Cellular metabolism of different life stages of marine teleosts during Ocean Acidification and Warming

Abstract

The anthropogenic emissions of greenhouse gases are causing an increase in atmospheric and oceanic temperatures. In the oceans, seawater temperature rises in parallel to the decrease in pH caused by the reaction of rising atmospheric CO2 with water. The combined phenomenon is known as Ocean Acidification and Warming (OAW). Rising temperatures and decreased water pH may induce adjustments in the energy budget of fish, requiring more energy for protein turnover and for ion and acid-base balance. Since most of these processes depend on the mitochondrial provision of ATP, this PhD project investigated if rising temperature and PCO2 affect the mitochondrial functioning with consequences for the energy budget and acclimation potential of the animals to climate changes. Mitochondria are specialised cellular organelles and their degree of specialisation may vary according to species and life-stage. To cover a broad range of this variability, this thesis analysed firstly the mitochondria of juvenile polar cod (Boreogadus saida) as polar fish and of juvenile Atlantic cod (Gadus morhua) from the Northeast Arctic population (NEAC) as temperate fish, which presently co- occur in the waters around Svalbard. Secondly, the mitochondria of juvenile NEAC were compared with the mitochondria of embryos of the same species (Øresund population) to assess the differences between life-stages. Lastly, mitochondria of Atlantic cod embryos were analysed together with the ones of Atlantic herring embryos (Clupea harengus) because of the different spawning behaviour of the two species (pelagic for Atlantic cod, benthic for herring). Polar cod and NEAC were acclimated for four months at combinations of temperature (polar cod: 0, 3, 6, 8°C; NEAC: 3, 8, 12, 16°C) and PCO2 (400 and 1170 μatm) at the end of which their cardiac mitochondrial respiration was tested. In addition, the lipid class composition in pooled cellular membranes and the capacity of a number of mitochondrial enzymes were analysed. Embryos of Atlantic cod and herring were incubated from fertilization to hatch at present and projected temperatures (Atlantic cod: 0, 3, 6, 9, 12°C; herring: 6, 10, 14°C) and PCO2 (400 and 1100 μatm). When the embryos reached the "50% eye pigmentation" developmental stage, whole-body mitochondrial functioning was assessed. Moreover, the hatching success, length at hatch and larval malformation rates were recorded. The mitochondrial parameters measured in all species were OXPHOS, proton leak, citrate synthase (CS) capacity and the capacity of the single components of the Electron Transport System (ETS) i.e., Complex I (CI), Complex II (CII) and Complex IV (CCO). Juvenile polar cod presented some stenothermal traits like the lack of adjustments of the membrane lipids, stable values of OXPHOS and ETS despite increasing temperatures and low values of CCO/ETS. The relation between OXPHOS and proton leak suggested an optimum temperature for ATP production in the 3-6°C range, while the proton leak increased dramatically at 8°C, which was not paired by OXPHOS, hence decreasing the ATP production and therefore the available energy in the cardiac cells. Since the heart plays a fundamental role in acclimation to temperature, the lower energy yield may be related to the higher mortality occurring at this temperature. Yet, polar cod mitochondria were not affected by elevated PCO2 besides the increase in CS activity, probably as compensatory response to overcome its inhibition. NEAC, on the other hand, displayed more eurythermal features like modifying the lipid components of the cellular membranes, high CCO/ETS and increasing OXPHOS and ETS with rising temperatures. Although proton leak also increased with temperature, the stable ATP production efficiency indicates the ability to control proton leak and ensure the required energy to the cellular processes in a broader range of temperatures. However, the cardiac mitochondria of NEAC were negatively impacted by incubation under elevated PCO2, especially in combination with the highest tested temperature (16°C). Individuals from that group presented lower OXPHOS, lower ETS and lower capacity of the ETS enzymes CI and CCO whereas the TCA cycle-related enzymes CS and CII were stimulated. Possibly, elevated PCO2 inhibited CS and CII which were up-regulated in order to compensate for the lower activity. If the compensation was just partial, the decrease in activity of the TCA cycle may have led to a decrease of the ETS activity and therefore of the OXPHOS capacity with negative consequences on the energy yield of the heart cells. In contrast to their juvenile conspecifics, Atlantic cod embryos possessed mitochondria with a narrower thermal window which were not sensitive to elevated PCO2. In fact, OXPHOS, ETS and CI increased with temperature until 9°C, where they reached a plateau, and CII presented the same capacity at control and high levels of CO2. However, the combination of high temperature (12°C) and elevated PCO2 exerted a negative effect at higher organizational levels, decreasing hatching success and hatchlings' length. Moreover, elevated PCO2 increased the larval malformation rates at all incubation temperatures. Similar trends were found in the embryonic mitochondria of Atlantic herring. In this species OXPHOS and CI increased with temperature until 10°C and then reached a plateau, while elevated PCO2 did not affect the mitochondrial functioning. While elevated PCO2, especially in combination with high temperatures, decreased the survival of Atlantic cod embryos, herring hatching success and hatchlings' size was only related to temperature, suggesting higher CO2-tolerance in this benthic spawner. In conclusion, with regard to the mitochondrial functioning, NEAC appeared more eurytherm and plastic in the range of temperatures projected for the waters around Svalbard at the end of the century. Despite their higher CO2- sensitivity they may outperform polar cod, displacing them or forcing them to retreat in the fjord bottom waters. The plasticity of juvenile NEAC was lowest at the embryonic level, where the thermal window was narrower and more susceptible to elevated PCO2, suggesting that embryos may be a bottle-neck for the population acclimation process. Moreover, tolerance to high CO2 may be related to the spawning behaviour, with benthic species being more tolerant than pelagic ones.