Aim and outline of the thesis

Although active organisms like fish and cuttlefish are believed to possess adequate capacities to cope with hypercapnia-induced acid-base disturbances, shifts in energy demand and intracellular pathways for acid-base regulation during chronic hypercapnia exposure may exacerbate the effects of rising seawater temperature on cellular and whole animal metabolism (Munday et al., 2012; Pörtner, 2012). The response of fish to warming or hypercapnia has until now mainly been investigated for temperate species, and no study analysed the interaction of warming and hypercapnia on Antarctic fish, such as Notothenia rossii, a representative for Antarctic notohenioids.

In light of the above-outlined physiological adaptations of Antarctic fish to their minimally fluctuating environment, it is highly questionable if N. rossii displays similar acclimation or acid-base regulation capacities as temperate fish. To predict the future fate of this unique group of fish, it is therefore important to identify the capacities of their circulatory and ventilatory system to supply tissues with sufficient oxygen during chronically elevated temperature and PCO2. Conversely, the capacity of mitochondria to produce energy aerobically is one of the mechanisms supporting and restricting performance including that of ventilation and circulation. The characterization of how exactly mitochondrial complexes of the ETS, enzymes or the mitochondrial structure of N. rossii respond to long-term elevated CO2 levels will therefore increase the knowledge about the capacity of N. rossii to respond or acclimate to rising temperature and PCO2. Measuring extra- and intracellular acid-base parameters after long-term cold and warm hypercapnia acclimation would finally help to elucidate if Antarctic fish possess the capacity to maintain the pH of their body fluids in an physiological optimum range, amongst others to ensure mitochondrial functioning.

However, the ability for warm acclimation has been reported to vary between Antarctic fish species (Podrabsky and Somero, 2006). The determination of mitochondrial characteristic in different Antarctic fish, but also in such species living in thermally more fluctuating habitats, would result in a better understanding on the capacities of Antarctic fish to cope with environmental challenges.

Mitochondrial respiration in Antarctic octopods has never been analysed, and other studies on cephalopod mitochondrial capacities are scarce (but see Mommsen and Hochachka,

1981; Oellermann et al., 2012). Moreover, knowledge on the effect of hypercapnia on cephalopod mitochondria is completely lacking. The determination of acute thermal effects and susceptibility towards warming and hypercapnia in two competing animal phyla, fish and cephalopods, may reveal unifying principles of metabolic responses to ocean warming and acidification in vertebrates and invertebrates. In case of the peculiar Antarctic ecosystem, such physiological knowledge will also help to elucidate if, and in favour of which species, the fragile ecological balance between benthic octopods and notothenioids might be put at risk by future climate change.

Overall, this thesis aims to shed light upon the capacities of the aerobic energy metabolism of marine fish and cephalopods from different latitudinal clines to respond to ocean acidification and warming. It will particularly focus on mitochondrial metabolism and capacities and consider the functional integration to higher hierarchical structures such as cellular and systemic levels by approaching the following three questions:

a) How does increased seawater PCO2 affect the thermal acclimation capacity of Antarctic fish?

Thermal tolerance and acclimation capacities are very low in most Antarctic teleost fish and sensitivities to combined stressors, i.e. higher PCO2 and temperature, are likely to be increased in cold-adapted animals (Pörtner, 2010). Little information exists on the acid-base regulating machinery in highly stenothermal Antarctic fish (Deigweiher et al., 2010), and nothing is known about mitochondrial responses to chronic environmental hypercapnia in teleost fish at all. To address the question above, publication I investigated the response of single complexes of the mitochondrial electron transfer system, and of the mitochondrial membrane structure in detail in warm- and hypercapnia acclimated Antarctic fish, N. rossii.

Routine metabolic rate (RMR), extra- and intracellular acid-base parameters and mitochondrial capacities are presented in publication II. Measurements of the aerobic enzymes, CS and COX, were performed in various tissues of acclimated N. rossii in publication III, to compare tissues-specific mitochondrial characteristics and responses towards warming and hypercapnia. It is hypothesized that Antarctic fish possess limited warm-acclimation capacities and that CO2 would exacerbate the effects of increased temperature at the mitochondrial, cellular and whole organism level.

b) Do high-Antarctic and sub-Antarctic/ Austral notothenioids display mitochondrial capacities of different thermal sensitivity, and in which way does this influence sensitivity to ocean warming and acidification?

This part of the study is dedicated to the question of whether mitochondrial capacities can serve as an indicator for the sensitivity of various notothenioid fish to warming or acidification. It is assumed that notothenioids living in thermally more fluctuating habitats may be more tolerant to changing environmental conditions than stenotherm notothenioids living in the cold and stable Antarctic Ocean. Therefore, sub-Antarctic and cold-eurytherm Austral species are expected to possess higher mitochondrial capacities with lower thermal sensitivities than their stenotherm relatives.

Besides the ability of an organism’s ventilatory and circulatory system to supply oxygen to respiring mitochondria at both low and high temperatures, mitochondrial capacities to produce ATP are believed to form the basis of the sensitivity of aerobic scope towards changing energetic demands, e.g. due to warming or elevated PCO2.

Thus, mitochondrial oxidative phosphorylation capacities and their ability to respond to acute thermal challenges are compared between Austral (Notothenia angustata), Sub-Antarctic (Lepidonotothen squamifrons), Sub-Antarctic (Notothenia rossii & Notothenia coriiceps) and High-Antarctic (Trematomus nicolai & Chionodraco hamatus) notothenioids as a possible indicator for metabolic flexibility towards future climate change (additional data).

Another focus lies on the elaboration of chronic hypercapnia tolerances of Antarctic versus Austral notothenioids. publication IV presents results from heart fibre respiration experiments of long-term hypercapnia acclimated N. angustata, which are compared to the hypercapnia tolerance of N. rossii.

c) Do mitochondrial acclimation and regulatory capacities to warming and hypercapnia provide competitive advantages to fish or cephalopods?

The overall goal of this part of the study is to compare metabolic capacities between (Antarctic) teleost fish and cephalopods. Ocean acidification may lead to an elevated energy demand for acid-base regulation by shifts in acid-base status and intracellular pathways (Pörtner, 2010). The sensitivity of cephalopods to ocean warming and acidification may vary, depending on several physiological characteristics, such as pH sensitivities (and

concentration) of their respiratory proteins. It is further related to the cephalopod’s thermal window, metabolic rate and mitochondrial capacities to sustain them during environmental challenges (Seibel and Walsh, 2001).

In this respect, it is hypothesized that octopods, which have the lowest metabolic rates and capacities among coleoid cephalopods, may be more sensitive to warming and hypercapnia than the more active cuttlefish. In publication V, aerobic metabolic capacities are therefore compared between two benthic octopods of different latitudinal origin (and thus putatively different thermal tolerance), the Antarctic Pareledone charcoti and the sub-tropical Eledone moschata, and then related to the well-studied bentho-pelagic cuttlefish Sepia officinalis.

Limitations in cephalopod physiology by different metabolic preferences, lower energy stores (O'Dor and Webber, 1986), and a less efficient acid-base regulation machinery, are expected to render cephalopods more susceptible to warming or acute and chronic hypercapnia exposure in comparison to fish.

The importance of different metabolic and acclimation capacities of Antarctic notothenioid fish and cephalopods will be discussed in light of the fragile balance between these ecotypically similar and competing groups of animals of different evolutionary origin.