Physiological impacts of warming and hypercapnia .1 Organismic level

Owing to the above-mentioned physiological adaptations for a life in permanently cold Antarctic waters, stenotherm species may prove to be particularly threatened by rising seawater temperature and PCO2. As cold stenothermal organisms generally possess extremely low metabolic rates and appear to have narrow thermal tolerance ranges (Clarke, 1991; Peck, 2005; Pörtner, 2006), even small increases in temperature could alter individual aerobic performance. For example, high enzymes quantities and high activation energies in mitochondria of Antarctic species cause a large increase in metabolic flux only at a small rise in temperature, and thereby contribute to the low upper critical temperatures in these species (Pörtner et al., 2000). Not only for cold-adapted species, a reduced capacity for aerobic performance at high temperatures has consequences for activity levels, growth rates, reproduction and ultimately limiting an organism’s thermal niche and geographical expansion, which in the end may endanger the whole population’s sustainability (Pörtner and

The OCLTT hypothesis states that metabolic performances are linked with an ectotherm’s ability to undergo physiological adjustments to temperature in order to maintain aerobic scope, and therefore define the whole-organism capacities to cope with thermal challenges, i.e. their acclimation capacities (c.f. figure 1.1). Although Antarctic marine organisms have extraordinarily narrow ranges over which temperature acclimation can occur, the thermal window, and thereby temperature limits, can be shifted by increasing heat tolerance in several species (Seebacher et al., 2005; Lowe and Davison, 2006; Franklin et al., 2007; Pörtner and Lannig, 2009). These acclimations are frequently based on incomplete metabolic compensations involving e.g. an increased net use of storage compounds such as carbohydrates and lipids, and metabolic rearrangements towards enhanced protein catabolism and reduced lipid biosynthesis (Brodte et al., 2006; Windisch et al., 2011). However, there is considerable variation in acclimation capacity among Antarctic fish species: some Antarctic notothenioids, such as Pagothenia borchgrevinki, possess a wide thermal window (Robinson and Davison, 2008), whereas the potential for acclimation is apparently low for many others, such as high Antarctic Trematomus species (Robinson and Davison, 2008; Enzor et al., 2013).

A recent study in different Antarctic invertebrate species (e.g. limpets, bivalves, ascidians or urchins) revealed highest tolerable acclimation temperatures of 1°C to 6°C over longer time periods, while other species like the brittle star Ophionotus victoria only tolerated acclimation temperatures 2°C above their habitat temperature (Peck et al., 2009). Also tropical species are predicted to have limited thermal acclimation capacities owing to their evolution in thermally stable environments (Munday et al., 2012). For example, some coral reef fishes of the Great Barrier Reef have greatly reduced aerobic scope at temperatures only a few degrees above summer mean temperatures, which highlights limited capacities particularly of stenothermal fish species to respond to a rapidly changing climate (Nilsson et al., 2010).

All these examples emphasize how various fish and invertebrate species may be differently sensitive to increases in water temperature. While some cold-adapted, more eurytherm temperate species may be able to shift or extend their geographical distribution range along a latitudinal cline towards the poles as a reaction towards a warming ocean, this is not possible for all species, particularly those already living at their limits in the Southern Ocean (Somero, 2010).

In light of the ongoing ocean acidification of warming oceans, the synergistic effects of both ocean warming and acidification have recently been found to reduce aerobic scope of marine ectotherms by further increasing their aerobic demand or suppressed efficiency of

oxygen supply (c.f. Pörtner, 2010; Pörtner, 2012). As a result, the capacity of an animal to increase its rate of aerobic energy turnover is likely to be reduced possibly even at temperatures within the optimal range of thermal tolerance (Pörtner and Farrell, 2008).

Thereby, the combination of these two stressors may further reduce the already very narrow thermal window of optimum performance in Antarctic species (c.f. figure 1.1), with consequences for activity levels, growth rates and probably population survival (Pörtner, 2010; Munday et al., 2012).

Due to the enhanced CO2 solubility in cold waters and body fluids, ocean acidification along with warming may become particularly threatening to polar ectotherms. Many stenotherm notothenioids live already close to their upper thermal tolerance limits. In line with this, it has been demonstrated that the already high thermal sensitivity of Antarctic Trematomus species and P. borchgrevinki can be enhanced by the synergistic impact of warming and elevated CO2 concentrations (Enzor et al., 2013). Similarly, it has been confirmed for tropical fish, which already live at the edge of their thermal range, that only moderately increased CO2 concentrations of 0.1 kPa can reduce whole animal aerobic scope (Munday et al., 2009).

Yet, the effects of elevated CO2 levels on adult marine fish have received little attention compared to those of rising temperature. Some previous studies revealed that most adult fish are not particularly vulnerable to ocean acidification, as they usually regulate intracellular pH (pHi) and, to various degrees, extracellular pH (pHe) by the accumulation of bicarbonate ions in body fluids, mediated through ion exchange via the gills in order to compensate for rising seawater PCO2 (Larsen et al., 1997; Pörtner et al., 1998; Brauner et al., 2004; Pörtner, 2005; Melzner et al., 2009). Hence acid-base and ion equilibria reach new steady state values and while tissue pHi may fully recover, blood pHe does not necessarily do the same (e.g. Sparus aurata, Michaelidis et al., 2007), which may cause specific, long-term shifts in metabolic equilibria (Deigweiher et al., 2010).

In the long run, the maintenance of permanently elevated bicarbonate levels for a new steady-state condition may represent a continuously higher energy demand of the animal for the maintenance of ion gradients via the cellular membranes. This could result in a higher fraction of metabolic energy needed for acid-base regulation. In line with this, fish show elevated activities of Na⁺/K⁺-ATPase (NKA) or Na⁺/HCO3- cotransporter under hypercapnia (Deigweiher et al., 2008), and NKA is considered a key enzyme involved in compensation of acid-base disturbances (Choe and Evans, 2003).

Marine invertebrates are hypothesized to be among the organisms most sensitive to ocean acidification, for example due to constraints in their metabolic rates, growth or calcification efficiencies (Jensen et al., 2000; Langenbuch and Pörtner, 2002; Michaelidis et al., 2005). Especially less mobile animal groups such as pteropods (Orr et al., 2005), echinoderms (Kurihara et al., 2004), bivalves (Kurihara et al., 2007) and particularly corals (Hoegh-Guldberg et al., 2007) are suggested to suffer more under hypercapnia than actively swimming animals with higher metabolic rates, such as crustaceans or cephalopods (Spicer et al., 2007; Gutowska et al., 2009; Melzner et al., 2009). Primary responses of the more sensitive marine organisms could be acid-base imbalances, which hamper calcification and the formation of calcareous shells, lead to metabolic depression (a condition expected to retard growth and reproduction), reduced activity, in severe cases to a loss of consciousness due to disruption of oxygen-transport mechanisms, and, if persistent, death (Reipschläger and Pörtner, 1996; Seibel and Walsh, 2001). For temperate crustaceans, experimental evidence is already available which indicates a narrowing of the thermal tolerance window of the edible crab Cancer pagurus and the spider crab Hyas araneus, by environmental hypercapnia (Metzger et al., 2007; Walther et al., 2009).

1.5.2 Mitochondrial level

With changing metabolic demand of an organism, e.g. under chronic hypercapnia or rising temperatures, the energy demand of tissues follows according to the metabolic role of the tissue.Previous studies suggest that whole animal thermal limits are mainly governed by capacity limitations of the circulatory system rather than a general failure of cellular energy metabolism, and that organelles cover a wider thermal tolerance window than those of the whole organism (Mark et al., 2002; Mark et al., 2005).

Rising standard metabolic rates and mitochondrial respiration during warming go hand in hand with higher leakiness of biological membranes (Hazel, 1995) and thus need an appropriate adjustment of aerobic capacities. Especially the persistent occurrence of mitochondrial proton leak plays a physiologically important role in thermal tolerance, as it can account for up to 20-25% of the whole animal basal metabolic rate (Brand, 2000;

Chamberlin, 2004). At high temperatures, excessive oxygen demand through enhanced proton leakage rates is followed by a rise of baseline oxygen demand, as observed in mitochondria of Antarctic bivalves and fish (Hardewig et al., 1999a; Pörtner et al., 1999). Such a drastically elevated mitochondrial oxygen demand, which is paralleled by progressively decreasing ATP

synthesis capacities, may at a certain point exceed the capacity of oxygen supply by the circulatory system and thus lead to a restriction or loss of aerobic mitochondrial metabolism (Pörtner, 2001, 2002b; Brand and Esteves, 2005).

As already pointed out, cold-adapted membranes frequently posses a high content of unsaturated fatty acids to maintain membrane fluidity in the cold (e.g. fish - Hazel, 1995;

cephalopods - Turner et al., 2005). On the other hand, the content of unsaturated fatty acids in the membranes of cold-adapted animals need to be reduced upon warming (Brand et al., 1994;

Porter et al., 1996; Brookes et al., 1998) – otherwise, membranes would become too fluid during warming, which may affect various membrane-associated proteins and processes, such as ETS complexes or the electrochemical proton gradient across the inner mitochondrial membrane (higher proton leakage, Brand et al., 1994; Porter et al., 1996; Lee, 2004).

Rising ambient temperatures can further lead to an increase in the production of reactive oxygen species (ROS) in cold adapted marine animals (Heise et al., 2003; Keller et al., 2004). It is postulated that higher ROS formation in the ETS can be prevented by controlled mild uncoupling by mitigating the proton motive force (Guderley, 2004).

Consequently, a control of proton leak would allow adjustments of the mitochondrial metabolism in response to temperature changes on the molecular level.

Functional responses to changes in tissue-specific aerobic energy demand include concomitant adjustments of it’s metabolic demand, such as shifts in substrate turnover (e.g.

seasonal shifts in glycogen and lipid usage in Arenicola marina, Sommer and Pörtner, 1999), or changes in mitochondrial abundance and/ or mitochondrial aerobic metabolism.

Mitochondrial adjustments can include changes in the activities of their enzymes such as citrate synthase (CS), which catalyses the first step of the TCA-cycle, and cytochrome c oxidase (COX), a mitochondrial trans-membrane protein and component of the electron transport system (ETS). The activities of these enzymes are thus commonly used as a parameter reflecting the metabolic responses to warming and hypercapnia (Guderley, 1998;

Windisch et al., 2011), and the changes in mitochondrial amount or structure: COX activities relate to mitochondrial membrane structure (Wodtke, 1981; O'Brien, 2011), and CS activity to the mitochondrial matrix volume (e.g. Hardewig et al., 1999b; Guderley and St-Pierre, 2002;

Guderley, 2004). To monitor the processes involved in temperature-related mitochondrial proliferation and their acclimation capacities, measurements of CS and COX activities have been used in several, eurythermal temperate and stenothermal Antarctic fish species (e.g. sea bass (Egginton and Sidell, 1989), cod (Lucassen, 2006) or trout (Battersby and Moyes,

temperature-related patterns of enzymatic responses are not necessarily the same in all species and particularly not in different tissue types (e.g. Dalziel et al., 2005; Hulbert et al., 2006).

Enzymatic responses to higher PCO2 are poorly studied in fish (tuna, Greco et al., 1982; sea bass, Michaelidis et al., 2007), and not at all in cephalopods.

Up to now, only few studies have demonstrated a compensation of mitochondrial oxygen demand in response to warm-acclimation, and they were conducted mostly on non-Antarctic fish (e.g. Dahlhoff and Somero, 1993; Sloman et al., 2008; but see Lannig et al., 2005). In contrast, stenotherm Antarctic fish analysed so far appear not to possess any mitochondrial compensation abilities in response to chronic warmth-exposure (Weinstein and Somero, 1998). Further studies on mitochondrial warm-acclimation capacities are rare, and data on the effects of chronic hypercapnia on mitochondrial capacities in both marine vertebrates and invertebrates are completely lacking so far.