Intertidal mussels regularly experience emersion-induced anoxia, in contrast to normoxic conditions experienced during submersion. We therefore hypothesized that acclimation to a tidal rhythm, as opposed to a rhythm of constant submersion, preconditions the proteome of the California mussel, Mytilus californianus, to respond differently to emersion-induced anoxia. Following acclimation, mussels either continued to receive the acclimation conditions (control) or were exposed to 100% nitrogengas (anoxia) during aerial emersion. We collected gill tissue for subsequent analysis of protein abundance with 2D gel electrophoresis and protein identification with tandem mass spectrometry. Relative to subtidally-acclimated mussels, tidally-acclimated mussels showed a greater propensity to respond to distrupted protein homeostasis during emersion through higher levels of several small heat shock protein isoforms, while they showed lower levels of several chaperones involved in redox-sensitive protein maturation in the endoplasmic reticulum during acute anoxia. Several metabolic proteins showed elevated levels in tidally-acclimated mussels, suggesting a compensatory response to reduced feeding times. However, changes in the abundance of several tricarboxylic acid cycle enzymes (e.g. aconitase, succinate dehydrogenase) suggest that tidally-acclimated mussels are also primed to sense reactive oxygen species (ROS) and limit their production, respectively. These findings are further supported by higher abundances of several aldehyde dehydrogenases and thioredoxin peroxidase, which function as scavengers of aldehydes and ROS, common products of lipid peroxidation. Finally, tidally-acclimated mussels are also more responsive to changes in cytoskeletal and vesicular trafficking dynamics in response to acute anoxia. Together, our analysis showed that proteostasis, energy metabolism, oxidative stress and cytoskeletal and trafficking processes are all involved in priming tidally-acclimated mussels to respond more dynamically to acute emersion-induced anoxia in Mytilus gill.

The intertidal zone is a dynamic environment that fluctuates with the 12.4-h tidal and 24-h light/dark cycle to predictably alter food availability, temperature, air exposure, wave action, oxygen partial pressure, and osmotic conditions. Intertidal sessile bivalves exhibit behavioral or physiological changes to minimize the persistent challenges of fluctuating environmental conditions, such as adjusting gaping behavior and heart rate. At the cellular level, transcriptomic studies on mussels’ baseline circadian and circatidal rhythms have determined that the circadian rhythm is the dominant transcriptional rhythm. However, as proteins reflect the basic molecular phenotype of an organism and their abundance may differ greatly from that of mRNA, these methods could fail to detect important cyclical changes in the proteome that cope with the regular stress of tidal rhythms. For this study, we acclimated intertidal Mytilus californianus to circadian (12:12 h light/dark cycle) and circatidal (6:6 h tidal cycle) conditions in a tidal simulator and sampled gill tissue from mussels every 2 h for 48 h for proteomic analysis. Approximately 86% of the proteins that were detected exhibited rhythmicity over the time course. The circadian cycle primarily determined the cyclic abundance of energy metabolism proteins, pivoting around the transition to the nighttime high tide. The tidal cycle contributed to alterations in cytoskeletal components, ER protein processing and vesicular trafficking, extracellular matrix and immune proteins, and oxidative stress and chaperoning proteins. We also found evidence that post-translational modifications may be important for driving these rhythms, as acetylation and phosphorylation motifs were enriched in the rhythmic proteins and we identified rhythms in elements of methylation, mitochondrial peptide processing, and acylation. These dynamic changes in proteins across numerous functional categories indicate that the combination of circadian and tidal cycles drive complex cellular changes to coordinate processes in a changing environment. This variation clearly shows that differential expression studies and biomonitoring efforts cannot assume a static baseline of cellular conditions in intertidal mussels.

Introduction California sea lion (Zalophus californianus) health is severely compromised by domoic acid toxicosis, which occurs in high levels during harmful algal blooms of Pseudonitzschia australis along the coast of California. Current diagnostic protocols are often inconclusive due to a 2-48 hour window of detectability within the urinary, circulatory, and gastric systems (Cook, et al. 2011 and Monte, Pers Comm, 2012). Past studies suggest that Z. californianus, with domoic acid toxicosis, commonly display abnormal behaviors (Goldstein, et al. 2008). However, many of these abnormal behaviors are also associated with other diagnoses and are therefore unreliable as diagnostic indicators. This study fills in a knowledge gap relating to abnormal behavior types and their correlation to domoic acid toxicosis and helps solve the problem of current, inconclusive, diagnostic protocols. In this study, my objectives were to identify abnormal behaviors correlated to domoic acid toxicosis, create a diagnostic ethogram, determine the applicability of the method in the field, and determine the applicability of triage based on the relationship between abnormal behaviors and domoic acid levels. Methods I conducted focal animal continuous scans (continuous observation of a single animal at a time, for a set period) with continuous data entry, on animals admitted to the Marine Mammal Center (main study location during 2011-2013) and the Marine Mammal Care Center (comparison location, 2013). I conducted my observations from behind a blind to prevent both human habituation and behavioral influence of the observer. Observations lasted between 10-15 minutes (10 minutes per pen in 2011, 15 minutes per animal in 2012-2013). Subjects were selected based on an admit date no later than 7 days from the observation date. I conducted focal animal continuous scans at Pier 39, a haul out location, in the San Francisco Bay. Animals included in the study had identifying marks or were isolated from other animals (making them easy to identify). I observed animals once per observation day with a total observation period not exceeding 15 minutes per animal. I logged domoic acid levels in feces, urine, and serum (collected by veterinary staff and analyzed with liquid chromatography and bioassays for the presence of domoic acid). I then compared these results to the types and severity of abnormal behaviors displayed by the domoic acid toxicosis sample. Results Results from data collected at the Marine Mammal Center suggest that head weaving (Wilcoxon, p Results from the Pier 39 study suggest that behavioral criteria may be applicable for ruling out domoic acid toxicosis in groups of animals. However, I did not test the method during times of harmful algal blooms. Therefore, the applicability of the method for use as a diagnostic tool in the field is unknown and further research is required. Results for the triage study were inconclusive. The number of animals that tested positive for domoic acid was small and not suitable for statistical analysis. I suggest further research into triage abilities. Conclusion Based on the results of these studies, I can conclude that behavioral analysis offers a reliable diagnostic tool for rescued Z. californianus. Practitioners can use behavioral diagnostic criteria with confidence for the diagnosis of domoic acid toxicosis in Z. californianus.

The ability to acclimate to changing temperature has consequences for the biogeographic range of a species and their potential for surviving ocean warming. Using a tide simulator, which recreates tidal conditions by controlling water levels, water and air temperatures, light levels, and food availability, we explored how temperature and tidal zone (i.e. intertidal and subtidal) influences the abundance of sirtuin 5 (SIRT5) protein in a California native mussel (Mytilus californianus). We compared how gill tissue abundance of SIRT5, a key regulator of the cellular stress response and energy metabolism responded in M. californianus exposed to different temperature conditions (13, 16, 19 and 22°C) over a period of four weeks. Two SIRT5 isoforms, one a putative cytosolic form and the other a mitochondrial form were found to be expressed in mussel gill tissue. The mitochondrial isoform increased during acclimation to warm temperatures. This finding is the first to show how SIRT5 protein abundance changes with temperature acclimation. Surprisingly, we did not identify any differences in gill SIRT5 abundance between mussels from intertidal and subtidal locations. Our results suggest that characterizing the responses of SIRT isoforms may lead to a better understanding of the physiological diversity of sirtuins.