Cellular and organismal strategies for heat acclimation in a thermally variable environment
Keywords: Biology.

Using mussels as an experimental system, I have challenged previous beliefs concerning how quickly animals gain and lose heat tolerance, and how this heat-adaptive strategy fits into the larger ecological context of animals' responses to climate change. I explore how the California mussel, Mytilus californianus, heat acclimates—physiologically adjusts to hotter temperatures—to a thermally variable environment. In the eight chapters that comprise my dissertation, I explore how mussels gain and lose heat acclimation (i.e., heat acclimation kinetics) to better understand how these heat-tolerant animals adjust to, and survive, in the intertidal zone—an environment where extreme and intermittent heat events frequently occur. Until now, it was unknown how quickly mussels could heat acclimate and then maintain this heat-acclimated state in the absence of further heat stress. Moreover, it was unclear whether the heat-acclimation temperature affected the rates of gain and loss of heat acclimation. To a large extent, it was also unknown what physiological mechanisms play a role in both the gain and loss of heat acclimation. Here, I address these important and closely interrelated questions by exploring how quickly mussels gain and lose heat acclimation, the various environmental components that shape this heat-adaptive strategy, and the cellular and organismal responses responsible for the heat-acclimation phenotype. In my first studies, which focused on whole-organism thermal responses, I show that the rate at which mussels are heated affects their thermal performance. I demonstrate that heating rate is a key component of heat acclimation that was previously not considered and therefore should be a part of predictive models that estimate animal survival with heat stress. On the converse side, I show that the absence of heat stress leads to a very slow decline in thermal performance, but only in mussels that experienced large temperature variability in the field. I thus reveal that heating rate is an important component of heat acclimation for animals living higher on the shore that experience large daily fluctuations in body temperature and fast heating rates. Next, I explore the kinetics of the heat acclimation response: the gain and loss of heat acclimation after a single heat-stress bout. I demonstrate that mussels can quickly heat acclimate (within 24-48 h) after a single heat-stress bout, and then maintain this improved heat tolerance for 2-3 weeks in the absence of additional heat stress. Importantly, the kinetics of the heat acclimation response are dependent on the heat-acclimation temperature, such that exposure to a hotter temperature leads to slower gain and loss of the heat-acclimation phenotype. I show that this adaptive strategy in the lab mirrors what animals experience in the field, where the majority of heat events are separated by 1-2 days, but up to one-third of heat events are still separated by up to 22 days. Thus, it is likely that mussels have evolved the heat-adaptive strategy in which they rapidly gain, and slowly lose, the heat-acclimation phenotype as a result of the frequency of extreme- and intermittent-heat events typical of their habitat. Next, I explored responses at the organ level, using performance of the heart—an extremely temperature-sensitive organ—to examine additional details of heat stress and acclimation. I examined how heart rate changes with heat acclimation in single individuals across time. Importantly, I show that not all mussels respond to heat acclimation: some animals are more adaptable (i.e., physiologically plastic) to heat than others. I show that those that exhibit the heat-acclimation phenotype are able to remain at near-maximal heart rates for a longer period of time during heat stress compared to those that do not respond to heat acclimation. Those that respond to heat acclimation can also tolerate more time at temperatures above the heart's critical temperature (where heart rate starts to decline after reaching a maximum). As heart rate responses are closely linked to aerobic metabolism, these data suggest that aerobic and anaerobic pathways may be enhanced with heat acclimation (as seen in other marine ectotherms). I next investigated the potential cellular mechanisms behind this heat-adaptive phenotype by evaluating levels of specific proteins in single individuals as they gain and lose heat acclimation. I look at two proteins that are heavily involved in the heat-stress and heat-acclimation responses: heat shock protein 70 (HSP70) and phosphorylated p38 mitogen-activated kinase (Pp38-MAPK). I show that heat-acclimated mussels' HSP70 levels are elevated (compared to unacclimated mussels) after an extreme heat-stress bout in which all unacclimated mussels died, but heat-acclimated mussels survived. However, levels of Pp38-MAPK are similar between groups. As Pp38-MAPK aides in the upregulation of HSP70 through various downstream effectors, it may be that heat acclimation modifies some of these downstream effectors that are activated by Pp38-MAPK, therefore allowing for continued upregulation of HSP70 at hotter temperatures, and thereby protecting the cell from damage. Finally, another important component of my thesis is a methodological contribution to many common practices in the field of mussel ecophysiology. Through several different studies outlined in my thesis, I demonstrate that cardiac thermal performance tests—a commonly used test to assess thermal tolerance in mussels—may fail to provide insights into how thermal tolerance and performance are affected by heat acclimation. Moreover, I show that the indices used in this test are highly dependent on the experimental protocol utilized and are not linked to animal survival. Secondly, I establish the ability for researchers to repeatedly sample hemolymph (blood) of single individuals across time (using fluorescence-activated cell sorting, FACS) without causing mussel mortality. As there is large inter-individual variability in mussels' responses to heat acclimation, this technique allows scientists to track aspects of individuals' physiology across time. I demonstrate the importance of this methodology here in my protein work, and hope that in the near-future, these new methods will help pinpoint the mechanisms behind the heat-adaptive phenotype, and therefore provide a basis for predictions concerning identification of animals that are most likely to survive extremely hot temperatures with climate change