Impact of high CO_1tn2 concentrations on marine life [Elektronische Ressource] : molecular mechanisms and physiological adaptations of pH and ion regulation in marine fish = Auswirkungen erhöhter CO_1tn2-Konzentrationen auf das Leben im Meer / vorgelegt von Katrin Deigweiher

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• Biologie

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• Publié par: universitat_bremen
• Publié le: 01 janvier 2009
• Nombre de lectures: 35
• Poids de l'ouvrage: 9 Mo

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Impact of high CO concentrations on marine life: 2
Molecular mechanisms and physiological adaptations of
pH and ion regulation in marine fish


Auswirkungen erhöhter CO -Konzentrationen auf das Leben im Meer: 2
Molekulare Mechanismen physiologischer Anpassungen der
pH- und Ionenregulation mariner Fische




Dissertation
zur Erlangung des akademischen Grades
- Dr. rer. nat. -




dem Fachbereich 2 Biologie/Chemie
der Universität Bremen


vorgelegt von
Katrin Deigweiher
Diplom-Biochemikerin




Bremen 2009










Gutachter:

1. Gutachter: Prof. Dr. Hans-Otto Pörtner
Alfred-Wegener-Institut für Polar- und Meeresforschung,
Am Handelshafen 12, 27570 Bremerhaven


2. Gutachter: Prof. Dr. Sørge Kelm
Universität Bremen, Fachbereich 2, Biochemie
Postfach 33 04 40, 28334 Bremen


Prüfer: Dr. Magnus Lucassen
Alfred-Wegener-Institut für Polar- und Meeresforschung,
Am Handelshafen 12, 27570 Bremerhaven


Prüfer: Prof. Dr. Reimer Stick
Universität Bremen, Fachbereich 2, Zellbiologie
Leobener Straße NW2 A3290, 28359 Bremen


Tag des Promotionskolloquiums: 15. April 2009 CONTENTS
Table of contents
List of abbreviations _______________________________________________________iii
List of figures____________________________________________________________ iv
Summary ________________________________________________________________ v
Zusammenfassung ________________________________________________________ vii
1 Introduction ________________________________________________ 1
1.1 Ocean acidification __________________________________________________ 1
1.2 Past and future CO concentrations _____________________________________ 2 2
1.3 Coping with acidification______________________________________________ 4
1.4 Acid-base regulation in the fish gill ______________________________________ 5
1.5 Energy maintenance _________________________________________________ 7
1.6 Concept of the thesis 9
2 Materials & methods________________________________________ 11
2.1 Animals__________________________________________________________ 11
2.2 Hypercapnia acclimation experiment____________________________________ 13
2.3 Whole animal respiration_____________________________________________ 14
2.4 Isolated perfused gill respiration _______________________________________ 14
2.4.1 Isolated perfused gill preparations __________________________________ 14
2.4.2 Oxygen consumption measurements ________________________________ 15
2.4.3 Application of inhibitors__________________________________________ 16
2.5 Molecular biology __________________________________________________ 16
2.5.1 RNA isolation _________________________________________________ 16
2.5.2 Cloning and sequencing of bicarbonate transporters_____________________ 17
2.5.3 mRNA quantification by real-time PCR ______________________________ 18
2.5.4 Whole cell and membrane extracts __________________________________ 19
2.5.5 Protein quantification by Western Blotting____________________________ 19
+ +2.5.6 Na /K -ATPase activity assay _____________________________________ 19
2.6 Suppression subtractive hybridization ___________________________________ 20
2.7 Statistics _________________________________________________________ 22
i CONTENTS
3 Publications _______________________________________________ 23
I Acclimation of ion regulatory capacities in gills of marine fish under environmental
hypercapnia _______________________________________________________ 25
II Hypercapnia induced shifts in gill energy budgets of Antarctic notothenioids _____ 39
III Differential gene expression in gills of marine eelpout under hypercapnia ________ 73
4 Additional results__________________________________________ 103
4.1 Isolated, perfused eelpout gill respiration _______________________________ 103
5 Discussion _______________________________________________ 105
5.1 Metabolic consequences of hypercapnia ________________________________ 105
5.2 Gill energy turnover 106
5.3 The molecular transport machinery ___________________________________ 108
5.4 Tanscriptomic analyses _____________________________________________ 113
5.5 Conclusions & perspectives _________________________________________ 115
6 References _______________________________________________ 119
7 Appendix ________________________________________________ 131
7.1 Primer list used for sequencing of NBC1 and AE1________________________ 131
7.2 List of upregulated genes in eelpout gills under hypercapnia _________________ 133
7.3 List of downregulated genes in eelpout gills under hypercapnia ______________ 139
Danksagung ___________________________________________________________ 147
Erklärung gem. § 5 (1) Nr. 3 PromO_________________________________________ 149


ii ABBREVIATIONS
List of abbreviations
- -AE Cl /HCO -exchanger (Anion Exchanger) 3
ATP Adenosine triphosphate
AWI Alfred Wegener Institute
CCS Carbon Capture and Storage
DIC Dissolved Inorganic Carbon
DTT Dithiothreitol
EST Expressed Sequence Tag
+HA H -ATPase
IPCC Intergovernmental Panel on Climate Change
ITR Inverted Terminal Repeats
MAPK Mitogen-Activated Protein Kinase
MHC Major Histocompatibility Complex
MRC Mitochondrion-rich Cell
+ -NBC Na /HCO (bicarbonate)-cotransporter 3
+ +NHE Na /H -exchanger
+ +NKA Na /K -ATPase
+ + -NKCC Na /K /2Cl -contransporter
PCO CO partial pressure 2 2
ppm parts per million
PCR Polymerase Chain Reaction
RLM-RACE RNA Ligase Mediated - Rapid Amplification of cDNA Ends
RT Reverse Transcription
SMR Standard Metabolic Rate
TCA TriCarboxylic Acid
Tris 2-Amino-2-hydroxymethyl-propane-1,3-diol


iii FIGURES
List of figures
Figure 1-1: Anthropogenic CO emission scenarios___________________________________2 2
Figure 1-2: Glacial - Interglacial ice core data _______________________________________3
Figure 1-3: Methods of ocean storage _____________________________________________4
Figure 1-4: Fish gill anatomy ____________________________________________________5
Figure 1-5: Gill ion transport in teleost fishes6
Figure 2-1: Fish species distribution pattern11
Figure 2-2: Sampling areas of fish species _________________________________________13
Figure 2-3: Total RNA sample from Z. viviparus gill tissue _____________________________16
Figure 2-4: Scheme of bicarbonate cotransporter sequences ___________________________18
Figure 2-5: Suppression subtractive hybridization reaction scheme ______________________21
Figure 4-1: Size comparison of isolated perfused gill respiration measurements____________103
Figure 4-2: Perfusion efficiency ________________________________________________103
Figure 5-1: Working model for ion transport regulation under hypercapnia_______________112

iv SUMMARY
Summary
The world’s oceans serve as a buffer system for atmospheric CO concentrations. However, the 2
buffer capacity of the oceans is limited, and the imbalance caused by the additional
anthropogenic CO input has already led to a measurable acidification of the oceans. Certainly, 2
these physicochemical changes affect marine organisms and their ecosystems. Within limits, fish
are able to acclimate to an elevated CO concentration (hypercapnia) and the accompanying pH 2
decrease by regulating their internal ion composition and acid-base parameters. The aim of this
thesis was to study the impact of CO on the mechanisms of ion regulation and on energy 2
metabolism, as well as the patterns of genetic regulation during acute (24 hours) and long-term
(six weeks) acclimation to hypercapnia (10,000 ppm CO) in marine fish. The experiments 2
focused on the gills, where over 90 % of the ion regulation takes place.
Elevated CO concentrations have no obvious impact on the standard metabolic rate of 2
the whole animal, as demonstrated in the North Sea eelpout Zoarces viviparus. The resting rate,
which was evaluated from oxygen consumption measurements, remained stable over four days of
hypercapnic incubation. Furthermore, no unusual behavioral or other stress indicators were
detectable, suggesting a perfect acclimation capacity of the fish - at least for short time periods.
Compensatory capacity of energy metabolism was demonstrated in isolated gills of the
North Sea eelpout and of two Antarctic nototheniid species, Notothenia coriiceps and Gobionotothen
gibberifrons. In this thesis, a setup for analysis of metabolic rates in isolated gills was established to
enable measurements of their oxygen consumption under hypercapnic conditions. Although
metabolic rate remained constant, energy allocation shifted significantly in the gills of the
+ +notothenioids. With specific inhibitors the energy demand for ion regulation (Na /K -ATPase),
protein and RNA biosynthesis could be evaluated. All three processes required more energy
under hypercapnia. The extra costs may be covered by an increase in mitochondrial efficiency
and energy s