Conclusions & perspectives

At first glance, marine fishes seemed to be perfectly capable of dealing with high CO₂ concentrations, since neither visible stress symptoms such as unusual activity or opercular flare nor any disturbance in their metabolic rates were detectable at organismic and tissue level.

Nevertheless, a closer analysis of the fishes’ gills revealed a large number of energetic, cellular and genomic modifications during hypercapnia acclimation.

Significant shifts in the energy budget of isolated gills from the Antarctic notothenioids occurred with increasing energy demands for ion regulation, translation and transcription under elevated CO₂ concentrations. Full external pH compensation was mimicked by the experimental design, thus the higher energy demand in the isolated gill system most likely represents the early regulatory phase of the time series defined in publication I. A shift in energy allocation towards ion regulation seemed to be required for establishing the new ion equilibrium in the gills.

Accordingly, capacities of Na⁺/K⁺-ATPase in common eelpout gills were found elevated under hypercapnia within the same time frame and also long-term, when a new steady state can be assumed. These findings indicate a common principle of hypercapnia response in marine fish.

Therefore, similar shifts to those found in the Antarctic fish seem likely in the eelpout gill energy turnover, although they could not be experimentally assessed due to methodological restrictions.

Moreover, assessing total enzymatic capacities and mRNA expression levels seem to be suitable tools for predicting functional tissue activities, as demonstrated for Na⁺/K⁺-ATPase.

The regulation of ion transport was not restricted to the sodium pump. A number of other ion transporters directly involved in proton or bicarbonate exchange have been found regulated at the transcriptional level. However, in contrast to Na⁺/K⁺-ATPase, these enzymes were all transporters that depend on ion gradients and cannot directly utilize ATP to increase their transport activity. Although post-translational modifications of these molecules may help in adapting their transport rates (e.g. by conformational changes, which can enable or disable entry of ions into the transport channel), their regulation by alternating mRNA content and finally

present thesis confirmed their presence and role for hypercapnia acclimation in gills of marine teleost fish for the first time.

The working model for hypercapnia acclimation (presented in publication I) was expanded by the differential gene expression analysis in the eelpout gills in the early regulatory phase (publication III), where the apical Na⁺/H⁺-exchanger isoform 2 (NHE2) and two V-type H⁺-ATPase (HA) isoforms, as well as Cl^- channels have been found upregulated after 24 hours of hypercapnia. Furthermore, differentially expressed gene sequences of carbonic anhydrase (CA) have been identified. The apparent adjustment of carbonic anhydrases by individually expressed isoforms stresses the importance of these enzymes for the hypercapnic response. Together, the observed changes at the transporter/enzyme level seem to aid pH recovery and the adjustment of HCO₃^- and Cl^- concentrations. At the presumed new steady state after six weeks, higher Na⁺/K⁺ -ATPase (NKA) and NBC1 levels were necessary to meet the requirements of the changed ion and acid-base equilibrium, while AE1 and NHE1 were back at control levels (Figure 5-1).

Certainly, the acclimation process was not limited to ion and acid-base regulation. The transcriptome study revealed a number of other processes in the gills that are modified under hypercapnia. The direct stress response proteins, as well as the high number of immune defense related proteins were indicating a general stress state of the gill tissue. The expression pattern also demonstrated enhanced activity for cell communication and maintenance, transcription, translation and protein degradation as well as shifts in metabolic pathways. Within mitochondria, the gene expression pattern of enzyme complexes of the respiratory chain indicated functional adjustments rather than a directed up- or downregulation, representing an unchanged overall ATP production. This is in line with the stable oxygen consumption rates of the isolated gills under hypercapnia in this study and is confirmed by the unchanged mitochondrial capacities found in the gills of Z. viviparus after 14 days of hyercapnia (Penno, 2006). However, for the metabolic fluxes in carbohydrate and lipid pathways the picture was different. Gluconeogenesis was favored over glycolysis, probably to fuel the pentose phosphate shunt. The generation of NADPH and RNA/DNA ribose components by this pathway may support the increased transcription activity, as well as general biosyntheses of metabolites that rely on NADPH.

Additionally, tricarboxylic acid cycle seemed downregulated, leading to a reduction of NADH production. Certainly, these processes still need to be confirmed by functional studies (e.g. by

eelpouts. As the shifts towards gluconeogenesis and pentose phosphate shunt consumes rather than produces energy, elevated mitochondrial efficiencies (e.g. by reducing the proton leak) have to be postulated. Other processes, which would occur under normal, well-fed conditions (e.g.

lipid synthesis), may also be reduced. The observed modifications of the energy and metabolic turnover are probably not limited to the gills. Shifts in energy metabolism of muscle tissues from aerobic to anaerobic energy production have been reported after 2 to 3 weeks of hypercapnia acclimation in marine eelpout and seabream (Penno, 2006; Michaelidis et al., 2007). A permanent redistribution of the animal’s total energy budget may thus be necessary under long-term elevated CO₂ concentrations.

It remains to be examined in future studies, whether stenothermal polar fishes respond more strongly to hypercapnia than eurythermal fish. With respect to the increased energy demand for ion regulation and underlying translational and transcriptional activity, the data sets from temperate eelpout and Antarctic notothenioid gills indicate a common picture. However, a direct comparison of the energy budgets was not possible, as the eelpout gills turned out to be less suitable for isolated gill respiration measurements (due to their small size). Another fish model will have to be used to assess the gill energy allocation under hypercapnia in eurythermal species.

For example, an adequate model species with appropriate gill size would be the Atlantic cod Gadus morhua, which has already been studied intensively with regard to environmental changes (Johansen & Pettersson, 1981; Larsen et al., 1997; Pörtner et al., 2001; Lucassen et al., 2006). In addition to this, gill Na⁺/K⁺-ATPase activity and protein level were found increased by 100 % under long-term hypercapnia acclimation in this species [6,000 ppm CO₂ (Melzner et al., 2008)].

Meaningful comparative studies rely on suitable model organisms. In this respect comparison of the present data of common eelpout should be extended to the confamilial Antarctic eelpout P. brachycephalum. This is of particular interest as whole animal respiration measurements had originally implied a more pronounced reaction of P. brachycephalum to hypercapnia, indicated by increased whole animal metabolic rates (Burgard, 2004). Moreover, the genetic similarity of these confamilial eelpout species is very high (96 - 99 % DNA sequence identity), such that most of the mRNA and protein quantification studies developed for Z. viviparus can be directly transferred to P. brachycephalum. Therefore, the enormous set of cDNA sequences obtained by the differential gene expression study in Z. viviparus can be used for transcriptional studies in both eelpout species. These genes can be used for completing the model of gill ion regulation under hypercapnia in marine fishes and reveal possible differences between

sensitivity of an organism. Moreover, the candidate genes found in fish may also serve as reliable markers for CO₂ sensitive processes in other marine (invertebrate) species, with probably lower regulatory capacities.

It must be considered that the CO₂ concentration of 10,000 ppm used consistently in this thesis is much higher than the expected environmental concentrations of about 1,000 ppm by the end of this century and also the maximal value of 1,900 ppm by the year 2300 (IPCC, 2007).

However, significantly higher concentrations than 1,900 ppm occur at least locally at sites with volcanic activity in the deep-sea or in some shallow water habitats. Near carbon storage sites extremely high CO₂ concentrations can be expected as well. However, due to the unratable risks of ocean storage for the marine environment, it has been prohibited at least for the North-East Atlantic by the OSPAR commission (http://www.ospar.org).

The present thesis was designed as a pilot study providing essential mechanisms of hypercapnia responses for the first time in marine fish at molecular to organismic level, including the identification and characterization of bicarbonate transporters, the energy budgeting in isolated gills and the construction of differential cDNA libraries. The regulatory processes enabling acclimation to elevated CO₂ concentrations will be based on the same mechanisms, albeit that the operating expense will depend on the magnitude of the disturbance. On the basis of this thesis further studies on the key processes will have to reveal to what extent hypercapnia acclimation will affect the fishes under realistic scenarios. In addition, marine ecosystems are challenged by both ocean acidification and climate warming. The combined effects of several stressors may reduce the animals’ tolerance windows (Pörtner & Farrell, 2008) and the resilience they would otherwise display against one stressor alone. Current studies indicate that common molecular mechanisms of stress responses may explain these synergistic effects. Experiments on the combined effects of CO₂ stress at elevated temperature are necessary for an understanding of the impacts of global change on marine fauna. Indeed, in studies on the crab Cancer pagurus the impact of hypercapnia led to a narrowing of the temperature tolerance window (Metzger et al., 2007). The data presented for fish in this thesis provide a useful framework for future investigations.