Mechanistische Untersuchungen zur Physiologie der CO_1tn2-Toleranz bei Cephalopoden [Elektronische Ressource] = Mechanistic studies on the physiology of CO_1tn2 tolerance in cephalopods / vorgelegt von Marian Yong-An Hu

Hu Maxwell

Sujets

• Biologie

Informations

• Publié par: christian-albrechts-universitat_zu_kiel
• Publié le: 01 janvier 2011
• Nombre de lectures: 14
• Poids de l'ouvrage: 11 Mo

Extrait

Mechanistische Untersuchungen zur Physiologie der CO 2
Toleranz bei Cephalopoden

Mechanistic studies on the physiology of CO tolerance in 2
cephalopods






Dissertation
Zur Erlangung des akademischen Grades
Dr. rer. nat.

Mathematisch Naturwissenschaftliche Fakultät der Christian-Albrechts-Universität zu Kiel
Vorgelegt von

Marian Yong-An Hu

Kiel 2011




























Gutachter:
1. Gutachter: Prof. Dr. Frank Melzner
2. Gutachter: Prof. Dr. Markus Bleich
Mündliche Prüfung abgelegt am 21.04.2011
Zum Druck genehmigt: 21.04.2011





Contents I

Table of contents

Summary _________________________________________________________________III
Zusammenfassung__________________________________________________________V

1. Introduction ___________________________________________________________ 1
1.1 . Ocean acidification _______________________________________________________1
1.2 . Biological impacts _________________________________________________________3
1.3 . Acid-base regulation _______________________________________________________5
1.4 . Cephalopods _____________________________________________________________8
1.5. The cephalopod gill_______________________________________________________9
1.6. Ontogeny-dependent sensitivities towards hypercapan?i________________________11
1.7. Questions and research hypotheses__________________________________________13

2. Methods _____________________________________________________________ 17
2.1 . Animals and experimental design___________________________________________17
2.1.1. General experimental setup_________________________ ____________________________18
2.1.2. Determination of the seawater carbonate system _____ ______________________________19
2.2. Determination of PVF abiotic parameters ___________________________________19
2.3. Oxygen consumption measurements________________________________________20
2.4 . Biochemical and molecular techniques _____________________________________21
2.4.1. Enzyme activity and protein levels________________________________________________21
2.4.2. Molecular cloning __________________________________ ___________________________22
2.4.3. Quantitative real time PCR______________________________________________________23
2.5. Histological methods______________________________________________________24
2.5.1 . Classical histology and immunocytochemistry ________ ______________________________24
2.5.2. In situ hybridization____________________________________________________________26
2.5.3 . Vital dye staining_____________________________________________________________27
2.5.4. Scanning electron microscopy _______________________ ____________________________28
2.6. Scanning ion-selective electrode technique__________________________________28
+
2.6.1. Measurement of surface H gradients_________________________________________ ____28
+
2.6.2. Measurement of apparent H fluxes ____________________________________________ __29





II Contents

3. Publications __________________________________________________________ 31
Publication 1 _________________________________________________________________33
Elevated seawaterp CO differentially affects branchial acid-base tranrstpeors over the course of 2
development in the cephalopoSde pia officinalis_____________________________________33
Publication 2 __________________________________________________________________85
New insights into ion regulation of cephalopod umscosll: a role of epidermal ionocytes in acid-
base regulation during embryogenesis____________________________________________85
Publication 3 ________________________________________________________________125
Localization of ion-regulatory epithelia in embr yaonsd hatchlings of two cephalopods _____1 25

4. Discussion___________________________________________________________ 161
4.1 . Differential response of ontogenetic stages_________________________________163
4.2 . Why are early stages more sensitive?_____________________________________168
4.2.1. The cephalopod egg: a naturally hypercapnic envimernont __________________________ 168
4.2.2. Ontogeny of ion-regulatory epithelia in early ssta_g_e______________________________ 169
4.3 . The cephalopod gill: site of acid-base regulatio_n____________________________170
+ +
4.3.1 . Role of the gill Na/K-ATPase in acid-base regulation ______________________________ 170
+ +
4.3.2. Response of gill Na/K-ATPase to CO stress ______________________________________ 172 2
4.3.3 . Candidate genes for acid-base regulation in theh acelopod gill_______________________ 174
4.4 . Acid-base regulation in gill epithelia: juvenilensd aadults _______________________177
4.5. Acid-base regulation: embryonic stages____________________________________179
4.6. Conclusions and future directions_________________________________________18 3

5. References __________________________________________________________ 187



Summary/Zusammenfassung I II

Summary

Elevated environmental CO concentrations (hypercapnia) are a stressor that has lately 2
received considerable attention: anthropogenic CO emissions are predicted to lead to a rise in 2
surface ocean pCO from 0.04 kPa up to 0.08 - 0.14 kPa within this century. The increased 2
hydration of CO changes seawater chemistry, causing a drop in ocean pH. This phenomenon 2
has been termed “ocean acidification” (OA). Changes in aquatic CO partial pressure affect 2
the physiology of all water breathing animals as the CO concentration in body fluids will 2
increase as well in order to maintain a substantial outward directed diffusion gradient for CO . 2
Among the aquatic taxa some have been identified as rather sensitive species (e.g. less active
calcifying species such as corals or echinoderms) whereas others (many active species such as
adult fish and cephalopods) can tolerate high CO concentrations over long exposure times. It 2
was shown that more tolerant organisms share the ability to compensate for a hypercapnia
induced acidosis by actively accumulating bicarbonate and eliminating protons from their
body fluids. This process requires the presence of an acid-base regulating machinery
consisting of a variety of ion transporters and channels.
Using in situ hybridization and immuno histochemical methods, the present work
+ + + + -demonstrates that Na /K -ATPase (NKA), a V-type-H -ATPase (V-HA), and Na /HCO 3
cotransporter (NBC) are co-localized in NKA-rich cells in the gills of cephalopods.
Furthermore, mRNA expression patterns of these transporters and selected metabolic genes
were examined in response to moderately elevated seawater pCO (0.16 and 0.35 kPa) over a 2
time-course of six weeks in different ontogenetic stages. Our findings support the hypothesis
that the energy budget of adult cephalopods is not significantly compromised during long-
term exposure to moderate environmental hypercapnia. However, the down regulation of ion-
regulatory and metabolic genes in late stage embryos, taken together with a significant
reduction in somatic growth, indicates that in contrast to adult cephalopods early life stages
are challenged more severely by elevated seawater pCO . This increased sensitivity of 2
cephalopod early life stages could be due to two primary reasons. The first is related to gill
development: similar to the situation in fish and decapod crustaceans, the cephalopod gill is
the most important site for ion-regulatory processes. During larval development, rudimentary
gill structures occur at stage 20, and differentiate over the course of embryonic development
as well as after hatching. This differentiation indicates that gas exchange and ion regulatory
capacity might be fully activated only after leaving the protective egg capsule. This could
partially explain the higher susceptibility of embryonic stages to environmental hypercapnia.
IV Summary/Zusammenfassung

The second reason for a higher sensitivity is due to the oviparous type of development in
cephalopods. Cephalopod embryos are exposed to very low egg fluid pO values (<6 kPa, ca. 2
28% air saturation) and high pCO values (>0.3 kPa) under control conditions during the final 2
phase of embryonic development. This is due to increasing metabolic rates and the egg casing
acting as a diffusion barrier for dissolved gases. The present work demonstrates that
environmental pCO is additive to the natural accumulation of CO in the perivitelline fluid 2 2
(PVF). This almost linear increase of PVF pCO is necessary in order to conserve the CO 2 2
diffusion gradient across the egg capsule that drives excretion of metabolic CO to the 2
seawater. Thus, alterations in environmental pCO create a greater challenge to the 2
developing embryo in comparison to juveniles or adults.
Despite the lack of adult-like high capacity ion regulatory epithelia (e.g. gills or
kidneys) the present work demonstrates for the first time t