Simulated and Field Environmental Effects on the Transcriptome and Metabolome of Mussel Mytilus californianus

Mussels of the genus Mytilus are distributed world-wide and are commercially cultured as a food source for humans. They are also an important ecological species that provide substrate for hundreds of invertebrate and vertebrate organisms as well as an energy source for a variety of marine species. Because of their commercial and ecological importance many studies have been conducted to understand aspects of their physiology. The dominant species on north-western rocky shorelines of North America is Mytilus californianus. As a sessile species M. californianus must endure fluctuations in temperature, salinity, food and oxygen due to the ebb and flood of the tide. During periods of low tide, mussels are exposed to the terrestrial environment where they cannot feed or breathe oxygen and are exposed to temperature fluctuations as a result of solar radiation, cloud cover, wave splash and wind shear. Mussels counteract these stresses by closing their valves to avoid dessication, and switching to anaerobic ATP-producing pathways as well as depressing their metabolism. Thus, M. californianus is well adapted to the highly variable environment of the intertidal zone. Using microarray-based gene expression profiling and metabolite screens, we performed a series of experiments aimed at understanding the fundamental mechanisms driving physiology in an intertidal marine mollusc. Experiments were performed in a custom built aquarium that simulated the intertidal zone, including precision control of tide, solar radiation, day:night cycles, and food levels. In our first experiment, we subjected mussels to balanced cycles of aerial emergence and submergence at constant temperature. Our findings revealed that >40% of the transcriptome exhibited rhythmic gene expression and that depending on the specific tidal conditions 80-90% of the rhythmic transcripts followed a circadian pattern of expression pattern with a period of 24-26 hr, while <2% followed a tidal pattern 10-14hr. Our data indicate that the circadian 24 hr cycle is the dominant driver of rhythmic gene expression in this intertidal inhabitant despite the profound environmental and physiological changes associated with aerial exposure during tidal emergence. Metabolite profiles of the same samples revealed that 24 metabolites oscillated significantly with a 12 hr period that was linked to the tidal cycle. These data confirmed the presence of alternating phases of fermentation and aerobic metabolism and highlight a role for carnitine conjugated metabolites during the anaerobic phase of this cycle. We also observed mussels that spontaneously open and close their valves in constant submerged conditions and a comparison of the expression and metabolite abundances revealed a close similarity in gene expression and utilization of metabolic pathways between subtidal and intertidal physiology as it relates to valve gape state. Lastly, we subjected mussels to an extreme environment that consisted of cycles of long aerial emergence periods combined with a daily heat stress. Surprisingly, the molecular phenotype was notably different from that observed under our more benign conditions, suggesting that M. californianus has a highly flexible physiology that allows it to make acute and complex cellular adjustments that allow it to buffer intense fluctuations in the often unpredictable environment within the intertidal zone. These experiments provide new insights and interpretations of intertidal physiology that can be used as a reference source for comparative studies of rhythmic biology in other organisms.