Understanding species-specific physiological tolerances to environmental extremes is key in determining the factors that contribute to regulating species distribution. This understanding will aid in determining which species will manage to thrive in a changing global climate. According to the IPCC (2013) it is expected that, in the coming years, many different types of abiotic factors will change as a result of global climate change. The intertidal habitat is a model habitat for studying environmental extremes as it is located at the interface between the marine and terrestrial environments, making it one of the most stressful marine habitats. It is characterized by a 24 hr light: dark cycle and a 12.4 tidal ebb and flow that exposes animals inhabiting this habitat to a wide array of aerial-associated stressors such as changes in temperature, aerial exposure, low oxygen or hypoxic conditions and desiccation stress. Sessile organisms such as marine mussels of the genus Mytilus, are an ideal study species for studying physiological tolerance at the environmental extremes of the intertidal habitat. In particular, M. trossulus and M. galloprovincialis are an excellent study system for examining physiological tolerance at environmental extremes due to the recent change in biogeographic range of both species. M. galloprovincialis, a native of the Mediterranean, has been taking over the coast of California and has been displacing the heat sensitive native M. trossulus. The effects of salinity stress and heat stress on the physiologies of these species have been investigated by Braby and Somero (2006a and 2006b), Tomanek and Zuzow (2010) and Tomanek et al. (2012). The results of these studies indicate that the invasive M. galloprovincialis is more heat tolerant but is sensitive to hyposalinity while the reverse is true for the native M. trossulus.. The next logical environmental stress to study is low tide or aerial-emersion as both species can be found both tidally and subtidally. According to Grieshaber et al. (1994) and Müller et al. (2012) Mytilus edulis mussels have mechanisms for mitigating aerial-emersion hypoxia; however, very few studies have been performed using the study system of M. trossulus and M. galloprovincialis in regard to aerial-emersion hypoxia. This study aimed to observe the responses of both M. trossulus and M. galloprovincialis to aerial-emersion hypoxic stress or low tide. The study also looked to see if the recent habitat history (tidal or subtidal) can play a role in the response of the mussels to hypoxia. The results of the experiment indicate that the invasive M. galloprovincialis may be less sensitive to hypoxic stress when compared to the native M. trossulus. This difference in sensitivity may be due to the difference in mechanisms of energy metabolism proteins and proteostasis proteins used to mitigate the effects of hypoxic stress. Moreover, tidal acclimation appears to better prepare the mussels for subsequent aerial exposure in both species, possibly based upon the principles of stress-hardening outlined by Kültz (2005).

The giant acorn barnacle, Balanus nubilus, is a resident of the subtidal and low intertidal rocky shoreline on the Pacific Coast of North America (Alaska to Baja California). B. nubilusis notable for having the largest muscle fibers in the animal kingdom; fiber diameters that can exceed 3mm in adults! At such extreme sizes these muscle cells may be at risk for insufficient oxygen delivery to mitochondria owing to low SA:V ratios and long intracellular diffusion distances. Oxygen limitation to these muscles may be further exacerbated during low tide air exposure (emersion) or environmental hypoxia events, which are increasing in frequency and duration along the world’s coastlines. We are interested in characterizing the internal oxygen conditions of B. nubilus during air emersion and anoxia so that we can ultimately investigate the physiologic mechanisms by which B. nubilus maintains function in their giant muscle fibers during environmental oxygen limitation. To this end, we examined the effects of air emersion and anoxia on 1) hemolymph gas, pH and ion levels, 2) oxygen consumption rates (MO2; emersion only), and 3) respiratory behaviors (e.g., cirri beating). In the first experiment, we measured hemolymph pO2, pCO2, pH and ion ([Na+], [Cl-], [K+], [Ca2+]) concentrations at 0, 3, 6 and 9h of exposure to air emersion, anoxic immersion and normoxic immersion (control). Next, we compared the average MO2 of barnacles held in water and air for 6h at three common temperatures (10, 15, or 20°C) using intermittent (aquatic) and closed-system (air) respirometry. Lastly, we investigated the respiratory behaviors (% time operculum open, %time testing, % time pumping, % time cirri beating, cirri beat frequency, opercular pulse frequency) of B. nubilusduring acute (6h) exposure to air emersion, anoxic immersion and normoxic immersion (control). Our data revealed that hemolymph pO­2 was significantly decreased in the anoxic barnacles by 3h and remained significantly depressed relative to the normoxic control for 9h. The air-exposed barnacles, however, maintained hemolymph oxygen levels that were intermediate to the control and anoxia barnacles for the entire experiment, achieving levels that were significantly lower than normoxic barnacles only by 9h. We also found that oxygen consumption rates for B. nubilus held at ecologically realistic temperatures were similar in air and water. From these data we conclude that B. nubilus is relatively adept at taking up oxygen from the environment while out of the water, which is common for certain barnacle species, and that air emersion represents a relatively mild environmental stress for this species (at least from a gas-exchange perspective). Efficient aerial gas-exchange by the giant acorn barnacle is likely facilitated by seawater pools stored in the mantle cavity, which can directly take up oxygen from the air and make it accessible to soft tissues and gill-like structures on the inside of the shell. This strategy, however, would require complementary behaviors aimed at oxygenating the mantle cavity fluid (e.g, aperture opening, cirri extensions to facilitate mixing), and this is exactly what we see. In our behavior experiment we found that air-exposed barnacles (and, more surprisingly, anoxic barnacles) spent significantly more time with their cirri extended than our control animals, who engaged more in an aperture pumping behavior with their cirri retracted. These behavioral preferences existed even though there were no significant differences in the total time spent with their aperture open (regardless of the behavior occurring while open) between any of the treatments. There were also interesting findings in the ion data. While there were no significant treatment effects on [Na+], [Cl-], or [Ca2+], we did observe significantly higher [K+] by 6h in both the emersion and anoxic groups relative to the normoxic group. We predict that this change in [K+] is likely attributable to its role in acid-base buffering. There was a strong correlation between pCO2 levels and pH across all treatments; however, decreases in pO2 levels in the hemolymph, which corresponded with increases in pCO2 levels, had only minimal effects on the hemolymph pH, indicating a well-buffered system. In conclusion, we found that air exposure does not inhibit aerobic metabolism in B. nubilus, owing largely to efficient aerial oxygen uptake and perhaps also effective acid base-buffering. We therefore predict that muscle function would be preserved during periods of low-tide emersion. Anoxia, on the other hand led to a significant decline in hemolymph oxygen content, which suggests that environmental hypoxia is likely to diminish functionality of their giant muscle fibers. In a parallel study, we intend to investigate the plastic response of B. nubilus muscle fibers to the same conditions (air emersion and anoxic immersion).

Salinity and oxygen availability have long been recognised as important factors influencing animal physiology and therefore species distribution. The maintenance of appropriate cellular ion levels is critical for many essential physiological processes, but at the same time is energetically expensive. Since hypoxia is likely to impose aerobic limitations for ATP generation, the maintenance of salt and water homeostasis could be at risk during hypoxia. The amphidromous inanga (Galaxias maculatus) is well known for its salinity tolerance and its life cycle that involves several salinity related migrations. During these migrations inanga also frequently encounters hypoxic waters, and therefore must maintain energy homeostasis when aerobic metabolism may be compromised. The present study has investigated behavioural, physiological, biochemical and molecular mechanisms by which inanga tolerate changes in salinity and hypoxia. After 14 days of acclimation to salinities ranging from freshwater to 43‰, inanga showed physiological acclimation. This was evident by no changes in metabolic rates or energy expenditures through this salinity range. Energy balance seemed to be tightly and efficiently controlled by changes in the proportion of protein and lipids used as energy substrate. No mortalities and only minor changes in plasma osmolality also indicated salinity acclimation. The remarkable osmoregulatory capacity of inanga was also evidenced after a seawater challenge. The osmotic balance of inanga was only disrupted during the first 24 hours after the challenge, evidenced by an increase in plasma osmolality and plasma Na+, and a decrease in muscle water content. These physiological changes were correlated with changes at the molecular level. Different isoforms of the catalytic subunit of the Na+,K+-ATPase (NKA) were isolated, partially sequenced and identified in inanga. Phylogenetic analysis grouped inanga isoforms (α-1a, α-1b, α-1c) with their respective homologues from salmonids. Patterns of mRNA expression were also similar to salmonids, with α-1a being downregulated and α-1b being up-regulated following seawater challenge. Previous to this study, NKA isoform switching was reported to occur only in salmonids and cichlids. The presence of NKA subunits that change with environmnetal salinity in inanga indicates that this isoform switching phenomenon is much more widespread among teleost lineages than previously thought. Aiming to elucidate the hypoxia tolerance of inanga, oxygen consumption rate as a function of decreasing external PO2 was evaluated. At no point did inanga regulate oxygen consumption, suggesting that this species is an oxyconformer. This is the first robust demonstration of the existence of oxyconforming in fish. Evaluation of the scaling relationship between oxygen consumption and fish size in normoxia, showed that the exponent of this relationship fell within the range previously reported for fish. However, in hypoxic conditions the scaling relationship was less clear suggesting different size-related mechanisms for tolerating hypoxia. Analysis of the aerobic and anaerobic metabolism of small and large fish, showed that smaller inanga were able to sustain aerobic metabolism for longer than larger inanga, which instead relied on anaerobic metabolism for extending their survival. This knowledge is likely to be of value for the conservation of this iconic fish species, by incorporating these size related differences in hypoxia tolerance in streams management. In light of the unusual oxyconforming response of inanga, a study examining the behavioural responses of this species to declining dissolved oxygen was performed. Inanga did not display a behaviour that might reduce energy expenditure during oxygen limitation; instead swimming activity and speed were elevated relative to normoxia. As hypoxia deepened inanga leaped out of the water, emersing themselves on a floating platform. Once emersed, fish exhibited an enhanced oxygen consumption rate compared to fish that remained in hypoxic water. Although this emersion behaviour was hypothesised to be of physiological advantage, both aquatic hypoxia and emersion resulted in similar physiological and biochemical consequences in inanga. While in hypoxic water oxygen availability seemed to be the limiting factor, in air failure of the circulatory system was hypothesised to be the cause of a similar metabolic signature to that found in aquatic hypoxia. Overall, inanga seemed to be not particularly well adapted to tolerate aquatic hypoxia. In light of the increasing likelihood of anthropogenic-induced hypoxia in inanga habitats, this is likely to have negative consequences for the future of inanga populations in the wild. Although this study provides the mechanisms behind the exceptional salinity tolerance of inanga, its susceptibility to hypoxia is likely to impose further constraints for the osmoregulatory processes that guarantee inanga survival during life cycle migrations. The results of the present study are relevant for understanding and managing the fishery of this economically- and culturally important fish species.

The design of an underwater observation vessel is the main objective during the thesis. An innovative approach will be applied in terms of modernization and design of the chosen topic. This work shows the author‘s creativity and ability to execute the required task in a specific time frame based on the appropriate level for a thesis. It reveals the steps that lead to an aesthetically balanced object from a functional product. Ergonomic standards and contemporary materials have been one of the basic knowledge.