHROME and PHOTOLYASE proteins. Dots on branch indicate that the node supports a value ≥0.97 (aLRT). Bathymodiolus azoricus proteins are highlighted in dark blue. Green: Lophotrochozoa; light blue: Euteleostomi; purple: Insecta; red: Crustacea; yellow: outgroup.

Fig. 5. Temporal expression profiles of Bathymodiolus azoricus canonical circadian clock transcripts.

Expression at the hydrothermal vent (a–h, blue plots) and in the laboratory under L:D 12:12 (i–p, orange plots). Transcriptomic data, n = 5 mussels/time point. a, i, bmal. b, j Clock. c, k, period. d, l, timeout (MN611451). e, m, timeless. f, n, photolyase. g, o, cry1 (MN611453). h, p, cry2. RAIN and ABSR results are shown when one of the analyses returned significant results or results very close to the significant threshold (FDR ≤ 0.05). Whisker plot central line: median; lower and upper hinges: first and third quartiles; whiskers: 1.5× interquartile range; points: outliers.