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F A C U L T Y O F S C I E N C E<br />

U N I V E R S I T Y O F C O P E N H A G E N<br />

PhD <strong>the</strong>sis<br />

Caroline Methling<br />

<strong>Cardio</strong>-<strong>respiratory</strong> <strong>Physiology</strong> <strong>of</strong> <strong>the</strong><br />

<strong>European</strong> <strong>Eel</strong> (Anguilla anguilla) in<br />

Extreme Environments<br />

Academic advisors: John F. Steffensen and Peter V. Skov<br />

Submitted: 15/01/2013<br />

Marine Biological Section, Department <strong>of</strong> Biology<br />

PhD School <strong>of</strong> Science, Faculty <strong>of</strong> Science,<br />

University <strong>of</strong> Copenhagen<br />

Denmark


Cover illustration:<br />

Section <strong>of</strong> map and quote from Schmidt, J. 1909. Ferskvandsaalenes<br />

(Anguilla) udbredning i verden. 1. Det Atlantiske Ocean og<br />

Tilgrænsende Omraader. En Bio-Geografisk Studie. D. Kgl. Danske<br />

Vidensk. Selsk. Skrifter, 7. Række, Naturvidensk. Og Ma<strong>the</strong>m. Afd.<br />

Viii. 3


Contents<br />

Committee 3<br />

Enclosed publications and manuscripts 3<br />

Preface 4<br />

Summary 5<br />

Resumé 7<br />

Objectives <strong>of</strong> <strong>the</strong> <strong>the</strong>sis 9<br />

Introduction 11<br />

1. The <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla) 11<br />

1.1 Distribution and Phylogeny 11<br />

1.2 Life History and Ecology 12<br />

1.3 Morphological and Physiological Specializations 14<br />

1.3.1 Hypoxia exposure 14<br />

1.3.2 Aerial exposure 14<br />

1.4 Silvering 15<br />

2. Energetics 16<br />

2.1 Partition <strong>of</strong> Energy 16<br />

2.2 Nutrients and Energy Metabolism 17<br />

2.2.1 Feeding Metabolism 18<br />

2.3 Measuring Metabolic Rate 19<br />

3. The Fish Heart 20<br />

3.1 Anatomy 20<br />

3.2 Cardiac Function 22<br />

3.3 Cardiac Performance and Control 23<br />

4. Swimming and Migration 25<br />

4.1 Why fish Migrate 25<br />

4.2 Capacity for Migration 25<br />

4.2.1 Swimming Performance 25<br />

4.2.2 Cost <strong>of</strong> Swimming 26<br />

4.2.3 Energy Utilization 27


5. Temperature 28<br />

5.1 Effect on Metabolism and Aerobic Scope 29<br />

5.2 Oxygen and Capacity Limited Thermal Tolerance 30<br />

5.2.1 Temperature Extremes 31<br />

5.3 Effects on Cardiac Morphology and Function 31<br />

5.4 Effects on Cardiac Performance 32<br />

6. Hypoxia 33<br />

6.1 Definition and Occurrences 33<br />

6.2 Effects on Ventilation and Oxygen Transport 33<br />

6.3 Effects on Cardiac Performance 34<br />

6.4 Effects on Metabolism and Aerobic Scope 35<br />

6.5 Interactive Effects <strong>of</strong> Hypoxia and Temperature 36<br />

7. Hypercapnia 37<br />

7.1 Effects on Blood pH and Oxygen Carrying Capacity 38<br />

7.2 Effects on Cardiac Function 38<br />

7.3 Effects on Metabolism and Aerobic Scope 39<br />

7.3.1 Feeding Metabolism 40<br />

Conclusions and Perspectives 41<br />

References 44<br />

Paper I<br />

Paper II<br />

Paper III<br />

Paper IV<br />

Co-author statements


COMMITTEE<br />

Chairman: Bent Vismann, Marine Biological Section, University <strong>of</strong> Copenhagen, Denmark<br />

Peter G. Bushnell, University <strong>of</strong> Indiana South Bend, South Bend, Indiana, USA<br />

Christel Lefrançois, Université de La Rochelle, France<br />

ENCLOSED PUBLICATIONS AND MANUSCRIPTS<br />

Paper I:<br />

Paper II:<br />

Paper III:<br />

Paper IV:<br />

Methling, C., Skov, P.V. and Steffensen, J.F., 2012. The influence <strong>of</strong> temperature<br />

and hypoxia on oxygen consumption in <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla) over a<br />

wide range <strong>of</strong> temperatures. (Draft manuscript)<br />

Methling, C., Steffensen, J.F., and Skov, P.V., 2012. The temperature challenges on<br />

cardiac performance in winter-quiescent and migration-stage eels Anguilla anguilla.<br />

Comparative Biochemistry and <strong>Physiology</strong> A-Molecular & Integrative <strong>Physiology</strong><br />

163, 66-73.<br />

Methling, C., Pedersen, P.B., Steffensen, J.F., and Skov, P.V., 2012. Tolerance<br />

towards hypercapnia does not preclude a negative impact on metabolism and<br />

postprandial processes in <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla). (Draft manuscript)<br />

Methling, C., Tudorache, C., Skov, P.V., and Steffensen, J.F., 2011. Pop Up<br />

Satellite Tags Impair Swimming Performance and Energetics <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

(Anguilla anguilla). Plos One 6.<br />

3


PREFACE<br />

The work presented in this <strong>the</strong>sis was undertaken during my enrolment as a PhD student at <strong>the</strong> PhD<br />

school <strong>of</strong> Science, Department <strong>of</strong> Biology, University <strong>of</strong> Copenhagen. The PhD scholarship was<br />

funded by Danish Agency for Science and Innovation, The Elisabeth and Knud Petersen<br />

Foundation, and <strong>the</strong> Faculty <strong>of</strong> Science, University <strong>of</strong> Copenhagen. I was based at Marine<br />

Biological Section (MARS) Helsingør, where I performed <strong>the</strong> majority <strong>of</strong> <strong>the</strong> work. From August<br />

2011 to March 2012 I visited <strong>the</strong> National <strong>Institut</strong>e <strong>of</strong> Aquatic Resources, Section for Aquaculture,<br />

Technical University <strong>of</strong> Denmark in Hirtshals, where <strong>the</strong> remaining part <strong>of</strong> <strong>the</strong> experimental work<br />

was conducted.<br />

First and foremost I would like to thank my academic advisors on this project, Pr<strong>of</strong>essor John F.<br />

Steffensen (principal supervisor, MARS) and Associate pr<strong>of</strong>essor Peter V. Skov (DTU); John for<br />

his help and ingenuity in <strong>the</strong> technical department and for <strong>the</strong> pleasant social ga<strong>the</strong>rings with<br />

colleagues, Peter for helpful discussions, constructive criticism on manuscripts and for having a<br />

positive outlook and a great sense <strong>of</strong> humor. I would also like to thank <strong>the</strong> people in <strong>the</strong> fish<br />

physiology group at MARS especially Maria F. Steinhausen, Jon C. Svendsen and Bjørn Tirsgaard<br />

for inspirational discussions, plenty <strong>of</strong> c<strong>of</strong>fee and help whenever needed. A thank is also due to all<br />

<strong>the</strong> people at MARS and DTU aqua for <strong>the</strong> kind help and advice, and letting me borrow equipment<br />

that made everything work out in <strong>the</strong> end. A warm thanks to all <strong>the</strong> fellow students at DTU aqua for<br />

making me feel most welcome, making Hirtshals a home away from home and making my stay<br />

both enjoyable and rewarding.<br />

Finally heartfelt thanks to all my family and friends for <strong>the</strong>ir encouragement, sympathy and interest.<br />

To Anders for all your help, never-ending support and for being forbearing through times when I<br />

was ei<strong>the</strong>r absent or absent minded, it meant everything to me.<br />

Caroline Methling<br />

Copenhagen, January 2013<br />

4


SUMMARY<br />

The main objective <strong>of</strong> this PhD <strong>the</strong>sis was to study <strong>the</strong> cardio-<strong>respiratory</strong> capabilities <strong>of</strong> <strong>the</strong><br />

<strong>European</strong> eel (Anguilla anguilla) under extreme conditions. Three environmental conditions were<br />

studied i.e. temperature, dissolved oxygen and carbon dioxide, while a fourth condition was<br />

physiological and focused on <strong>the</strong> impressive spawning migration <strong>of</strong> A. anguilla.<br />

Ambient temperature influences <strong>the</strong> rate <strong>of</strong> most biological functions including metabolic processes,<br />

which in turn determines <strong>the</strong> overall metabolic capacity. In Paper I it is demonstrated that A.<br />

anguilla has a wide <strong>the</strong>rmal optimum as absolute aerobic scope (MS ABS ) was constant between<br />

10°C and 30°C, and eels were able to maintain a high oxygen uptake, even at <strong>the</strong> highest<br />

temperature studied (30°C). Still, <strong>the</strong> scope for o<strong>the</strong>r activities was considerably reduced as aerobic<br />

metabolism could only be increased about 3 fold at <strong>the</strong> highest temperature. Resting and maximum<br />

oxygen consumption (MO 2 ) and thus MS ABS were significantly suppressed at 0°C in accordance<br />

with <strong>the</strong> observed torpidity <strong>of</strong> eels at low temperatures. The ability to regulate MO 2 during hypoxia<br />

was assessed by determination <strong>of</strong> <strong>the</strong> critical O 2 partial pressure (P CRIT ) at 0, 10, 20 and 30°C and<br />

P CRIT was found to be positively correlated with temperature. Excess post-hypoxic oxygen<br />

consumption (EPHOC) was quantified after 2 hours <strong>of</strong> severe hypoxia exposure and also increased<br />

with temperature. The duration <strong>of</strong> EPHOC was about 3 times shorter at 0°C, than at 10, 20 and<br />

30°C. The fraction <strong>of</strong> MS ABS utilized for recovery was elevated at <strong>the</strong> temperature extremes,<br />

indicating that hypoxia narrows <strong>the</strong> <strong>the</strong>rmal tolerance <strong>of</strong> A. anguilla.<br />

In Paper II it is demonstrated that temperature also influenced <strong>the</strong> contractile properties <strong>of</strong><br />

ventricular muscle in vitro, in that <strong>the</strong> time-course <strong>of</strong> contraction, and thus maximum attainable<br />

heart rate in vivo, greatly depended on ambient temperature. The relative ventricular mass was<br />

increased after long term acclimation to 0°C and 10°C compared to individuals acclimated to 20°C,<br />

indicative <strong>of</strong> a compensatory mechanism for <strong>the</strong> limitation in heart rate at low temperature in A.<br />

anguilla. The force <strong>of</strong> contraction and myocardial power production increased after an acute<br />

decrease in ambient temperature from 20°C to 10°C (mimicking <strong>the</strong> vertical movements performed<br />

during <strong>the</strong> spawning migration). This may serve to <strong>of</strong>fset <strong>the</strong> depressant effect on heart rate and thus<br />

ensure adequate cardiac performance when diving to cooler depths. Fur<strong>the</strong>rmore, <strong>the</strong> individual<br />

contribution <strong>of</strong> three different sarcolemmal Ca 2+ channels (L-type, NCX and SOCE) to <strong>the</strong><br />

generation <strong>of</strong> force also depended on ambient temperature.<br />

5


Elevations in CO 2 partial pressure (hypercapnia) is a common phenomenon in aquaculture facilities<br />

<strong>of</strong> A. anguilla. In Paper III it is demonstrated, that when exposed to a constant high level <strong>of</strong><br />

hypercapnia (60mmHg), eels took a longer time (22%) to digest a meal size <strong>of</strong> fixed proportions<br />

(0.5% body weight) compared to eels held under normocapnic conditions, while eels exposed to<br />

oscillating CO 2 partial pressures (20-60mmHg) had a reduced post-prandial ammonia excretion.<br />

This suggests that depending on <strong>the</strong> specific conditions, hypercapnia may limit <strong>the</strong> appetite and feed<br />

intake as it takes longer time to process a meal, and/or that a smaller amount <strong>of</strong> <strong>the</strong> dietary energy<br />

and nitrogen content will be allocated to growth due to a reduced absorption /assimilation<br />

efficiency. Regardless, <strong>the</strong>se results demonstrate that hypercapnia adversely affects <strong>the</strong> postprandial<br />

processes in A. anguilla.<br />

Pop-up satellite archival tags (PSATs) have recently been applied in attempts to follow <strong>the</strong> oceanic<br />

spawning migration <strong>of</strong> A. anguilla. Due to <strong>the</strong> size <strong>of</strong> <strong>the</strong>se tags, it is likely that <strong>the</strong>ir hydraulic drag<br />

constitutes an additional cost during swimming, which may have implications for successful<br />

migration. In Paper IV, migration stage eels were subjected to swimming trials at increasing<br />

speeds <strong>of</strong> 0.3 - 0.9 body lengths s -1 , first without and subsequently with, a scaled down PSAT<br />

dummy attached, while rates <strong>of</strong> oxygen consumption (MO 2 ) were measured. The tag increased MO 2<br />

during swimming and elevated <strong>the</strong> minimum cost <strong>of</strong> transport (COT min ) by 26%. Standard (SMR)<br />

and active metabolic rate (AMR) as well as aerobic metabolic scope remained unaffected, which<br />

suggests that <strong>the</strong> observed effects were caused by increased drag. Swimming with a tag decreased<br />

<strong>the</strong> critical swimming speed (U crit ) and also altered swimming kinematics as verified by significant<br />

changes to tail beat frequency (f), body wave speed (v) and Strouhal number (St). The results<br />

demonstrate that energy expenditure, swimming performance and efficiency all are significantly<br />

affected in migrating eels fitted with external tags.<br />

6


RESUMÉ<br />

Det overordnede formål med denne PhD var at studere den europæiske åls (Anguilla anguilla)<br />

hjerte -og respiratoriske formåen under ekstreme forhold. Fokus blev lagt på tre forskellige<br />

miljømæssige faktorer: temperatur, iltforhold samt kuldioxidforhold. En fjerde faktor var<br />

fysiologisk, og var i relation til den europæiske åls imponenrende lange gydevandring.<br />

Omgivelsernes temperatur påvirker de fleste biologiske funktioner inklusiv metabolske processer,<br />

hvilket har betydende indflydelse på det metabolske råderum. I manuskript I bliver det påvist at A.<br />

anguilla har et bredt temperatur-optimum, i at med det absolutte aerobe metabolske råderum<br />

(absolute metabolic scope, MS ABS ) var uforandret mellem 10°C og 30°C, samt at ålene var i stand<br />

til at opretholde et konstant højt iltforbrug (mass-specific oxyegn consumption rate, MO 2 ), selv ved<br />

den højeste eksperimentelle temperatur (30°C). Med det sagt, vil råderummet for andre metabolske<br />

aktiviteter dog være stærkt begrænsede da st<strong>of</strong>skiftet kun ville kunne blive forøget med omtrent en<br />

faktor 3 ved 30°C. Iltforbruget ved hvile, samt ved maximal aktivitet, og defor også MS ABS , var<br />

betydeligt nedsat ved 0°C, hvilket er i god overenstemmlese med observationer af ål der går i en<br />

dvalelignende tilstand ved lave vandtemperaturer. Evnen til at regulere iltoptaget under hypoksi ved<br />

forskellige temperaturer, blev vurderet ved at fastsætte værdier for den kritiske iltspænding (critical<br />

oxygen partial pressure, P CRIT ) ved 0, 10, 20 samt 30°C, og det blev konstateret at der er en positiv<br />

sammenhæng mellem P CRIT og temperatur. Iltforbruget efter at have været udsat for 2 timers svært<br />

hypoksiske forhold, steg i forhold til iltforbruget ved hvile. Omfanget af dette øgede iltforbrug<br />

(excess post-hypoxic oxygen consumption, EPHOC) afhæng også af vandtemperaturen og var<br />

yderligere forøget ved de højere temperaturer. Ved 0°C, forblev iltforbruget kun forhøjet i ca 1/3 af<br />

tiden det var forhøjet ved de øvrige temperaturer (10, 20 samt 30°C). Procentdelen af det<br />

metabolske råderum der blev udnyttet i forbindelse med det forøgede iltforbrug afhang også af<br />

temperaturen og var således størst ved ekstremerne. Dette peger på, at hypoksiske forhold vil<br />

indskrænke den europæiske åls temperatur tolerance spektrum.<br />

I manuskript II bliver det påvist at temperaturen også har indflydelse på visse af<br />

hjertemuskulaturens kontraktionsegenskaber in vitro. Navnlig var kontraktionsperioden, og dermed<br />

den maximale slagfrekvens in vivo, under kraftig inflydelse af vandtemperaturen. Det<br />

hjertesomatiske indeks var forøget hos ål der var akklimeret til 0°C og 10°C, i forhold til ål der var<br />

akklimeret til 20°C. Dette kan opfattes som en måde hvorpå den europæiske ål kan kompensere for<br />

den begrænsede maximale hjerteslagsfrekvens ved lave temperaturer. Kraftudviklingen i det<br />

7


ventrikulære hjertevæv blev forøget, efter temperaturen blev akut sænket fra 20°C til 10°C (hvilket<br />

skulle simulere temperaturforholdene under de neddykninger ålene foretager sig under deres<br />

gydevandring). Denne forøgelse af kraftudviklingen kan tolkes som en kompensation for den lavere<br />

slagfrekvens og dermed være medvirkende til at sikre hjertefunktionen når ålene dykker ned på<br />

koldere vand. Temperaturen viste sig derudover også at have inflydelse på hvormeget 3 særskilte<br />

Ca 2+ kanaler (L-type, NCX samt SOCE), lokaliseret i den ydre cellemembran, bidrog til den<br />

samlede krafudvikling i det ventrikulære hjertevæv.<br />

Forøget CO 2 partial tryk (hyperkapni) er et <strong>of</strong>te forekommende fænomen i åleopdrætsanlæg. I<br />

manuskript III bliver det påvist, at ål der var udsat for et konstant højt CO 2 niveau (60mmHg), var<br />

længere tid (22%) om at fordøje et måltid svarende til 0,5% af kropsvægten, end ål der gik under<br />

normale CO 2 forhold. Ål der var udsat for svingende CO 2 partial tryk (20-60mmHg), havde en<br />

nedsat ammonium udskillelse after indtagelse af måltidet. Disse resultater tyder på, at afhængigt af<br />

de specifikke hyperkapniske forhold, kan et forøget CO 2 niveau medvirke til at reducere ædelysten<br />

samt fødeindtaget, da det tager længere tid at fordøje det enkelte måltid, eller medvirke til at en<br />

mindre andel af måltidets energi -samt kvælst<strong>of</strong>sindhold bliver allokeret til vækst, på grund af en<br />

reduceret absorbtions/ assimilerings effektivitet. Uanset hvad, viser resultaterne at hyperkapniske<br />

forhold kan have en ugunstig indflydelse på fordøjelsesprocesserne hos den europæiske ål.<br />

I et nyligt forsøg på at følge ålen på dens gydevandring i Atlanterhavet, blev der anvendt såkaldte<br />

Pop-up satellite tags (PSATs). Grundet størrelsen på denne type tags kan det tænkes, at den ekstra<br />

vandmodstand udgør en potentiel energetisk meromkostning, der kan have betydning for om<br />

hvorvidt gydevandringen kan fuldføres. I manuskript IV blev blankål sat til at svømme ved<br />

stigende svømmehastigheder svarende til 0,3-0,9 kropslængder i sekundet. Denne svømmetest blev<br />

foretaget først uden, denæst med en PSAT-replikat påhæftet, alt imens ålens iltforbrug blev målt.<br />

Når ålene svømmede med det påhæftede tag, havde de et forøget iltforbrug, hvilket betød at<br />

mindsteomkostningen ved at at svømme en given længde (minimum cost <strong>of</strong> transport, COT min ) blev<br />

forøget med 26%. Iltforbruget ved hvile og ved maximal aktivitet, samt det aerobe metabolske<br />

råderum ændrede sig ikke, hvilket tyder på at merforbruget af ilt var en direkte effekt af den<br />

forøgede vandmodstand. Den kritikske svømmehastighed (U crit ) var lavere med et påhæftet tag,<br />

mens andre kinematiske variable såsom haleslagsfrekvens, hastigheden af kropsbølgen samt<br />

Strouhal nummeret alle var forøgede. Disse resultater viser, at energiforbruget,<br />

svømmeegenskaberne samt effektiviteten påvirkes når blankål svømmer med et påhæftet tag.<br />

8


OBJECTIVES OF THE THESIS<br />

The <strong>European</strong> eel (Anguilla anguilla) has attracted considerable scientific as well as economic<br />

attention for a long time, which is reflected in <strong>the</strong> quite extensive literature on this species.<br />

Anguillid eels are most fascinating fishes as <strong>the</strong>y have adapted to living in a broad range <strong>of</strong><br />

environments, <strong>the</strong>y are able to survive extreme conditions, and <strong>the</strong>y undertake <strong>the</strong> longest spawning<br />

migrations <strong>of</strong> any known species <strong>of</strong> fish. The drastic population decline that has occurred during <strong>the</strong><br />

past decades fur<strong>the</strong>r emphasizes <strong>the</strong> importance <strong>of</strong> increasing our understanding <strong>of</strong> <strong>the</strong><br />

ecophysiology <strong>of</strong> A. anguilla in both natural surrounding and in aquaculture settings. Thus <strong>the</strong> main<br />

objective <strong>of</strong> this PhD <strong>the</strong>sis was to study basic physiological functions i.e. cardio-<strong>respiratory</strong><br />

physiology in A. anguilla under environmental extremes and in relation to <strong>the</strong> spawning migration.<br />

The first part <strong>of</strong> this <strong>the</strong>sis focuses on ambient temperature, and how this affects two major<br />

physiological functions i.e. whole animal oxygen uptake and cardiac function. The span <strong>of</strong><br />

temperatures tolerated by A. anguilla is impressively wide, ranging from approximately -1°C to<br />

more than 30°C. Although A. anguilla is a thoroughly studied species, no single study has so far<br />

examined <strong>the</strong> effect <strong>of</strong> temperature on basic and active metabolic rate, and aerobic metabolic scope<br />

over a broader range <strong>of</strong> temperatures including <strong>the</strong> extremes. Thus, one <strong>of</strong> <strong>the</strong> objectives <strong>of</strong> <strong>the</strong> first<br />

study (Paper I) was to examine basal and maximal oxygen consumption rates when acclimated to a<br />

wide range <strong>of</strong> temperatures (0°C-30°C), in order to estimate metabolic capacity i.e. aerobic scope as<br />

a function <strong>of</strong> temperature.<br />

The main objective <strong>of</strong> <strong>the</strong> second study (Paper II) was to examine cardiac function in vitro during<br />

two different temperature challenges imposed on A. anguilla in its natural surroundings. One<br />

approach simulates overwintering at very low temperatures, while a second mimics <strong>the</strong> temperature<br />

fluctuations <strong>the</strong>y experience during <strong>the</strong>ir spawning migration, when making diel vertical migrations.<br />

Myocardial ventricular contractions were studied in terms <strong>of</strong> duration, contractility at different<br />

pacing frequencies, and under adrenergic stimulation. This was studied after long term acclimation<br />

to 0°C representative <strong>of</strong> winter conditions, at 10°C and 20°C and during acute temperature changes<br />

<strong>of</strong> ± 10°C, representing conditions during vertical migrations. In addition, <strong>the</strong> cellular Ca 2+ cycling<br />

mechanisms were studied after long term acclimation by selective Ca 2+ channel blockade.<br />

The second part <strong>of</strong> this <strong>the</strong>sis focuses on environmental hypoxia. During hypoxic events, it is<br />

essential for long term survival that enough oxygen can be extracted from <strong>the</strong> surrounding water to<br />

sustain minimum metabolic requirements. A. anguilla is considered a very hypoxia-tolerant species,<br />

but <strong>the</strong>re is considerable variation in <strong>the</strong> estimates <strong>of</strong> <strong>the</strong> precise oxygen tension (P CRIT ) where<br />

9


oxygen uptake sustaining <strong>the</strong> basal metabolic requirements becomes compromised. Some <strong>of</strong> this<br />

variation is likely caused by differences in experimental temperature as P CRIT is expected to be<br />

temperature dependent. Therefore, <strong>the</strong> second objective <strong>of</strong> Paper I was to estimate <strong>the</strong> critical<br />

oxygen partial pressure, P CRIT and to examine how this is related to ambient temperature across <strong>the</strong><br />

entire range (i.e. 0°C to 30°C).<br />

The third part <strong>of</strong> this <strong>the</strong>sis focuses on hypercapnia. As previous studies have demonstrated that <strong>the</strong><br />

A. anguilla is quite tolerant to elevated CO 2 per se, and because severe hypercapnia almost<br />

exclusively occurs in aquaculture facilities, <strong>the</strong> objective <strong>of</strong> this part <strong>of</strong> <strong>the</strong> study was to examine<br />

how hypercapnia affected <strong>the</strong> postprandial processes <strong>of</strong> eels in terms <strong>of</strong> post- feeding oxygen<br />

consumption and nitrogen excretion (Paper III).<br />

The final focus <strong>of</strong> this <strong>the</strong>sis is on swimming metabolism in relation to <strong>the</strong> oceanic spawning<br />

migration <strong>of</strong> A. anguilla. To date, <strong>the</strong>re have been no observations <strong>of</strong> spawning adults or eggs in <strong>the</strong><br />

Sargasso Sea, which is considered <strong>the</strong> spawning area <strong>of</strong> <strong>the</strong> <strong>European</strong> eel. A recent telemetry study<br />

using externally attached Pop-up satellite tags (PSAT´s) tracked migrating eels as far as 1000 km<br />

<strong>of</strong>f <strong>the</strong> coast <strong>of</strong> Ireland swimming towards <strong>the</strong> Sargasso Sea; however <strong>the</strong> mean cruising speed was<br />

lower than generally assumed. Valuable information about migration routes and various physical<br />

parameters can be attained using PSAT´s however <strong>the</strong> physical dimensions <strong>of</strong> <strong>the</strong> tag may constitute<br />

an additional metabolic cost for <strong>the</strong> animal and reduce swimming efficiency. Thus <strong>the</strong> objectives <strong>of</strong><br />

<strong>the</strong> fourth study (Paper IV) were to estimate <strong>the</strong> potential metabolic costs <strong>of</strong> swimming with an<br />

externally attached PSAT by measuring oxygen consumption at a range <strong>of</strong> swimming speeds, and to<br />

examine <strong>the</strong> effect <strong>of</strong> <strong>the</strong> tag on kinematic variables and swimming efficiency.<br />

10


INTRODUCTION<br />

In <strong>the</strong> following, I will give a general introduction into <strong>the</strong> biology <strong>of</strong> <strong>the</strong> <strong>European</strong> eel; give an<br />

introduction into <strong>the</strong> areas <strong>of</strong> fish physiology that have been <strong>the</strong> focus points <strong>of</strong> this PhD <strong>the</strong>sis, i.e.<br />

energetics, cardiac function and swimming performance; and provide a general overview <strong>of</strong> how<br />

environmental factors, namely temperature, dissolved oxygen and carbon dioxide, affect said<br />

physiological functions in fish. The main results <strong>of</strong> <strong>the</strong> attached publications/ manuscripts are also<br />

presented and discussed in relation to <strong>the</strong> current literature on <strong>the</strong> subject.<br />

1. THE EUROPEAN EEL (ANGUILLA ANGUILLA)<br />

1.1 Distribution and Phylogeny<br />

The <strong>European</strong> eel (Anguilla anguilla, Linneaus) can be found all over Europe and North Africa<br />

(Figure 1.1). To <strong>the</strong> north, <strong>the</strong>re are documented catches as far as <strong>the</strong> North Cape and in <strong>the</strong> North<br />

East as far as <strong>the</strong> Murmansk Coast and Kola Bay (Sorokin and Konstantinov, 1960). <strong>Eel</strong>s have been<br />

caught as far south as <strong>the</strong> coast <strong>of</strong> Morocco and <strong>the</strong> Canary Islands. The south eastern distribution<br />

limit reaches <strong>the</strong> Black Sea and <strong>the</strong> western limit as far as <strong>the</strong> Azores and Iceland (Schmidt, 1923).<br />

The <strong>European</strong> eel belongs to <strong>the</strong> genus Anguilla, which is a monophyletic group, comprised <strong>of</strong> 15<br />

species (Minegishi et al., 2005). The <strong>European</strong> eel is most closely related to American eel (Anguilla<br />

rostrata, LeSueur), that inhabits <strong>the</strong> western part <strong>of</strong> <strong>the</strong> North Atlantic. The common ancestor <strong>of</strong> <strong>the</strong><br />

A. anguilla and A. rostrata is believed to be <strong>the</strong> Japanese eel (Anguilla japonica) with <strong>the</strong> speciation<br />

event taking place around 3 million years ago by migration through <strong>the</strong> American Isthmus (Panama)<br />

(Lin et al., 2001).<br />

A. anguilla and A. rostrata are very closely related and <strong>the</strong> main morphological character that<br />

distinguishes <strong>the</strong> two Anguilla species is <strong>the</strong> number <strong>of</strong> vertebrae, ranging from 103 -110 in A.<br />

rostrata and from 110-119 in A. anguilla (Boëtius, 1980). However, variations in haemoglobins,<br />

transferrins and several allozymes have also been identified (Fine et al., 1967). The occurrences <strong>of</strong><br />

hybrids around Iceland have been documented in number <strong>of</strong> studies (Avise et al., 1990; Albert et<br />

al., 2006), which demonstrates that <strong>the</strong> two species are not completely isolated from each o<strong>the</strong>r in<br />

terms <strong>of</strong> reproduction. The population structure <strong>of</strong> <strong>the</strong> <strong>European</strong> eel has been studied since <strong>the</strong> late<br />

1960´s and some controversy exits regarding <strong>the</strong> observed heterogeneity or <strong>the</strong> differentiation<br />

within <strong>the</strong> species (van Ginneken and Maes, 2005). Observations on variations in allozymes led to<br />

<strong>the</strong> hypo<strong>the</strong>sis that A. anguilla reproduce randomly as one large population according to <strong>the</strong> concept<br />

11


<strong>of</strong> <strong>the</strong> panmixia. This was disputed later on by molecular genetic studies, which demonstrated subtle<br />

but distinct geographical genetic variations. Most recently, <strong>the</strong> hypo<strong>the</strong>sis <strong>of</strong> panmixia has gained<br />

new support from more extensive molecular studies (Pujolar et al., 2006; Maes et al 2006). These<br />

recent studies also demonstrate that genetic variation in <strong>the</strong> <strong>European</strong> eel is larger on <strong>the</strong> temporal<br />

scale than on <strong>the</strong> geographical scale. Based on several genetic and morphological characters within<br />

<strong>the</strong> arrivals <strong>of</strong> young eels (i.e. glass eels), it was observed that <strong>the</strong>re is considerable variation<br />

between <strong>the</strong> cohorts from different years, and even between recruitment waves within one cohort<br />

(Maes et al., 2009; Pujolar et al., 2009).<br />

Figure 1.1 Geographical distribution <strong>of</strong> <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla).<br />

The distribution is indicated by <strong>the</strong> dark shaded areas. Black line: trajectory <strong>of</strong> <strong>the</strong> drift <strong>of</strong> leptocephali larvae<br />

towards continental Europe based on a Lagrangian particle simulation (Bonhommeau et al., 2010). Markers<br />

represent pop-up positions <strong>of</strong> Pop-up satellite archival tags (PSAT´s) attached to silver eels that were<br />

released from Galway (Ireland) indicated by arrow (Aarestrup et al., 2009). Modified from Tesch (2003).<br />

1.2 Life History and Ecology<br />

The life cycle <strong>of</strong> <strong>the</strong> <strong>European</strong> eel is complex. It consists <strong>of</strong> many developmental stages which has<br />

fascinated scientists since ancient Greece. Anguilla anguilla is a catadromous semelparous species,<br />

living in freshwater, but returning to <strong>the</strong> ocean to spawn, with a single spawning migration during<br />

its life cycle. Owing to <strong>the</strong> dedicated effort <strong>of</strong> <strong>the</strong> Danish biologist Johannes Schmidt at <strong>the</strong><br />

beginning 20 th century, <strong>the</strong> spawning area <strong>of</strong> both <strong>the</strong> <strong>European</strong> eel and American eel was identified<br />

as <strong>the</strong> nor<strong>the</strong>rn Sargasso Sea in <strong>the</strong> Atlantic Ocean (Schmidt, 1923). He reached this conclusion<br />

after having backtracked migrating larvae, observing that as he went fur<strong>the</strong>r west, <strong>the</strong> larvae became<br />

progressively smaller. Consequently <strong>the</strong> smallest larvae were found in <strong>the</strong> Sargasso Sea. These<br />

larvae measured 5mm, which is not much larger than <strong>the</strong> 3-4 mm at time <strong>of</strong> hatching, making <strong>the</strong><br />

12


Sargasso <strong>the</strong> most likely site <strong>of</strong> spawning (Tesch, 2003). However since <strong>the</strong>re are no documented<br />

observations <strong>of</strong> spawning adults or eggs, this remains to be unequivocally proven.<br />

After hatching, eel larvae develop a leaf like shape and grow to about 70-80 mm in length (Figure<br />

1.2). These larvae are called lepthocephali as <strong>the</strong>y were originally thought to be a separate species<br />

<strong>of</strong> fish. The lepthocephali drift with <strong>the</strong> oceanic currents towards continental Europe (Bonhommeau<br />

et al., 2010; Munk et al., 2010), (Figure 1.1). The duration <strong>of</strong> this larval migration is still subject to<br />

some debate. Depending on <strong>the</strong> methods <strong>of</strong> determination, it has been estimated to be between 7-9<br />

months or as long as 2 years (Bonhommeau, 2010). Recently it was discovered, that lepthocephali<br />

larvae preferentially feed on gelatinous zooplankton (Riemann et al 2010). When <strong>the</strong>y reach <strong>the</strong><br />

continental slope, <strong>the</strong>y undergo <strong>the</strong> first metamorphosis into glass eels. They gradually attain <strong>the</strong><br />

slender eel shape, but still lack pigmentation and are <strong>the</strong>refore characteristically transparent. Glass<br />

eels enter rivers mouths and estuaries and during this migration towards inland water bodies, <strong>the</strong>y<br />

begin to develop <strong>the</strong> brown, yellow and dark green pigmentation characteristic <strong>of</strong> <strong>the</strong> continental or<br />

yellow stage eels. These early juvenile eels are also referred to as elvers. During <strong>the</strong> continental<br />

stage, eels inhabit streams, rivers, ponds, lakes, brackish as well as marine areas. This extensive<br />

range <strong>of</strong> habitats and large distribution area categorizes <strong>the</strong> <strong>European</strong> eel as both euryhaline and<br />

eury<strong>the</strong>rmal. They live near <strong>the</strong> bottom, under stones, in crevices and caves, and in muddy<br />

sediments. The diet <strong>of</strong> <strong>the</strong> yellow stage eel is quite diverse and consists <strong>of</strong> a wide range <strong>of</strong> benthic<br />

invertebrates, larvae, crustaceans and fish. During winter, eels in <strong>the</strong>ir natural environment reduce<br />

Figure 1.2 Ontogeny <strong>of</strong> <strong>European</strong> eel (A. anguilla)<br />

showing <strong>the</strong> major life stages<br />

Modified from Dekker (2000).<br />

or cease activity, become torpid and are<br />

known to bury <strong>the</strong>mselves in muddy<br />

sediments (Bruun, 1963). In <strong>the</strong> laboratory,<br />

eels stop feeding and reduce <strong>the</strong>ir activity<br />

below 8°C (Nyman, 1972). The duration <strong>of</strong><br />

<strong>the</strong> continental growth stage varies with <strong>the</strong><br />

geographical location and also differs between<br />

male and females. Males remain in <strong>the</strong> yellow<br />

stage until <strong>the</strong>y are between 2-15 years (body<br />

length 30-46 cm) and females until <strong>the</strong>y are<br />

between 4-20 years (body length 50-100 cm)<br />

(Tesch, 2003). At <strong>the</strong> end <strong>of</strong> <strong>the</strong> yellow phase,<br />

eels undergo a second metamorphosis where<br />

13


<strong>the</strong>y develop into silver -or migration stage eels, which is also <strong>the</strong> stage <strong>of</strong> sexual maturation. At<br />

this point eels cease to feed. The downstream spawning migration takes place from late spring to<br />

early winter, and is mainly governed by lunar phases. The knowledge <strong>of</strong> migration routes has so far<br />

been confined to <strong>the</strong> continental shelf (Tesch, 1978; 1989; 1995; Fricke 1995; McCleave 1999).<br />

However, a recent telemetry study successfully tracked eels as far as 1.027 Km from Galway,<br />

Ireland (Figure 1.1) (Aarestrup et al, 2009). In <strong>the</strong> same study, Aarestrup and co-workers (2009)<br />

also discovered that migrating eels perform diel vertical migrations <strong>of</strong> up to 400 meters.<br />

1.3 Morphological and Physiological Specializations<br />

Anguillid eels possess some unique morphological and physiological features among <strong>the</strong> teleosts.<br />

Some <strong>of</strong> <strong>the</strong>se characteristics allow <strong>the</strong> eel to survive in extreme environmental conditions like<br />

severe shortage <strong>of</strong> oxygen (hypoxia) and prolonged aerial exposure.<br />

1.3.1 Hypoxia exposure<br />

When <strong>the</strong> oxygen content <strong>of</strong> <strong>the</strong> surrounding water is reduced, <strong>the</strong> <strong>European</strong> eel makes <strong>respiratory</strong><br />

adjustments to maintain an adequate oxygen uptake, while at <strong>the</strong> same time reducing <strong>the</strong> amount <strong>of</strong><br />

oxygen required. To maintain an adequate supply <strong>of</strong> oxygen, A. anguilla increases <strong>the</strong> ventilatory<br />

volume (Chan 1986; Peyraud-Waitzeneeger and Soulier 1989; Cruz-Neto and Steffensen, 1997),<br />

which is a typical response observed in fish. To fur<strong>the</strong>r support oxygen supply, A. anguilla can<br />

effectively extract oxygen from <strong>the</strong> surrounding water as <strong>the</strong> O 2 -carrying molecules (haemoglobins)<br />

<strong>of</strong> A. anguilla are characterized by having a very high oxygen affinity (i.e. low P50) (Laursen et al.,<br />

1985). This enables eels to achieve blood oxygen saturation, even in water that is too O 2 -deficient<br />

for most o<strong>the</strong>r species. The <strong>European</strong> eel is even able to survive without oxygen in <strong>the</strong> water<br />

(anoxia) for a few hours (van Warde et al., 1983). This is possible because eels are able to suppress<br />

<strong>the</strong>ir oxygen demands. Experimentally exposing A. anguilla to anoxia, caused a 70% reduction in<br />

whole animal metabolic rate (van Ginneken et al., 2001). This large reduction in overall energy<br />

demands may partially result from a hypo-metabolic state in <strong>the</strong> liver as <strong>the</strong> energy (ATP)<br />

production was reduced by 85% in liver cells exposed to anoxia while <strong>the</strong> energy status was<br />

stabilized at a new lower level (Busk and Boutilier, 2005).<br />

1.3.2 Aerial exposure<br />

The ability to survive being out <strong>of</strong> water for hours to days must be considered extraordinary for a<br />

water breathing fish. Several features enable <strong>the</strong> <strong>European</strong> eel to survive aerial exposure. Unlike in<br />

14


most o<strong>the</strong>r teleost fishes, <strong>the</strong> opening <strong>of</strong> <strong>the</strong> operculum is reduced to a narrow vertical slit, which<br />

serves to keep <strong>the</strong> gills moist when out <strong>of</strong> water. The buccal cavity is filled with air and <strong>the</strong><br />

extraction <strong>of</strong> oxygen progresses at a slow rate (Berg and Steen, 1965). The skin is quite thick<br />

compared to o<strong>the</strong>r fish and has a considerable capacity for mucus secretion, which helps keep <strong>the</strong><br />

skin moist and prevents dehydration. Out <strong>of</strong> water, <strong>the</strong> skin is able to support its own O 2<br />

requirements by cutaneous oxygen uptake (Berg and Steen, 1965) which reduces <strong>the</strong> demand for<br />

brancial O 2 uptake (Le Moigne et al., 1986). Ano<strong>the</strong>r extraordinary feature is <strong>the</strong> pneumatic duct,<br />

which is <strong>the</strong> anterior part <strong>of</strong> <strong>the</strong> swimbladder. The pneumatic duct in eels is relatively large and has<br />

a re-absorptive function, meaning it can function as oxygen reservoir (Tesch 2003). As <strong>the</strong><br />

branchial gas exchange is restricted outside <strong>of</strong> water, <strong>the</strong>re will also be a gradual build up <strong>of</strong> CO 2 ,<br />

which causes an acidification <strong>of</strong> <strong>the</strong> blood (Hyde and Perry 1987; Hyde et al., 1987). Blood acidosis<br />

disturbs <strong>the</strong> function <strong>of</strong> <strong>the</strong> heart in fish (Driedzic and Gesser, 1994). However, <strong>the</strong> heart <strong>of</strong><br />

<strong>European</strong> eel is more resistant to acidosis compared to o<strong>the</strong>r fish (Nielsen and Gesser, 1984), which<br />

<strong>the</strong>n enables eels to survive aerial exposure.<br />

1.4 Silvering<br />

The metamorphosis from continental (yellow) eels to migrating (silver) eels represent a suite <strong>of</strong><br />

morphological and physiological changes that reflect <strong>the</strong> change from being sedentary and bottom<br />

dwelling to being active and pelagic, pre-adapt <strong>the</strong>m for life in <strong>the</strong> open ocean and enable <strong>the</strong>m to<br />

undertake <strong>the</strong> 5- 6000km oceanic journey back to <strong>the</strong> Sargasso Sea.<br />

Some noticeable external changes occur during silvering process, and <strong>the</strong>se are commonly used to<br />

identify silver stage eels. The pigmentation on <strong>the</strong> ventral side changes from yellow/ dark green to a<br />

lighter almost white coloration with a metallic hue, hence <strong>the</strong> name silver eels. The dorsal side<br />

darkens and this increases <strong>the</strong> contrast to <strong>the</strong> ventral side. This coloration is similar to <strong>the</strong> countershading<br />

<strong>of</strong> o<strong>the</strong>r pelagic species. The pectoral fins become elongated, <strong>the</strong> eye diameter increases,<br />

and <strong>the</strong> skin thickens. Significant changes also occur in <strong>the</strong> sensory systems. The yellow eel relies<br />

heavily on sense <strong>of</strong> smell and has an unprecedented olfactory sensitivity among fishes (Teichmann<br />

1959). However, this regresses in silver eels. In addition to <strong>the</strong> increase in eye size, <strong>the</strong> number <strong>of</strong><br />

rod cells increases and <strong>the</strong>re is a change <strong>of</strong> retinal pigments. Toge<strong>the</strong>r, <strong>the</strong>se changes improve <strong>the</strong><br />

absorption <strong>of</strong> light at greater depths <strong>of</strong> water (Durif et al., 2009). Ano<strong>the</strong>r indication that silver eels<br />

are adapted to <strong>the</strong> deep sea is a thickening <strong>of</strong> <strong>the</strong> swimbladder wall and an increased capacity for<br />

gas excretion. Migrating eels do not feed, and <strong>the</strong> alimentary tract degenerates, but its osmoregulatory<br />

function is retained. To fuel <strong>the</strong> trans-Atlantic journey silver eels have increased body fat<br />

15


deposits and have an average 20% body fat (Svedang and Wickstrom, 1997). During <strong>the</strong> silvering<br />

process in female eels <strong>the</strong> gonads begin to grow, but fully developed gonads have only been<br />

observed in eels after hormonal treatments. The migrating eels that leave <strong>the</strong> <strong>European</strong> shores can<br />

<strong>the</strong>refore be considered to be in a state <strong>of</strong> pre-puberty and only reach full sexual maturation<br />

somewhere along <strong>the</strong> oceanic journey (Rousseau et al 2009).<br />

2. ENERGETICS<br />

2.1 Partition <strong>of</strong> Energy<br />

Intake <strong>of</strong> energy is fundamental to growth and reproduction in all living organisms. The energy<br />

obtained from food is partitioned into all <strong>the</strong> different bodily processes e.g. muscle activity,<br />

biochemical reactions and moving molecules against gradients. This partitioning can be expressed<br />

by this basic energy balance equation (sec. Winberg, 1956; Elliot, 1976):<br />

I = (R s + R a + R f ) + (G s + G g ) + (E f + E u + E n )<br />

This states that <strong>the</strong> intake (I) <strong>of</strong> energy from ingested foods is distributed between <strong>the</strong> following<br />

fractions:<br />

1) Respiration i.e. standard metabolism (R s ), active metabolism (R a ), and feeding metabolism (R f ).<br />

2) Growth i.e. somatic growth (G s ) and reproductive growth (G g ).<br />

3) Excretion i.e. feces (E f ), urine (E u ) and ammonia (E n ).<br />

Standard metabolism (R s ) represents <strong>the</strong> minimal energy requirements to maintain bodily functions<br />

in a resting and unfed state. This includes ventilation, circulation, maintenance <strong>of</strong> ion gradients,<br />

protein repair, production <strong>of</strong> hormones etc. In o<strong>the</strong>r words, standard metabolism represents <strong>the</strong><br />

energy requirement <strong>of</strong> a fish that is nei<strong>the</strong>r in a state <strong>of</strong> growth nor degeneration. Active metabolism<br />

(R a ) represents <strong>the</strong> energy requirements that are above standard metabolism such as swimming and<br />

o<strong>the</strong>r activities. Feeding metabolism (R f ), also termed specific dynamic action (SDA), represents<br />

<strong>the</strong> energy required to process and assimilate food (see below). Finally, faeces represent <strong>the</strong> loss <strong>of</strong><br />

energy from food items that were not digested, and urine and ammonia represents <strong>the</strong> loss <strong>of</strong> energy<br />

from <strong>the</strong> breakdown <strong>of</strong> absorbed nutrients that were not assimilated (Brett and Groves, 1979).<br />

16


2.2 Nutrients and Energy Metabolism<br />

Quantifying foods in terms <strong>of</strong> energy units (Joule) is a convenient abstraction to use in bioenergetic<br />

models. However, energy is not a nutrient in itself, but a property <strong>of</strong> nutrients. In order to utilize <strong>the</strong><br />

energy contained in foods, it must be transferred to energy storing molecules (i.e. adenosine<br />

triphosphate, ATP) by <strong>the</strong> oxidation <strong>of</strong> <strong>the</strong> chemical bonds. The three main constituents <strong>of</strong> food<br />

(macronutrients) are proteins, lipids and carbohydrates. The difference in <strong>the</strong> type and number <strong>of</strong><br />

chemical bonds between <strong>the</strong>se nutrients gives a different yield <strong>of</strong> energy when metabolized. The<br />

mass specific energy yield <strong>of</strong> <strong>the</strong> macronutrients, when completely oxidized, is: 23.9kJ g -1 for<br />

proteins, 39.5kJ g -1 for lipids, and 17.5kJ g -1 for carbohydrates (Gnaiger and Bitterlich, 1984). Still,<br />

<strong>the</strong> energy yield does not relate to <strong>the</strong> quality <strong>of</strong> <strong>the</strong> food for <strong>the</strong> individual fish or a given species<br />

and depending on whe<strong>the</strong>r a fish is carnivorous, herbivorous, or omnivorous, it will have different<br />

nutritional requirements. Protein requirement <strong>of</strong> fish is high compared to o<strong>the</strong>r vertebrates and is<br />

also higher in carnivorous fish than in herbivorous or omnivorous species. As an example, <strong>the</strong><br />

optimum level <strong>of</strong> dietary protein has been estimated to be between 45-48% in eels (Anguilla spp.)<br />

that are carnivorous (Satoh, 2002), while it is between 30-35% in <strong>the</strong> omnivorous common carp<br />

(Cyprinus carpio) (Takeuchi et al., 2002). At <strong>the</strong> cellular level, <strong>the</strong> hydrolysis <strong>of</strong> ATP releases <strong>the</strong><br />

energy that can <strong>the</strong>n be used to perform <strong>the</strong> required metabolic or chemical work. Metabolism <strong>of</strong><br />

macronutrients is divided into: catabolism which refers to <strong>the</strong> breakdown <strong>of</strong> nutrients, and<br />

anabolism which refers to <strong>the</strong> syn<strong>the</strong>sis <strong>of</strong> e.g. proteins, lipids, waste products and more. Energy<br />

metabolism is <strong>the</strong>refore <strong>the</strong> sum <strong>of</strong> ATP consuming and ATP producing processes and metabolic<br />

rate is <strong>the</strong> turnover <strong>of</strong> ATP. The generation <strong>of</strong> ATP occurs ei<strong>the</strong>r aerobically through oxidative<br />

phosphorylation, or anaerobically through substrate-level phosphorylation. In oxidative<br />

phosphorylation, oxygen acts as <strong>the</strong> final electron acceptor in <strong>the</strong> electron transport chain and gives<br />

<strong>the</strong> highest yield. If <strong>the</strong>re is a shortage <strong>of</strong> oxygen, this pathway is inhibited and ATP production<br />

progresses via substrate-level phosphorylation with a lower yield (van den Thillart and van Raaij,<br />

1995).<br />

The use <strong>of</strong> lipids, proteins and carbohydrate as metabolic fuels depends on <strong>the</strong> metabolic state <strong>of</strong> <strong>the</strong><br />

fish. Thus whe<strong>the</strong>r <strong>the</strong> fish is resting, swimming at sustained speeds or maximum burst speed,<br />

starving or feeding, it utilizes <strong>the</strong>se substrates in varying proportions (Wood, 2001). For instance in<br />

resting non-fed rainbow trout (Oncorhynchus mykiss), lipid is <strong>the</strong> predominately used metabolic<br />

substrate (37-68%) followed by carbohydrate (20-37%), while proteins are used to a smaller degree<br />

(16-24%) (Lauff and Wood, 1996a). On <strong>the</strong> o<strong>the</strong>r hand, when fish are digesting a meal, <strong>the</strong><br />

17


oxidation <strong>of</strong> proteins increases especially if <strong>the</strong> meal is large and has a high protein content (Wood,<br />

2001). In rainbow trout performing aerobic exercise, lipid remains <strong>the</strong> main fuel source, but <strong>the</strong><br />

oxidation <strong>of</strong> carbohydrates increases when swimming at higher velocities (Lauff and Wood, 1996b).<br />

During exhaustive swimming, <strong>the</strong> employment <strong>of</strong> white muscle means an increased use <strong>of</strong><br />

endogenous carbohydrate (glycogen) as metabolic fuel (van den Thillart and van Raaij, 1995; Liew<br />

et al., 2012). Fur<strong>the</strong>rmore, environmental factors like temperature and dissolved oxygen (DO) can<br />

affect <strong>the</strong> use <strong>of</strong> metabolic substrates, with an increased use <strong>of</strong> carbohydrates at low temperatures<br />

(Kieffer et al., 1998; Alsop et al., 1999). Under conditions <strong>of</strong> low DO (hypoxia) or no O 2 (anoxia),<br />

ATP production predominately occurs by substrate-level phosphorylation with <strong>the</strong> use <strong>of</strong><br />

endogenous glycogen and high-energy phosphates (i.e. phosphocreatine, pCR) as substrates (van<br />

den Thillart and van Raaij, 1995).<br />

Metabolic rate<br />

2.2.2 Feeding Metabolism<br />

In all animals including fish, metabolic rate increases after ingesting a meal (Secor, 2009). This<br />

post-prandial increase in metabolic rate is most commonly referred to as specific dynamic action<br />

(SDA). Numerous physiological processes have been hypo<strong>the</strong>sized to contribute to SDA. These<br />

processes can be divided into pre-absorptive, absorptive and post-absorptive processes. Preabsorptive<br />

processes include: secretion <strong>of</strong> acid and digestive enzymes, peristalsis <strong>of</strong> <strong>the</strong> alimentary<br />

canal, protein catabolism and blood pH regulation. The post-absorptive processes include: amino<br />

acid deamination and oxidation, anabolic processes i.e. protein syn<strong>the</strong>sis, and syn<strong>the</strong>sis and<br />

excretion <strong>of</strong> urea (McCue, 2006). It has become <strong>the</strong> general consensus that protein syn<strong>the</strong>sis,<br />

Figure 2.1 Hypo<strong>the</strong>tical post-prandial metabolic pr<strong>of</strong>ile<br />

Scope<br />

Meal ingested<br />

Peak<br />

SDA<br />

Time to peak<br />

Standard metabolic rate<br />

Time post-feeding<br />

Duration<br />

Metabolic rate pre –and post feeding plotted against time. Commonly<br />

quantified parameters <strong>of</strong> <strong>the</strong> SDA pr<strong>of</strong>ile are indicated. Modified from<br />

Secor (2009).<br />

protein turnover and growth<br />

represent <strong>the</strong> largest<br />

components <strong>of</strong> <strong>the</strong> SDA<br />

response (Brown and Cameron,<br />

1991; McCue, 2006; Secor,<br />

2009). A generalized SDA<br />

response begins with a rapid<br />

increase in metabolic rate<br />

(Figure 2.1). After a varying<br />

amount <strong>of</strong> time, a peak is<br />

reached and metabolic rate begins<br />

18


to slowly decrease. The point where metabolic rate is no longer discernible from SMR marks <strong>the</strong><br />

end <strong>of</strong> <strong>the</strong> SDA response (Figure 2.1). The magnitude <strong>of</strong> <strong>the</strong> SDA response is quantified as <strong>the</strong> total<br />

energy expenditure above SMR for <strong>the</strong> duration <strong>of</strong> <strong>the</strong> response. Additional parameters <strong>of</strong> <strong>the</strong> SDA<br />

pr<strong>of</strong>ile typically quantified include: <strong>the</strong> postprandial metabolic peak, <strong>the</strong> scope <strong>of</strong> <strong>the</strong> peak, time to<br />

<strong>the</strong> peak and total duration. The SDA coefficient (C SDA ) expresses <strong>the</strong> amount <strong>of</strong> energy that is<br />

expended on digesting a meal relative to <strong>the</strong> energy content <strong>of</strong> that meal (Wea<strong>the</strong>rley 1976). The<br />

SDA coefficient is a useful parameter, as it allows for both inter -and intraspecific comparisons<br />

(Jobling, 1981).<br />

Some <strong>of</strong> <strong>the</strong> factors that are known to influence <strong>the</strong> SDA response pertain both to <strong>the</strong> meal, to <strong>the</strong><br />

state <strong>of</strong> <strong>the</strong> animal, and to environmental conditions. Increased meal size increases both <strong>the</strong> SDA<br />

duration and magnitude (Jobling and Davies, 1980; Fu et al., 2005a; Jordan and Steffensen, 2006;<br />

Wang et al., 2012). Diets high in protein have been observed to increase SDA (Beamish and<br />

Trippel, 1990; Ross et al, 1992; Fu et al., 2005b), but not in all species (Peres and Oliva-Teles,<br />

2001). In <strong>the</strong> aquaculture industry, it is <strong>of</strong> great importance that C SDA is minimized, as to avoid<br />

unnecessary waste <strong>of</strong> expensive feed and to maximize growth. Both <strong>the</strong> size <strong>of</strong> a meal and <strong>the</strong><br />

dietary protein content has been shown to be determinant <strong>of</strong> C SDA (Boyce and Clarke, 1997;<br />

Beamish and Trippel, 1990). The current emphasis is put on composition <strong>of</strong> <strong>the</strong> diet i.e. <strong>the</strong> relative<br />

amounts <strong>of</strong> proteins, lipids and carbohydrates, as a significant determinant <strong>of</strong> SDA (Secor, 2009).<br />

Body size may also influence SDA. The postprandial metabolic peak has been found to increase<br />

with body mass (Sims and Davies 1994; Boyce and Clarke, 1997), which is predictable considering<br />

<strong>the</strong> increase in absolute metabolism with increased mass. Increased body mass may prolong <strong>the</strong><br />

SDA response or have no effect, depending on species (Boyce and Clarke, 1997; Hunt von Herbing<br />

and White, 2002). The major environmental determinant <strong>of</strong> SDA is temperature, which is also <strong>the</strong><br />

most studied, while <strong>the</strong> effects <strong>of</strong> dissolved gasses (O 2 and CO 2 ) on SDA in fish have only received<br />

little attention (Secor, 2009). The effects <strong>of</strong> temperature, O 2 and CO 2 on post-prandial metabolism<br />

are fur<strong>the</strong>r discussed in chapters 5, 6 and 7.<br />

2.3 Measuring Metabolic Rate<br />

Metabolic rate is <strong>the</strong> turnover <strong>of</strong> ATP, but this cannot be measured in vivo, <strong>the</strong>refore one must<br />

resort to indirect measures. Heat production is directly proportional to ATP turnover and is <strong>the</strong> most<br />

precise measure, as it includes both aerobic and anaerobic processes. Whole animal oxygen<br />

consumption is however <strong>the</strong> most widely applied measure <strong>of</strong> metabolic rate in fish. Intermittent<br />

flow-through respirometry is one <strong>of</strong> <strong>the</strong> experimental approaches to measuring oxygen consumption<br />

19


in fish is. The principle <strong>of</strong> this technique is to measure <strong>the</strong> decrease in oxygen partial pressure (PO 2 )<br />

inside a closed respirometer holding a fish, and <strong>the</strong>n periodically flush <strong>the</strong> respirometer with water<br />

from <strong>the</strong> outside (Steffensen, 1989). This allows for several short and separate measurements <strong>of</strong><br />

mass specific oxygen consumption rate (MO 2 ) that can be derived from <strong>the</strong> following equation:<br />

MO 2 = V (dPO 2 /dt) α M -1<br />

where V is volume <strong>of</strong> <strong>the</strong> respirometer minus <strong>the</strong> volume <strong>of</strong> <strong>the</strong> fish, dPO 2 /dt is <strong>the</strong> decrease in PO 2<br />

within <strong>the</strong> set time (measurement period), α is oxygen solubility under <strong>the</strong> given conditions and M<br />

is <strong>the</strong> wet weight <strong>of</strong> <strong>the</strong> fish. The advantages <strong>of</strong> this technique is that <strong>the</strong>re is no build-up <strong>of</strong><br />

metabolic waste products, that <strong>the</strong>re is adequate oxygenation inside <strong>the</strong> respirometer chamber and<br />

sudden outbursts <strong>of</strong> activity are less likely to influence <strong>the</strong> overall measurement <strong>of</strong> metabolic rate.<br />

As <strong>the</strong> quality <strong>of</strong> <strong>the</strong> measurements depends on <strong>the</strong> steepness <strong>of</strong> <strong>the</strong> slope (dPO 2 /dt), it is important<br />

to choose <strong>the</strong> right respirometer to fish volume ratio, taking into account how much oxygen <strong>the</strong> fish<br />

consumes which in turns depends on activity levels and on temperature (see chapter 5). Standard<br />

metabolic rate (SMR) is measured while <strong>the</strong> fish is in a resting, non-feeding and non-reproducing<br />

state. Using intermittent flow-through respirometry reduces <strong>the</strong> risk <strong>of</strong> overestimating SMR due to<br />

sudden activity. Active metabolic rate (AMR) corresponds to <strong>the</strong> state where oxygen uptake is<br />

maximized and <strong>the</strong> capacities <strong>of</strong> <strong>the</strong> ventilatory and circulatory systems are fully exploited. The<br />

AMR <strong>of</strong> a fish can be measured by forcing a fish to swim at <strong>the</strong> maximum (aerobic) sustained<br />

swimming speed (see chapter 4) in a swimming respirometer. In some fish, <strong>the</strong> maximum oxygen<br />

uptake (MMR) occurs when recovering from exhaustive exercise (Schurmann and Steffensen,<br />

1997) and can be measured after chasing a fish to exhaustion. In some fish, MMR occurs when<br />

swimming at high speed while digesting a meal (Jourdan-Pineau et al., 2010). Routine metabolic<br />

rate (RMR) represents <strong>the</strong> various states <strong>of</strong> activity that lie between SMR and MMR. This includes<br />

moderate swimming activity and feeding (SDA).<br />

3. THE FISH HEART<br />

3.1 Anatomy<br />

The teleost heart consists <strong>of</strong> four chambers that are arranged in series. The heart is surrounded by a<br />

double membranous sac – <strong>the</strong> pericardium. The outer pericardial membrane adheres to <strong>the</strong><br />

surrounding tissues, and <strong>the</strong> space between <strong>the</strong> two membranes is filled with pericardial fluid. The<br />

venous blood enters <strong>the</strong> heart in <strong>the</strong> sinus venosus and is directed to <strong>the</strong> atrium through an opening<br />

20


guarded by <strong>the</strong> sinoatrial valve. From <strong>the</strong> atrium <strong>the</strong> blood is pumped into <strong>the</strong> ventricle and from <strong>the</strong><br />

ventricle into <strong>the</strong> bulbus arteriosus. The atrium and <strong>the</strong> ventricle are muscular contracting chambers<br />

and propel <strong>the</strong> blood through <strong>the</strong> vasculature. The sinus venosus and <strong>the</strong> bulbus arteriosus are<br />

cavities, which collects <strong>the</strong> blood and connects <strong>the</strong> heart to <strong>the</strong> vasculature (Figure 3.1). The bulbus<br />

arteriosus is highly compliant, dampens peak pressure and maintains a more constant flow. The<br />

opening between <strong>the</strong> ventricle and bulbus arteriosus is guarded by <strong>the</strong> bulboventricular valve that<br />

prevents <strong>the</strong> blood from flowing backwards into <strong>the</strong> ventricle (Santer, 1985; Olson and Farrell<br />

2006). Blood leaving <strong>the</strong> bulbus arteriosus, enters <strong>the</strong> ventral aorta, passes through <strong>the</strong> gills into <strong>the</strong><br />

dorsal aorta and from <strong>the</strong>re on to <strong>the</strong> rest <strong>of</strong> <strong>the</strong> body. Two different cardiac tissues can be discerned<br />

based on <strong>the</strong> arrangement <strong>of</strong> muscle fibres (cardiomyocytes), a spongy and a compact layer. In <strong>the</strong><br />

spongiosum, <strong>the</strong> muscle fibres are arranged in a mesh-like structure, which greatly increases <strong>the</strong><br />

Figure 3.1 Scanning electron micrograph <strong>of</strong> <strong>the</strong> heart from<br />

<strong>the</strong> Antarctic noto<strong>the</strong>nioid, Noto<strong>the</strong>nia coriiceps shown in<br />

cross section<br />

The heart consists <strong>of</strong> four chambers: <strong>the</strong> sinus venosus (not visible),<br />

<strong>the</strong> atrium (AT), <strong>the</strong> ventricle (V), and <strong>the</strong> bulbus arteriosus (BA).<br />

AV, atrio-venticular valve; BV, bulbo-ventricular valve. Arrow<br />

indicates blood flow. See text for details. Photograph by P.V. Skov.<br />

surface area that comes in<br />

contact with <strong>the</strong> blood. The<br />

compact myocardium (compacta)<br />

consists <strong>of</strong> circumferentially<br />

arranged<br />

cardiomyocytes<br />

encasing <strong>the</strong> spongiosum (Figure<br />

3.2). Oxygen supply to <strong>the</strong><br />

compact myocardium is achieved<br />

by a coronary circulation,<br />

derived ei<strong>the</strong>r from <strong>the</strong> efferent<br />

branchial vessels or a branch<br />

from <strong>the</strong> subclavian arteries. This<br />

ensures that <strong>the</strong> cardiomyocytes<br />

are supplied with oxygenated<br />

arterial blood, which o<strong>the</strong>rwise<br />

depends solely on <strong>the</strong> venous<br />

reserve <strong>of</strong> O 2 . A compact myocardium and concomitant coronary circulation is not present in all<br />

species, but occurs mostly in <strong>the</strong> more athletic species where a high cardiac performance is<br />

demanded (Farrell, 1991). In highly active species like tunas and sharks, <strong>the</strong> coronary arteries are<br />

also present in <strong>the</strong> spongiosum and to some extent in <strong>the</strong> atrium (Olson and Farrell, 2006). Based on<br />

<strong>the</strong>se anatomical features, four different types <strong>of</strong> ventricles have been characterized (Figure 3.2):<br />

21


The Type I ventricle consists solely <strong>of</strong> a spongy myocardium (spongiosum), lacks a compact layer<br />

(compacta) and a coronary circulation. The Type II ventricle consists <strong>of</strong> a spongiosum encased by a<br />

capillarized compacta. The Type III ventricle similarly consists <strong>of</strong> an inner spongiosum and an<br />

outer compacta, but in this type, capillaries are also present in <strong>the</strong> spongiosum. The type IV<br />

ventricle resembles <strong>the</strong> Type III but is characterized by having a relatively larger compacta and a<br />

more extensively capillarized atrium.<br />

Figure 3.2 Schematic representation <strong>of</strong><br />

<strong>the</strong> different types <strong>of</strong> ventricles in fish.<br />

The Type I ventricle consists solely <strong>of</strong> a spongy<br />

myocardium (spongiosum). The Type II<br />

ventricle consists <strong>of</strong> an inner spongiosum<br />

encased by an outer compact myocardium<br />

(compacta) with a coronary blood supply. The<br />

Type III ventricle consists <strong>of</strong> a spongiosum and<br />

a compacta, both capillarized. In <strong>the</strong> Type Iv<br />

ventricle, <strong>the</strong> compacta is relatively larger and<br />

<strong>the</strong> atrium receives coronary blood supply. See<br />

text for fur<strong>the</strong>r details. From Farrell and Jones<br />

(1992).<br />

3.2 Cardiac Function<br />

The function <strong>of</strong> <strong>the</strong> heart is to maintain a continuous circulation <strong>of</strong> blood through <strong>the</strong> vasculature.<br />

This ensures <strong>the</strong> supply <strong>of</strong> oxygen and nutrients to all active tissues, <strong>the</strong> removal <strong>of</strong> metabolic waste<br />

products and <strong>the</strong> transport <strong>of</strong> hormones and chemical signal molecules. In order to fulfil this<br />

function, <strong>the</strong> heart has to perform contractions at regular intervals. The heartbeat is initiated by a<br />

spontaneous electrical excitation <strong>of</strong> specialized pacemaker cells, located in <strong>the</strong> sinus venosus or<br />

sinoatrial junction. The excitation spreads to <strong>the</strong> atrium and subsequently to <strong>the</strong> ventricle causing<br />

<strong>the</strong>m to perform rhythmical contractions. The excitation event (i.e. <strong>the</strong> action potential, AP) is a<br />

series <strong>of</strong> ion fluxes across <strong>the</strong> cell membrane (sarcolemma, SL). The AP consists <strong>of</strong> a depolarizing<br />

phase followed by a repolarizing phase (Voranen et al., 2002; Behrs, 2002). The coupling between<br />

excitation and contraction (E-C coupling) begins with <strong>the</strong> depolarization event. This causes <strong>the</strong><br />

opening <strong>of</strong> Ca 2+ channels in <strong>the</strong> SL and a flux <strong>of</strong> Ca 2+ ions into <strong>the</strong> cell (Figure 3.3). Repolarization<br />

22


causes <strong>the</strong> Ca 2+ channels to close and <strong>the</strong> Ca 2+ ions to be transported back out <strong>of</strong> <strong>the</strong> cell via <strong>the</strong><br />

Na + /Ca 2+ exchanger (NCX). The source <strong>of</strong> Ca 2+ needed to activate <strong>the</strong> contractile apparatus<br />

(my<strong>of</strong>ibrils) can be both extra -and intracellular. In fish cardiomyocytes, <strong>the</strong> activator Ca 2+ is<br />

primarily <strong>of</strong> extracellular origin (Shiels et al., 2002). The majority <strong>of</strong> extracellular Ca 2+ enters <strong>the</strong><br />

cell via <strong>the</strong> L-type Ca 2+ channels, but some may enter through <strong>the</strong> NCX operating in reverse during<br />

<strong>the</strong> depolarizing phase (Shiels et al., 2002, Vornanen 1999, Paper II) (Figure 3.2). Ano<strong>the</strong>r mode<br />

<strong>of</strong> SL entry, termed store operated calcium entry (SOCE) has so far only been described in<br />

mammalian cardiomyocytes (Huang et al., 2006), but it may also play a role in <strong>the</strong> E-C coupling <strong>of</strong><br />

fish cardiomyocytes as demonstrated in Paper II). The release <strong>of</strong> Ca 2+ from intracellular stores is a<br />

secondary mechanism termed calcium induced calcium release (CICR) (Figure 3.3). The increase in<br />

free Ca 2+ activates receptors (ryanodine receptors, RyR) in <strong>the</strong> sarcoplasmatic reticulum (SR),<br />

which causes <strong>the</strong> SR to release Ca 2+ . During <strong>the</strong> relaxation process, Ca 2+ transporters located on <strong>the</strong><br />

SR membrane (SERCA) removes some <strong>of</strong> <strong>the</strong> free Ca 2+ by transporting it back into <strong>the</strong> SR (Tiitu<br />

and Vornanen, 2003)(Figure 3.3).<br />

Figure 3.3 Schematic representation <strong>of</strong><br />

Ca 2+ cycling in a fish cardiomyocyte<br />

during depolarization and repolarization<br />

Ca 2+ enters <strong>the</strong> cell through L-type channels<br />

and reverse mode Na + /Ca 2+ exchange (NCX) in<br />

<strong>the</strong> cell membrane (sarcolemma, SL. (Possibly<br />

also through SOCE). Ca 2+ can also be released<br />

from internal stores (sarcoplasmatic reticulum,<br />

SR) through calcium induced calcium release<br />

(CICR) activating ryanodine receptors (RyR).<br />

Ca 2+ is transported back out <strong>of</strong> <strong>the</strong> cell through<br />

<strong>the</strong> NCX and back into <strong>the</strong> SR through<br />

SERCA.MF, my<strong>of</strong>ibrils. See text for fur<strong>the</strong>r<br />

details. Modified from Vornanen et al. (2002).<br />

3.3 Cardiac Performance and Control<br />

Whenever <strong>the</strong>re is an increase in metabolic activity, e.g. when swimming or feeding, <strong>the</strong>re is an<br />

increased demand for oxygen to be delivered to <strong>the</strong> working tissues. To meet <strong>the</strong>se demands, <strong>the</strong><br />

heart must increase its performance. The measure <strong>of</strong> cardiac performance is cardiac output, which is<br />

<strong>the</strong> volume <strong>of</strong> blood expelled per unit <strong>of</strong> time. Cardiac output is thus determined by two factors,<br />

heart rate and stroke volume. As an example, exercising rainbow trout (Oncorhynchus mykiss)<br />

23


consumes 7.5 times more oxygen than when resting and to support <strong>the</strong> swimming musculature,<br />

heart rate is 1.6 times higher and cardiac output 3 times higher than at rest (Kiceniuk and Jones,<br />

1977). Heart rate is dictated by <strong>the</strong> intrinsic pacemaker rate, but can be modulated by extrinsic<br />

factors. These factors are ei<strong>the</strong>r blood borne substances like adrenaline, or direct innervations <strong>of</strong> <strong>the</strong><br />

autonomous nervous system. Autonomous control <strong>of</strong> heart rate is both inhibitory and excitatory<br />

(Taylor et al., 1999). For instance, lowering <strong>of</strong> <strong>the</strong> heart rate (bradycardia) is observed in some<br />

species exposed to low environmental O 2 (Farrell 2007) and is achieved by increasing <strong>the</strong><br />

cholinergic inhibitory tonus (See chapter 6). An increase in heart rate (tachycardia), for instance<br />

during swimming (Kiceniuk and Jones 1977) is achieved by ei<strong>the</strong>r decreasing <strong>the</strong> cholinergic<br />

inhibitory tonus or in some species by increasing <strong>the</strong> stimulatory adrenergic tonus (Farrell and Jones<br />

1992; Nilsson, 1983). Stroke volume is directly related to <strong>the</strong> end-diastolic volume and <strong>the</strong><br />

ventricular contractility. Contractility is defined as <strong>the</strong> force by which a muscle fibre can contract at<br />

a given length (Olson and Farrell 2006). The contractility <strong>of</strong> muscle fibres decreases when forced to<br />

contract at higher frequencies (Shiels et al.,2002; Paper II).This negative force-frequency response<br />

is partly caused by a frequency-dependent mismatch in <strong>the</strong> beat-to-beat Ca 2+ cycling (Shiels et al.,<br />

2002).<br />

Adrenaline (AD) can act as modulator <strong>of</strong> cardiac contractility, and thus cardiac performance which<br />

has been demonstrated in several species (Shiels et al., 2002, Skov et al., 2009; Paper II).<br />

Stimulation <strong>of</strong> β-adrenoceptors, associated with <strong>the</strong> L-type Ca 2+ channels in <strong>the</strong> SL, causes <strong>the</strong><br />

channels to stay open for an extended time. An increased adrenergic tonus can <strong>the</strong>refore cause a<br />

positive inotropic response (i.e. increase in force) by increasing <strong>the</strong> amount <strong>of</strong> Ca 2+ entering <strong>the</strong> cell<br />

(Vornanen ,1998;Bers, 2002). Besides increasing <strong>the</strong> Ca 2+ delivery, adrenergic stimulation can also<br />

increase <strong>the</strong> rate <strong>of</strong> recovery i.e. <strong>the</strong> Ca 2+ removal (Gesser, 1996). In summary, adrenergic<br />

stimulation can cause stronger and faster contractions to support a heightened cardiac performance.<br />

Thus increased levels <strong>of</strong> AD have been observed in plasma <strong>of</strong> exercising fish (Olson and Farrell<br />

2006). However, <strong>the</strong> effect <strong>of</strong> AD on cardiac performance may be more complex and depend on <strong>the</strong><br />

type <strong>of</strong> receptors that are expressed. For example in <strong>European</strong> eel (A. anguilla), AD causes<br />

bradycardia during winter when <strong>the</strong>re is a predominance <strong>of</strong> α-adrenoceptors, but tachycardia during<br />

summer, when <strong>the</strong>re is a predominance <strong>of</strong> β-adrenoceptors (Pennec and Peyraud ,1983;Peyraud-<br />

Waitzenegger et al. 1980). Also in A. anguilla, stimulation <strong>of</strong> a β-adrenoceptor subtype (β3-<br />

adrenoceptor) causes a negative inotropic response (Imbrogno et al., 2006).<br />

24


4. SWIMMING AND MIGRATION<br />

4.1 Why Fish Migrate<br />

In order to grow and reproduce, fishes must select an appropriate habitat, which, among o<strong>the</strong>rs, is<br />

optimal in terms <strong>of</strong> food quality and availability, temperature, and dissolved oxygen. Since aquatic<br />

environments are rarely stable in any or all <strong>of</strong> <strong>the</strong>se parameters, fishes must move from one place to<br />

ano<strong>the</strong>r in order to maximize growth and fitness. The habitats <strong>of</strong> fishes can be divided into<br />

functional types. Habitats that are suitable for feeding, habitats that <strong>of</strong>fer refuge from unfavourable<br />

conditions and habitats that are suitable for reproduction. The movements between <strong>the</strong>se types <strong>of</strong><br />

habitats can thus be categorized as, feeding migration, refuge migration and spawning migration<br />

(Northcote, 1978). The spatial distance between <strong>the</strong> types <strong>of</strong> habitats varies greatly. A fish may<br />

move a few meters up stream to a preferred temperature, or it may swim several thousand<br />

kilometres across <strong>the</strong> ocean to spawn. The type <strong>of</strong> migration <strong>the</strong>refore places different demands on<br />

<strong>the</strong> physiology and nutritional status <strong>of</strong> <strong>the</strong> individual fish.<br />

4.2 Capacity for Migration<br />

4.2.1 Swimming Performance<br />

Swimming speed and endurance are inversely correlated. At low swimming speeds, endurance<br />

depends on available energy stores for aerobic metabolism and at higher speeds, <strong>the</strong> metabolic<br />

capacity for anaerobic metabolism. The range <strong>of</strong> speeds by which a fish can propel itself has been<br />

divided into three categories (Brett 1964). Sustained swimming speeds refer to <strong>the</strong> range <strong>of</strong> speeds<br />

during which only aerobic red muscle is recruited. Fish can in <strong>the</strong>ory sustain <strong>the</strong>se speeds<br />

indefinitely, provided that dietary energy is available or that energy stores are sufficient. Beyond <strong>the</strong><br />

maximum sustained swimming speed (U MS ), fish begin to recruit anaerobic white muscle and as<br />

speed increases more and more white muscle is recruited. This range <strong>of</strong> speeds is termed prolonged<br />

swimming speeds. Fish can maintain prolonged swimming speeds until white muscle energy stores<br />

and metabolic substrates are depleted. The maximum prolonged swimming speed (U MP )<br />

corresponds to <strong>the</strong> speed where <strong>the</strong> oxygen uptake is maximized. The range <strong>of</strong> speeds above U MP is<br />

referred to as burst speeds. The maximum burst speed U MP is where endurance is minimal i.e.<br />

seconds or less. When experimentally forcing a fish to swim at increasing velocities i.e. by<br />

incremental increases in water velocity in an annular tank or a flume, <strong>the</strong> fish will fatigue at a point<br />

termed <strong>the</strong> critical swimming speed (U CRIT ) (Beamish 1978). U CRIT is a widely used measure <strong>of</strong><br />

prolonged swimming performance, as it is relatively easy to measure. However, U CRIT will also<br />

25


depend on how well a fish is able to swim under <strong>the</strong> experimental conditions and is <strong>of</strong> limited value<br />

for fish that are not motivated or able to swim steadily at high speeds.<br />

4.2.2 Cost <strong>of</strong> Swimming<br />

When a fish begins to swim faster, energy expenditure is increased. The metabolic rate during<br />

swimming is termed active metabolic rate (AMR). Active metabolic rate can <strong>the</strong>refore be described<br />

as a function <strong>of</strong> swimming speed (U) according to:<br />

AMR = a + bU x<br />

where a, b are constants and a is an estimate <strong>of</strong> <strong>the</strong> standard metabolic rate (SMR) (Videler, 1993).<br />

This power curve is commonly constructed by measuring <strong>the</strong> oxygen consumption at increasing<br />

velocities until U CRIT Figure 4.1A). The metabolic cost <strong>of</strong> swimming per unit distance (cost <strong>of</strong><br />

transport, COT) is given by <strong>the</strong> ratio <strong>of</strong> AMR over U:<br />

COT = AMR U -1<br />

The cost <strong>of</strong> transport is high at low speeds because <strong>of</strong> <strong>the</strong> time required to cover a distance and<br />

because SMR represents a large part <strong>of</strong> <strong>the</strong> total energy expenditure that is not used for swimming.<br />

Conversely, <strong>the</strong>re is an increased cost <strong>of</strong> swimming at high speeds, due to increased drag. The<br />

optimum swimming speed (U OPT ) is defined as <strong>the</strong> speed where <strong>the</strong> energetic cost <strong>of</strong> swimming is<br />

lowest and lies in <strong>the</strong> range <strong>of</strong> sustained speeds (Brett and Groves, 1979). Ma<strong>the</strong>matically U OPT can<br />

MO 2<br />

(mgO 2<br />

kg -1 h -1 )<br />

COT (mgO 2<br />

kg -1 km -1 )<br />

160<br />

120<br />

80<br />

40<br />

0<br />

80<br />

70<br />

60<br />

50<br />

40<br />

30<br />

A<br />

B<br />

SMR<br />

COT MIN<br />

U OPT<br />

U CRIT<br />

0.2 0.4 0.6 0.8 1.0<br />

Swimming speed (Bl s -1 )<br />

be expressed as <strong>the</strong> speed where COT is minimal<br />

(Figure 4.1B). For a migrating fish, cost <strong>of</strong> transport<br />

(COT) determines <strong>the</strong> distance that can be covered<br />

by <strong>the</strong> energy that is available. Thus having a low<br />

cost <strong>of</strong> transport infers being able to swim a long<br />

distance.<br />

Figure 4.1 Swimming energetics <strong>of</strong> <strong>European</strong> eel<br />

(A. anguilla) subjected to a U CRIT swim test.<br />

A: Mass specific oxygen consumption rate as a function<br />

<strong>of</strong> swimming speed according to: AMR = a + bU x ,<br />

where a and b are constants and a is an estimate <strong>of</strong> <strong>the</strong><br />

standard metabolic rate (SMR). Arrows indicate <strong>the</strong><br />

estimated SMR and <strong>the</strong> critical swimming speed, U CRIT<br />

B: Cost <strong>of</strong> transport (COT) calculated as COT = AMR<br />

U -1 , where U is swimming speed. Arrows indicate <strong>the</strong><br />

optimal swimming speed (U OPT ), where COT is minimal<br />

(COT MIN ). See Paper IV for fur<strong>the</strong>r details.<br />

26


It can be assumed that fish migrate at <strong>the</strong> most efficient speed (U OPT ) i.e. where COT is minimum.<br />

The <strong>European</strong> eel (A. anguilla) has one <strong>of</strong> <strong>the</strong> longest spawning migrations <strong>of</strong> any known species <strong>of</strong><br />

fish and must swim 5-6000km to reach <strong>the</strong> spawning site. In Paper IV, <strong>the</strong> swimming efficiency <strong>of</strong><br />

<strong>European</strong> eel was assessed by subjecting eels to a swim test, where water velocity was gradually<br />

increased to U CRIT . The optimal swimming speed was estimated to be 0.6 BL s -1 (body lengths per<br />

second) corresponding to 1.3 km h -1 . The minimum cost <strong>of</strong> transport (COT MIN ) <strong>of</strong> an eel weighing 1<br />

kg was estimated to be 0.57 kJ km -1 (Paper IV). A very similar result was obtained by Van<br />

Ginneken and co-workers (2005), who succeeded in making eels swim continuously at 0.5 body<br />

lengths per second for 173 days. Van Ginneken and co-workers estimated that <strong>the</strong> cost <strong>of</strong> transport<br />

at that speed was 0.62 kJ km -1 for a 1 kg eel. Toge<strong>the</strong>r <strong>the</strong>se studies demonstrate that <strong>the</strong> minimum<br />

cost <strong>of</strong> transport in <strong>European</strong> eel is very low -that is <strong>the</strong>y are very efficient swimmers from an<br />

energy conserving perspective. In comparison, different species <strong>of</strong> Pacific salmon (Oncorhynchus<br />

spp) make a spawning migration <strong>of</strong> about 1000 km up <strong>the</strong> Fraser River in British Columbia. The<br />

minimum cost <strong>of</strong> transport in <strong>the</strong>se salmonids is between 1.4- 2.1 kJ km -1 for a 1kg fish, with an<br />

optimum swimming speed <strong>of</strong> 0.8-1.2 BL s -1 (Lee et al., 2003a). Thus COT MIN is about 2-4 times<br />

higher in Pacific salmon (Oncorhynchus spp) than in <strong>European</strong> eel (A. anguilla). Precisely what<br />

makes eels more efficient swimmers than salmonids in terms <strong>of</strong> energy saving is not fully obvious.<br />

In fact, <strong>the</strong> propulsion efficiency <strong>of</strong> eels, using undulatations <strong>of</strong> a large part <strong>of</strong> <strong>the</strong>ir body for<br />

swimming (termed anguilliform swimming), is suggested to be less than in fish (e.g. salmonids) that<br />

undulate only <strong>the</strong> posterior part (carangiform swimming) (Videler 1993; Nauen and Lauder, 2002;<br />

Tytell and Lauder, 2004). The low efficiency infers that less force is directed to <strong>the</strong> forward<br />

movement and <strong>the</strong>refore contradicts <strong>the</strong> energy efficient swimming <strong>of</strong> eels. A partial explanation to<br />

<strong>the</strong> energy efficiency in eel may lie in an atypical muscle activity pattern. In <strong>the</strong> American eel<br />

(Anguilla rostrata), only <strong>the</strong> posterior part <strong>of</strong> <strong>the</strong> muscle fibres are activated when swimming at low<br />

speeds. The anterior part <strong>of</strong> <strong>the</strong> fibres is recruited with swimming speed is increased (Gillis et al.,<br />

1998).<br />

4.2.3 Energy Utilization<br />

A successful migration necessitates having enough energy to fuel working muscle. Lipids have <strong>the</strong><br />

highest oxycaloric value i.e. 39.5 kJ per gram (Gnaiger and Bitterlich, 1984), and fish that migrate<br />

long distances have a high tissue fat percentage. To illustrate, female <strong>European</strong> eel have an average<br />

somatic fat content <strong>of</strong> 20% (Swedang and Wickström, 1997). Based on this value and a COT <strong>of</strong><br />

27


0.57 kJ km -1 (Paper IV), it is possible to make a gross energy budget for a 1 kg female eel<br />

swimming 6000 km. The total fat reserve <strong>of</strong> a 1 kg eel would be 200 g which equals 7.9 MJ.<br />

Swimming at 1.3 km h -1 and using 0.57 kJ km -1 would require 3.4 MJ for <strong>the</strong> 6000 km journey. This<br />

corresponds to 86 grams <strong>of</strong> fat and 43% <strong>of</strong> <strong>the</strong> total fat reserves. However to complete <strong>the</strong> spawning<br />

run (migration and spawning), <strong>the</strong>re must also be energy reserves left for <strong>the</strong> development <strong>of</strong><br />

gonads and production <strong>of</strong> gametes. Having enough energy for both migration and gonadal<br />

maturation is pivotal for semelpareous species like Anguilla spp and Pacific salmon (Oncorhynchus<br />

spp), especially because <strong>the</strong>se species do not feed during <strong>the</strong> migration. Sockeye salmon (O. nerka)<br />

have different somatic lipid content depending on <strong>the</strong> distance to <strong>the</strong>ir spawning site. Populations<br />

that swim long distances, have higher somatic energy content when entering <strong>the</strong> river, than<br />

populations that spawn closer to <strong>the</strong> coast. However, regardless <strong>of</strong> distance to spawning site, female<br />

salmon allocate approximately equal amounts <strong>of</strong> <strong>the</strong>ir energy reserves to migration and gonadal<br />

growth (Crossin et al., 2004). There are no observations <strong>of</strong> spawning eels in <strong>the</strong> wild, so <strong>the</strong> amount<br />

<strong>of</strong> lipid reserves allocated to gonadal maturation in natural conditions is unknown. Artificial<br />

maturation by hormonal treatment suggests <strong>the</strong> fat energy reserves must be substantial for gonadal<br />

growth, and female eels with less than 16% body fat at maturation show very low reproductive<br />

capacity (Durif et al., 2006).<br />

5. TEMPERATURE<br />

Ambient temperature is unequivocally <strong>the</strong> most important physical factor in <strong>the</strong> aquatic<br />

environment. Given that most fish are ecto<strong>the</strong>rms, <strong>the</strong>y are very susceptible to changes in<br />

temperature. Temperature affects aquatic animals on different timescales. Acute effects are direct<br />

and occur within minutes to hours, like changes in heart rate and oxygen consumption. Acclimation<br />

effects occur over days to weeks and may be morphological, physiological and biochemical like<br />

changes heart size and/or expression <strong>of</strong> different protein is<strong>of</strong>orms. Finally, adaptation effects occur<br />

over generations via <strong>the</strong> process <strong>of</strong> natural selection acting on for example heat tolerance. A<br />

common way <strong>of</strong> quantifying direct effects <strong>of</strong> temperature is <strong>the</strong> van’t H<strong>of</strong>f equation:<br />

where R 2 is <strong>the</strong> rate <strong>of</strong> a physiological or biochemical process at a given temperature t 2 , and R 1 is<br />

<strong>the</strong> rate <strong>of</strong> that process at a different temperature t 1 . The Q 10 factor expresses <strong>the</strong> change in <strong>the</strong> rate<br />

<strong>of</strong> a process when temperature is increased by 10°C.<br />

28


5.1 Effect on Metabolism and Aerobic Scope<br />

Metabolic rate varies as function <strong>of</strong> temperature, with typical Q 10 values for standard metabolic rate<br />

(SMR) <strong>of</strong> fish varying between 2 and 3 (Holeton, 1974; Clarke and Johnston, 1999). It follows <strong>the</strong>n,<br />

that oxygen consumption at <strong>the</strong> SMR typically increases exponentially with temperature, until <strong>the</strong><br />

lethal temperature is approached (Figure 5.1A). Maximum oxygen consumption (MMR) as a<br />

function <strong>of</strong> temperature follows a different curve. In <strong>the</strong> low temperature range, MMR increases<br />

rapidly and faster that SMR with increasing temperatures, until it reaches a plateau and peaks.<br />

MMR <strong>the</strong>n begins to rapidly decrease as temperature increases fur<strong>the</strong>r (Figure 5.1A). Aerobic<br />

metabolic scope (MS) is defined as <strong>the</strong> difference between SMR and MMR, and, if plotted as a<br />

function <strong>of</strong> temperature, it typically follows a bell-shaped curve (Fry, 1947) (Figure 5.1B). This<br />

metabolic scope curve, also termed <strong>the</strong> ‘Fry curve’, demonstrates <strong>the</strong> linkage between <strong>the</strong> ability <strong>of</strong><br />

a fish to consume oxygen and its <strong>the</strong>rmal tolerance range. The aerobic metabolic scope represents<br />

<strong>the</strong> maximum amount <strong>of</strong> oxygen that can be made available for activities above those considered<br />

maintenance, such as feeding, swimming, growth, and reproduction. The temperature, at which<br />

aerobic metabolic scope is maximal, is referred to as <strong>the</strong> optimal temperature (T OPT ) (Figure 5.1B).<br />

As maximum metabolic rate is highest at T OPT , this temperature also represents <strong>the</strong> <strong>the</strong>rmal<br />

optimum for swimming performance and cardiac performance (Brett, 1971). The Fry curve remains<br />

a useful method to examine <strong>the</strong> temperature limits <strong>of</strong> individual performance, but it can also be<br />

applied in a broader ecological context when estimating performance on a population level.<br />

Oxygen consumption<br />

SMR<br />

MMR<br />

A<br />

Figure 5.1 Schematic <strong>of</strong> standard and<br />

maximum oxygen consumption and<br />

aerobic metabolic scope<br />

Aerobic scope<br />

T CRIT<br />

T P<br />

T OPT<br />

T OPT<br />

window<br />

T P<br />

B<br />

T CRIT<br />

A: standard (SMR) and maximum (MMR)<br />

oxygen consumption rates as a function <strong>of</strong><br />

temperature. B: Aerobic metabolic scope (MS)<br />

as function <strong>of</strong> temperature. T OPT , optimum<br />

temperature; T P , pejus temperatures; T CRIT ,<br />

critical temperatures. The T OPT window<br />

corresponds to <strong>the</strong> range <strong>of</strong> temperatures<br />

between <strong>the</strong> upper and lower T P . See text for<br />

details.<br />

Temperature<br />

29


Temperature constrains biochemical reactions (Hochochka and Somero, 2002) and this includes <strong>the</strong><br />

post-prandial processes in ecto<strong>the</strong>rmic animals (McCue 2006). The effect <strong>of</strong> temperature on postprandial<br />

metabolism has been studied in a wide range <strong>of</strong> temperate, subtropical and tropical fish<br />

species. The general observation is that temperature has little effect on <strong>the</strong> total amount <strong>of</strong> energy<br />

devoted to SDA (i.e. <strong>the</strong> SDA coefficient). However temperature significantly affects <strong>the</strong> SDA<br />

pr<strong>of</strong>ile with <strong>the</strong> SDA peak increasing and occurring faster and higher at higher temperatures and<br />

prolongation <strong>of</strong> <strong>the</strong> SDA response at decreasing temperatures (Lou and Xie, 2008; Perez-Casanova<br />

et al., 2010; Vanella et al., 2010).<br />

5.2 Oxygen and Capacity Limited Thermal Tolerance<br />

The Fry curve forms <strong>the</strong> basis for <strong>the</strong> concept <strong>of</strong> Oxygen and Capacity Limited Thermal Tolerance<br />

(OCLTT) that links <strong>the</strong> temperature dependent loss in aerobic capacity to <strong>the</strong> loss <strong>of</strong> performance<br />

on <strong>the</strong> cellular, organismal, and species level (Pörtner, 2001). The upper and lower temperatures<br />

where aerobic scope begins to decline are referred to as <strong>the</strong> pejus temperatures (T P ) (Pörtner, 2001).<br />

Between <strong>the</strong> upper and lower pejus temperatures, maximum aerobic scope is maintained and this<br />

range <strong>of</strong> temperatures is termed <strong>the</strong> <strong>the</strong>rmal tolerance window (Pörtner, 2001) (Figure 5.1B). The<br />

temperatures at which aerobic scope approaches zero are termed <strong>the</strong> upper and lower critical<br />

temperatures (T CRIT ) (Figure 5.1B). T CRIT represents <strong>the</strong> temperatures where survival becomes time<br />

limited (Pörtner, 2001; Pörtner, 2002; Pörtner and Farrell, 2008). The width <strong>of</strong> <strong>the</strong> <strong>the</strong>rmal tolerance<br />

window reflects <strong>the</strong> amplitude <strong>of</strong> temperature fluctuations in a species natural environment or<br />

habitat (Pörtner and Knust, 2007. Steno<strong>the</strong>rmal polar species and tropical species have narrow<br />

<strong>the</strong>rmal tolerance windows (Pörtner, 2000; Nilsson et al., 2009) while eury<strong>the</strong>rmal temperate<br />

species have a wide <strong>the</strong>rmal window. For example, aerobic scope was reduced by about 50% in two<br />

species <strong>of</strong> coral reef fish (Ostorhinchus cyanosoma and O. doederleini) at 31°C compared to <strong>the</strong><br />

mean annual temperature <strong>of</strong> 29°C, was reduced to nil at 33°C (Nilsson et al., 2009). In Paper I, <strong>the</strong><br />

aerobic scope <strong>of</strong> <strong>European</strong> eel (Anguilla anguilla) was estimated over a range <strong>of</strong> acclimation<br />

temperatures from 0°C to 30. A limitation in maximum oxygen consumption was not reached at<br />

30°C and <strong>the</strong>re was no significant reduction in aerobic scope between 10°C and 30°C. This<br />

demonstrates a wide <strong>the</strong>rmal tolerance window for <strong>European</strong> eel that, as hypo<strong>the</strong>sized, reflects <strong>the</strong><br />

broad range <strong>of</strong> temperatures this species may experience in its natural habitats. Environmental<br />

factors that constrain metabolism like dissolved oxygen and CO 2 , will limit <strong>the</strong> aerobic scope (Fry,<br />

1971). Hypoxia (low environmental O 2 ) and hypercapnia (high environmental CO 2 ) both reduce<br />

30


aerobic scope and narrow <strong>the</strong> <strong>the</strong>rmal tolerance window (Pörtner and Farrell, 2008). This is fur<strong>the</strong>r<br />

discussed in chapters 6 and 7.<br />

5.2.1 Temperature Extremes<br />

Central to <strong>the</strong> concept <strong>of</strong> OCLTT is that <strong>the</strong>rmal tolerance is set by oxygen limitations and that ATP<br />

production becomes anaerobic at both high and low temperature extremes (Pörtner, 2001). The<br />

capacity <strong>of</strong> <strong>the</strong> mitochondria to produce ATP decreases at both high and low temperatures and this<br />

lead to limitations in ventilation and circulation (Pörtner, 2002). The strategies to overcome <strong>the</strong><br />

limitations in ATP production at low temperature varies between species. For example, <strong>the</strong><br />

mitochondrial density or <strong>the</strong> mitochondrial enzyme efficiencies may increase after acclimation to<br />

low temperatures in some species (Guderley et al., 2004; St. -Pierre et al., 1998). This ensures that a<br />

certain rate <strong>of</strong> ATP production can be maintained to support cellular demands. Ano<strong>the</strong>r strategy to<br />

overcome <strong>the</strong> cold-induced limitation in mitochondrial capacity is to reduce cellular ATP demands<br />

instead <strong>of</strong> increasing production capacity as exemplified by anguillid eels. Cold acclimated eels<br />

(Anguilla spp) can increase aerobic scope at low temperatures by depressing metabolic rate, as<br />

evident by <strong>the</strong> large decrease in whole animal oxygen uptake with Q 10 values above 3.5 (Egginton<br />

and Johnston, 1984; Walsh et al., 1983; Paper I). In <strong>the</strong> o<strong>the</strong>r end <strong>of</strong> spectrum, mitochondrial ATP<br />

production becomes less efficient due to an increase in proton leakage at high temperature. This<br />

contributes to <strong>the</strong> increase in SMR and consequently reduction in aerobic scope (Hardewig et al.,<br />

1999; Pörtner, 2001)<br />

5.3 Effects on Cardiac Morphology and Function<br />

The effect <strong>of</strong> temperature on fish hearts includes changes in gross morphology, tissue composition<br />

and function. A common, but not universal response to seasonal changes in ambient temperature is<br />

a change in relative ventricular mass (RVM), which is expressed as a percentage <strong>of</strong> body mass.<br />

Temperature affects <strong>the</strong> viscosity <strong>of</strong> fluids including blood. For example, in <strong>the</strong> red blooded<br />

Antarctic Trematomus bernacchii blood viscosity is about 40% higher at 0°C than what it would be<br />

at 10°C (Axelsson, 2005). An increase in ventricle size is believed to represent a way to compensate<br />

for <strong>the</strong> increased stress on <strong>the</strong> heart that is associated with <strong>the</strong> increased blood viscocity (Graham<br />

and Farrell 1989). Cardiac growth (hypertrophy) has been observed in several species after cold<br />

acclimation e.g. common carp (Cyprinus carpio, Goolish, 1987), Goldfish (Carassius auratus,<br />

Tsukuda et al., 1985), sea raven (Hemitripterus americanus, Graham and Farrell, 1985), rainbow<br />

trout (O.mykiss, Graham and Farrell 1989; Klaiman et al., 2011) and <strong>European</strong> eel (A. anguilla,<br />

31


Paper II). Not all species increase RVM during cold acclimation e.g. crucian carp (Carassius<br />

carassius, Matikainen and Vornanen, 1992), white perch (Morone americana) and yellow perch<br />

(Perca flauescens, Sephton and Driedzic, 1991). Depending on <strong>the</strong>ir activity levels during <strong>the</strong> cold<br />

season, temperate species can be categorized as being ei<strong>the</strong>r cold active or cold dormant. Species<br />

like white perch, yellow perch and rainbow are cold active and maintain feeding activities during<br />

winter. Sea raven, crucian carp and eels, on <strong>the</strong> o<strong>the</strong>r hand display minimal activity during winter.<br />

However no clear correlation between hypertrophy and activity levels have been established<br />

(Driedzic et al., 1996). The relative amounts <strong>of</strong> spongy and compact myocardium can also be<br />

influenced by temperature as recently observed in male rainbow trout (Klaiman et al., 2011).<br />

Temperature also influences <strong>the</strong> Ca 2+ cycling in fish cardiocytes as <strong>the</strong> contribution to force<br />

development <strong>of</strong> Ca 2+ originating from <strong>the</strong> sarcoplasmatic reticulum (SR) increases after cold<br />

acclimation in some species (Shiels et al., 2002). Also in A. anguilla, temperature influences <strong>the</strong><br />

relative significance <strong>of</strong> <strong>the</strong> NCX channel and <strong>the</strong> SOCE in bringing Ca 2+ into <strong>the</strong> cell, with<br />

increased contribution at high temperature (Paper II).<br />

5.4 Effects on Cardiac Performance<br />

Temperature has a direct effect on <strong>the</strong> intrinsic pacemaker rate, with Q 10 values around 2 (Gollock<br />

et al., 2006). In order to maintain activities at <strong>the</strong> temperature extremes, compensatory mechanisms<br />

are necessary. Fish can compensate for <strong>the</strong> depressant effect <strong>of</strong> low temperature by reducing <strong>the</strong><br />

inhibitory cholinergic control <strong>of</strong> heart rate (Seibert, 1979). During an acute decrease in temperature,<br />

cardiac performance (i.e. cardiac output) may be maintained as <strong>the</strong> cold-induced bradycardia can<br />

cause an increase <strong>the</strong> contractile strength (Shiels et al., 2002). This mechanism is <strong>of</strong> great adaptive<br />

value for fish that make regular excursion into deeper colder waters to forage or to escape predators.<br />

An increase in contractility was observed in <strong>European</strong> eel (A. anguilla) after an acute drop in<br />

temperature from 20°C to 10°C (Paper II), which may serve to protect cardiac output during<br />

vertical migrations. Cold active species may have different strategies to cope with low<br />

environmental temperatures than cold dormant species. In cold active rainbow trout (O. mykiss)<br />

acclimatization to low temperature resets <strong>the</strong> pacemaker to a higher rate, so that heart rate is higher<br />

than expected from a typical Q 10 . Maintaining a high heart rate in <strong>the</strong> cold is possible because <strong>the</strong><br />

duration <strong>of</strong> <strong>the</strong> contraction is reduced along with <strong>the</strong> time needed for restitution between<br />

contractions (Driedzic et al., 1996; Aho and Vornanen, 1999; Aho and Voranen, 2001). This<br />

positive cold compensation allows rainbow trout (O. mykiss) to remain active during winter. The<br />

complete opposite acclimatory response is observed in <strong>the</strong> winter dormant crucian carp (C.<br />

32


carassius). Cold acclimated crucian carp have a lower heart rate (i.e. Q 10 > 3), increased duration <strong>of</strong><br />

contraction and a decreased rate <strong>of</strong> restitution (Matikainen and Vornanen, 1992; Tiitu and<br />

Vornanen, 2001). This negative compensation is believed to be a way to conserve energy during<br />

winter as <strong>the</strong>se fish remain dormant and do not eat.<br />

The maximal heart rate in many active fish is around 120 beats per minute (Farrell 1991). As <strong>the</strong><br />

intrinsic heart rate increases with temperature, <strong>the</strong> scope for increasing <strong>the</strong> cardiac output via fur<strong>the</strong>r<br />

increases in heart rate will progressively become limited (Steinhausen et al., 2008). Acclimatization<br />

may reset <strong>the</strong> pacemaker to a lower rate (Haverinen and Vornanen, 2007), however when maximal<br />

heart rate is reached, cardiac output will decrease upon fur<strong>the</strong>r increases in temperature. This is due<br />

to <strong>the</strong> fact that stroke volume is ei<strong>the</strong>r insensitive to changes in temperature (Gollock et al., 2006;<br />

Steinhausen et al., 2008) or decreases at warm temperatures (Axelsson et al., 1992; Sandblom and<br />

Axelsson, 2007).<br />

6. HYPOXIA<br />

6.1 Definition and Occurrences<br />

Hypoxia can be defined as <strong>the</strong> level <strong>of</strong> dissolved oxygen (DO), at which oxygen-requiring<br />

physiological functions become compromised. In o<strong>the</strong>r words, hypoxia is a shortage <strong>of</strong> oxygen.<br />

Low levels <strong>of</strong> DO occur naturally in many aquatic systems. In <strong>the</strong> marine environment, oceanic<br />

oxygen minimum zones (OMZs) represent <strong>the</strong> largest areas <strong>of</strong> low DO. OMZs develop in high<br />

productivity areas when decomposing material sinks to <strong>the</strong> bottom (Helly and Levin, 2004). Low<br />

DO in marine coastal areas occurs in deep basins or in systems that become stratified like <strong>the</strong> Baltic<br />

Sea, Kattegat, Osl<strong>of</strong>jord and <strong>the</strong> Black Sea (Fonselius, 1969; Baden et al., 1980; Karlson et al.,<br />

2002; Kideyes, 2002). In freshwater systems, low DO occurs in stratified lakes, lakes with ice<br />

cover, and in streams and ponds with high organic content (Graham, 2006). Increased nutrient loads<br />

from human activities have increased <strong>the</strong> occurrence <strong>of</strong> which aquatic systems become hypoxic and<br />

have increased <strong>the</strong> frequency and duration <strong>of</strong> hypoxic events in systems that are already naturally<br />

prone to hypoxia. Hypoxia from anthropogenic eutrophication is considered to be one <strong>of</strong> <strong>the</strong><br />

greatest threats to aquatic ecosystems on a global scale (Diaz and Breitburg, 2009).<br />

6.2 Effects on Ventilation and Oxygen Transport<br />

Given that hypoxia is a constantly re-occurring phenomenon in many aquatic habitats, fishes have<br />

developed a wide array <strong>of</strong> anatomical, physiological and behavioural traits to overcome this<br />

adversity. Examples <strong>of</strong> behavioural responses to hypoxia include aquatic surface respiration (ASR)<br />

33


and air breathing, and many sub-tropic and tropic species, frequently exposed to low DO, have<br />

developed anatomical specializations that fur<strong>the</strong>r facilitate <strong>the</strong>se modes <strong>of</strong> respiration (Kramer and<br />

McClure, 1982; Graham, 1997). When exposed to low DO in <strong>the</strong>ir environment, fish <strong>the</strong>y can<br />

employ several mechanisms along <strong>the</strong> oxygen transport cascade to ensure adequate O 2 is delivered<br />

to <strong>the</strong> tissues (Fritsche and Nilsson, 1993). Starting at gills, a typical response is an increase in<br />

ventilation (hyperventilation) to increase <strong>the</strong> flow <strong>of</strong> water, which can be achieved by ei<strong>the</strong>r<br />

increasing <strong>the</strong> ventilation frequency and/or <strong>the</strong> volume (Perry et al., 2009). Hyperventilation<br />

increases <strong>the</strong> transfer <strong>of</strong> O 2 from <strong>the</strong> water to <strong>the</strong> blood by increasing <strong>the</strong> O 2 gradient between blood<br />

and water. However at some point (level <strong>of</strong> DO), <strong>the</strong> metabolic cost <strong>of</strong> <strong>the</strong> increased ventilation will<br />

exceed <strong>the</strong> benefits and hyperventilation becomes energetically unfavourable. Oxygen uptake can<br />

also be increased by increasing <strong>the</strong> area <strong>of</strong> <strong>the</strong> gill that is perfused (Booth 1979). Hyperventilation<br />

also raises <strong>the</strong> pH value <strong>of</strong> <strong>the</strong> blood as more CO 2 is removed from <strong>the</strong> blood (see chapter 7). This in<br />

turn leads to an increase in <strong>the</strong> O 2 -binding affinity <strong>of</strong> <strong>the</strong> haemoglobins. The O 2 -carrying capacity <strong>of</strong><br />

<strong>the</strong> blood can be fur<strong>the</strong>r increased by releasing more red blood cells from <strong>the</strong> spleen <strong>the</strong>reby<br />

increasing <strong>the</strong> haemoglobin concentration in <strong>the</strong> blood (Wells and Weber, 1990). An increased O 2 -<br />

extraction by <strong>the</strong> tissues increases <strong>the</strong> partial pressure gradient between arterial and venous blood<br />

which in turns increases <strong>the</strong> gradient between venous blood and <strong>the</strong> water. When above<br />

mechanisms are fully exploited and <strong>the</strong> O 2 -content <strong>of</strong> <strong>the</strong> blood declines, an adequate supply to <strong>the</strong><br />

tissues can still be maintained by increasing <strong>the</strong> cardiac output.<br />

6.3 Effects on Cardiac Performance<br />

A common initial response <strong>of</strong> water breathing fish exposed to hypoxia is a decrease in heart rate.<br />

This response is unique to fish and is termed reflex bradycardia (Taylor 1992). Reflex bradycardia<br />

is triggered by O 2 -chemoreceptors located around <strong>the</strong> gill and in <strong>the</strong> buccal cavity, and it is<br />

mediated by an increased inhibitory cholinergic tonus from <strong>the</strong> autonomous nervous system<br />

(Burleson et al., 1992). There are several conceivable benefits to slowing down <strong>the</strong> heart rate when<br />

<strong>the</strong> oxygen supply becomes limited. The main adaptive value <strong>of</strong> reflex bradycardia is believed to be<br />

a protection <strong>of</strong> <strong>the</strong> heart from hypoxia (Farrell, 2007). This can be achieved via several<br />

mechanisms. For one, it increases <strong>the</strong> retention time <strong>of</strong> <strong>the</strong> blood in <strong>the</strong> lumen, making more time<br />

for oxygen exchange between <strong>the</strong> blood and myocardium. This is <strong>of</strong> particular benefit for species in<br />

which <strong>the</strong> heart relies solely on <strong>the</strong> venous reserve <strong>of</strong> oxygen (see chapter 3). Oxygen delivery to<br />

<strong>the</strong> myocardium via <strong>the</strong> coronary circulation (if present) may also benefit from <strong>the</strong> extended period<br />

between contractions as peak coronary blood flow occurs during diastole. Reflex bradycardia may<br />

34


also increase <strong>the</strong> strength <strong>of</strong> contraction (contractility) via <strong>the</strong> negative force-frequency response<br />

(Shiels et al., 2002). The extended diastolic period allows more time for filling, which increases <strong>the</strong><br />

end-diastolic volume. An increase in end-diastolic volume toge<strong>the</strong>r with an increase in contractility<br />

facilitates an increase in stroke volume (Olson and Farrell 2006; Farrell, 2007). It has also been<br />

suggested that reflex bradycardia safeguards <strong>the</strong> extraction <strong>of</strong> oxygen at <strong>the</strong> gills (Taylor 1985).<br />

However in at least two species i.e. Atlantic cod (G. morhua) and <strong>European</strong> eel (A. anguilla), reflex<br />

bradycardia does not improve <strong>the</strong> ability to regulate oxygen uptake, suggesting <strong>the</strong> response does<br />

not necessarily benefit oxygen transfer at <strong>the</strong> gills (McKenzie et al., 2009; Iversen et al., 2010). The<br />

hypoxia-induced bradycardia will inherently cause a decrease in cardiac output, unless accompanied<br />

by a compensatory increase in stroke volume. Not surprisingly, a general cardiac response to<br />

hypoxia is an increase in stroke volume (Gamberl and Driedzic, 2009). At what level <strong>of</strong> hypoxia<br />

this increase in stroke volume occurs, and how well it compensates to maintain cardiac output<br />

varies between species. Thus it may occur at a lower, higher or similar oxygen partial pressure<br />

(PO 2 ) than <strong>the</strong> reflex bradycardia and <strong>the</strong> compensation may be fully or partial (Sandblom and<br />

Axelsson, 2005; Axelsson et al 2002; Agnisola et al., 1999; Iversen et al., 2010). The overall<br />

outcome is that some species <strong>of</strong> fish are able to regulate cardiac output down to a low PO 2 , while in<br />

o<strong>the</strong>r species cardiac output conforms to <strong>the</strong> ambient PO 2 (Gamperl and Driedzic, 2009).<br />

6.4 Effects on Metabolism and Aerobic Scope<br />

If confined to environments where <strong>the</strong> oxygen partial pressure (PO 2 ) progressively declines, <strong>the</strong> vast<br />

majority <strong>of</strong> fish species can regulate <strong>the</strong>ir oxygen uptake by making some <strong>of</strong> <strong>the</strong> behavioural,<br />

ventilatory and circulatory adjustments mentioned above. In this range <strong>of</strong> oxygen pressures, fish are<br />

referred to as oxygen regulators, due to <strong>the</strong>ir ability to regulate uptake in <strong>the</strong> face <strong>of</strong> declining<br />

oxygen levels. However, this constant oxygen uptake, corresponding to <strong>the</strong> minimal metabolic<br />

demands (SMR), can only be maintained down to a certain oxygen level (Figure 6.1). This point is<br />

termed <strong>the</strong> critical partial pressure (P CRIT ) <strong>of</strong> oxygen, below which all behavioural and physiological<br />

adaptations are exhausted, and oxygen uptake depends entirely on PO 2 , why fish become oxyconformers.<br />

The P CRIT for <strong>the</strong> maximum oxygen consumption rate (MMR) is much higher than for<br />

SMR and <strong>the</strong>refore conform to ambient PO 2 at higher levels (Figure 6.1). The rationale behind this<br />

is that exercising fish have no fur<strong>the</strong>r means <strong>of</strong> increasing oxygen uptake as <strong>the</strong> possible<br />

adjustments in ventilation and circulation have already been made (Farrell and Richards, 2009).<br />

Aerobic metabolic scope will continue to decrease with PO 2 and eventually at P CRIT , <strong>the</strong>re will no<br />

scope left.<br />

35


Figure 6.1 Hypo<strong>the</strong>tical schema <strong>of</strong><br />

oxygen consumption as a function <strong>of</strong><br />

ambient O 2 partial pressure (PO 2 )<br />

Oxygen consumption<br />

P CRIT<br />

SMR<br />

The scope for o<strong>the</strong>r routine activities like swimming<br />

and feeding will also decrease with increasing<br />

severity <strong>of</strong> hypoxia (Claireaux and Lagardere, 1999),<br />

(Figure 6.1). Some species <strong>of</strong> fish respond to<br />

hypoxia by decreasing <strong>the</strong>ir spontaneous swimming<br />

activity (Nilsson et al., 1993; Schurmann and<br />

Steffensen, 1997; Dalla via et al., 1998; Dominici et<br />

al., 2000; Behrens and Steffensen, 2007). Also, fish<br />

exposed to hypoxia show reduced appetite and<br />

reduced food intake (Brett 1979). For example,<br />

Atlantic cod (Gadus morhua), immediately<br />

regurgitates stomach content if acutely exposed to<br />

hypoxia (Claireaux et al., 2000). These responses<br />

and changes in routine activity levels may be viewed<br />

as an attempt to conserve aerobic scope (Claireaux<br />

and Lefrancois, 2007).<br />

The effects <strong>of</strong> hypoxia on post-prandial processes in fish are less well studied. In Atlantic cod (G.<br />

morhua), digesting a meal corresponding to 5% <strong>of</strong> <strong>the</strong> body mass in hypoxia, decreased <strong>the</strong> postprandial<br />

metabolic peak and prolonged <strong>the</strong> duration <strong>of</strong> <strong>the</strong> SDA response (Jordan and Steffensen,<br />

2007). In Nile tilapia (Oreochromis niloticus) hypoxia exposure reduced <strong>the</strong> assimilation efficiency<br />

(Tsadik and Kutty, 1987. Also in carp (Cyprinus carpio), assimilation efficiency was reduced and<br />

faecal losses increased when exposed to severe hypoxia (Zhou et al., 2001). A limited nutrient<br />

absorption during hypoxia could be <strong>the</strong> consequence <strong>of</strong> a reduced gastrointestinal blood flow<br />

(Axelsson, 1991). Regardless <strong>of</strong> <strong>the</strong> mechanisms behind, reduced feed intake, reduced assimilation<br />

efficiency and increased faecal losses may serve to minimize <strong>the</strong> postprandial O 2 requirements and<br />

maximize aerobic scope.<br />

MMR<br />

Routine Activity<br />

Ambient PO 2<br />

Aerobic<br />

scope<br />

Solid line represents standard metabolic rate<br />

(SMR), short-dash line represents routine<br />

metabolic rate and long-dash line represents<br />

maximum metabolic rate. Arrow indicates<br />

<strong>the</strong> critical PO 2 i.e. P CRIT , where oxygen<br />

consumption is no longer regulated but<br />

conforms to ambient PO 2 . See text for fur<strong>the</strong>r<br />

details.<br />

6.5 Interactive Effects <strong>of</strong> Hypoxia and Temperature<br />

The solubility <strong>of</strong> oxygen decreases with increasing temperatures. Meanwhile, standard metabolic<br />

rate (SMR) increases with temperature (see chapter 5). To compensate for <strong>the</strong>se changes, and in<br />

order to maintain adequate O 2 supply, fish must increase <strong>the</strong> transfer <strong>of</strong> oxygen from <strong>the</strong> water to<br />

<strong>the</strong> blood, e.g. by increasing <strong>the</strong> ventilation. However, <strong>the</strong> amount <strong>of</strong> water that can be transported<br />

across <strong>the</strong> gills is limited by <strong>the</strong> capacity <strong>of</strong> <strong>the</strong> branchial pump (Farrell and Steffensen, 1987). This<br />

36


infers that if ambient hypoxia occurs at a higher temperature, fish will have a reduced capacity to<br />

compensate by increasing ventilation, and any increase in temperature will aggravate <strong>the</strong> state <strong>of</strong><br />

hypoxia. P CRIT is a representative measure <strong>of</strong> <strong>the</strong> capacity to extract O 2 from <strong>the</strong> water, and it can<br />

<strong>the</strong>refore be expected that P CRIT will increase with temperature. Indeed, a positive correlation<br />

between P CRIT and temperature has been described for several species <strong>of</strong> fish (Fernandes and<br />

Rantin, 1989; Schurmann and Steffensen, 1997; Valverde, 2006; Corkum and Gamberl, 2009;<br />

Nilsson et al., 2010), and for <strong>European</strong> eel (A. Anguilla) exposed to a gradual stepwise hypoxia at<br />

0°C, 10°C, 20°C and 30°C, P CRIT increased exponentially with temperature (Paper I). In some<br />

species however, such a clear positive correlation is not observed or has to a higher degree<br />

depended on <strong>the</strong> range <strong>of</strong> temperatures studied (Ott et al., 1980; Cerezo and Garcia, 2004; Barnes et<br />

al 2011). A positive correlation between P CRIT and temperature infers that <strong>the</strong> switch to anaerobic<br />

metabolism occurs at a higher PO 2 when temperature is increased. In relation to <strong>the</strong> concept <strong>of</strong><br />

Oxygen and Capacity Limited Thermal Tolerance (OCLTT) (Pörtner and Farrell, 2008; see also<br />

chapter 5), this corresponds to a narrowing <strong>of</strong> <strong>the</strong> <strong>the</strong>rmal tolerance window as that <strong>the</strong> upper<br />

critical temperature is reduced.<br />

7. HYPERCAPNIA<br />

Hypercapnia refers to an increase in <strong>the</strong> partial pressure <strong>of</strong> CO 2 (PCO 2 ). The amount <strong>of</strong> CO 2<br />

physically dissolved in water is in equilibrium with atmospheric CO 2 . Dissolved CO 2 reacts<br />

chemically with water to form carbonic acid, which dissociates and produces bicarbonate while<br />

releasing a proton:<br />

CO 2(gas) ↔ CO 2(dissolved) + H 2 O ↔ H 2 CO 3 ↔ HCO - 3 + H +<br />

As a result, an increase in PCO 2 will cause a decrease in <strong>the</strong> pH value <strong>of</strong> <strong>the</strong> water. The magnitude<br />

<strong>of</strong> <strong>the</strong> pH change, at any given PCO 2 , depends on <strong>the</strong> buffer capacity <strong>of</strong> <strong>the</strong> system. The bicarbonate<br />

ion (HCO - 3 ) acts as a buffer and in HCO - 3 rich water, an increase in PCO 2 will have a smaller effect<br />

on pH. Hypercapnia may occur naturally in aquatic environments with a limited air-to-water surface<br />

due to <strong>the</strong> respiration <strong>of</strong> different biota. Although <strong>the</strong> PCO 2 in freshwater surfaces is generally<br />

lower than 1mmHg, it can increase rapidly and in some cases reach values <strong>of</strong> 60mmHg in warm and<br />

densely vegetated stagnant water bodies (Ultsch, 1996). Hypercapnia also frequently occurs in<br />

recirculating aquaculture systems, particularly under high degrees <strong>of</strong> water re-use and high rearing<br />

densities, due to an accumulation <strong>of</strong> expired CO 2 (Crocker et al., 2000; Steffensen and Lomholt<br />

1990).<br />

37


7.1 Effects on Blood pH and Oxygen Carrying Capacity<br />

Carbon dioxide is a small molecule that can readily diffuse across <strong>the</strong> gill epi<strong>the</strong>lia into <strong>the</strong> blood,<br />

where carbonic acid is formed. Thus, <strong>the</strong> immediate consequence <strong>of</strong> environmental hypercapnia is a<br />

decrease in <strong>the</strong> pH value <strong>of</strong> plasma. The increased proton load can be buffered by plasma HCO - 3 or<br />

by non-bicarbonate buffers (e.g. plasma proteins). Carbon dioxide will also diffuse into <strong>the</strong> red<br />

blood cells, where it binds to haemoglobin (Hb). When H + binds to Hb, it causes a conformational<br />

change that reduces <strong>the</strong> affinity for oxygen i.e. <strong>the</strong> Bohr effect. Ano<strong>the</strong>r effect <strong>of</strong> H + on Hb O 2 -<br />

affinity, which is unique to fish haemoglobin, is <strong>the</strong> Root effect (Root, 1931). The Root effect is<br />

characterized by <strong>the</strong> inability <strong>of</strong> haemoglobin to become fully saturated even at high oxygen<br />

tensions. Via <strong>the</strong> Bohr and Root effects, hypercapnia ultimately results in a reduced O 2 -carrying<br />

capacity and possibly hypoxaemia depending on <strong>the</strong> severity <strong>of</strong> <strong>the</strong> acidosis (Gallaugher and<br />

Farrell, 1998). Fish are able to fur<strong>the</strong>r compensate for <strong>the</strong> acidosis by excreting H + or accumulating<br />

HCO - 3 in exchange for Cl - ions via <strong>the</strong> gills (Heisler, 1984; 1993). The tolerance to hypercapnia<br />

varies between species and is reflected in <strong>the</strong> buffering capacity and in <strong>the</strong> ability to stabilize pH<br />

(Ishimatsu et al., 2005; Melzner et al., 2009a). A general response <strong>of</strong> many fishes to hypercapnia is<br />

an increase in ventilation (volume and/or frequency), elicited via chemoreceptors externally located<br />

around <strong>the</strong> gill. This has been demonstrated in wide range <strong>of</strong> species (Crocker et al 2000;<br />

McKendry et al., 2001; Perry and McKendry 2001; Gilmour et al., 2005; Boijink et al., 2010). The<br />

effects <strong>of</strong> hypercapnia can be both acute and chronic. Acute effects on respiration, circulation and<br />

metabolism may ultimately reduce growth rates and Darwinian fitness. Long term effects may<br />

change species distributions via avoidance behaviour or reduced swimming activity (Pörtner et al.,<br />

2004; Ishimatsu et al., 2005).<br />

7.2 Effects on Cardiac Function<br />

The effect on hypercapnia on cardiac function in fish depends on species, <strong>the</strong> severity <strong>of</strong> <strong>the</strong><br />

acidosis and <strong>the</strong> exposure time. For example, acute exposure to mild hypercapnia (5-10mmHg)<br />

causes bradycardia in rainbow trout (O. mykiss) (Perry et al., 1999), tachycardia in <strong>European</strong> eel (A.<br />

anguilla), (McKenzie et al., 2003), and has no effect on heart rate in yellowtail (Seriola<br />

quinqueradiata), (Lee et al., 2003b). More severe hypercapnia (~20mmHg) causes tachycardia in<br />

white sturgeon (Acipenser transmontanus), (Crocker et al., 2000), but a PCO 2 above 40mmHg<br />

causes bradycardia in eels (McKenzie et al., 2003). From <strong>the</strong>se observations, <strong>the</strong>re does not appear<br />

to be a uniform chronotropic response among fish exposed to hypercapnia. Cardiac stroke volume<br />

on <strong>the</strong> o<strong>the</strong>r hand, increases in almost all species examined, including those that show bradycardia.<br />

38


The increase in stroke volume may fully or partially compensate for any decreases in heart rate. For<br />

most species however, <strong>the</strong> overall result is a decrease in cardiac output (Crocker et al 2000;<br />

McKendry et al., 2001; Perry and McKendry 2001; Lee et al., 2003; Gilmour et al., 2005; Boijink et<br />

al., 2010). While stroke volume is not reduced during exposure to elevated CO 2 in vivo, it has been<br />

observed in several species, that cardiac contractility decreases following hypercapnic acidosis and<br />

this loss <strong>of</strong> contractile strength is likely caused by H + ions directly affecting <strong>the</strong> Ca 2+ cycling in <strong>the</strong><br />

myocardial cells (Driedzic and Gesser 1994). As contractility is one <strong>of</strong> <strong>the</strong> determinants <strong>of</strong> stroke<br />

volume (see chapter 3), <strong>the</strong> loss <strong>of</strong> myocardial strength in vitro suggest <strong>the</strong> existence <strong>of</strong><br />

compensatory mechanisms, when fish are exposed to hypercapnia.<br />

7.3 Effects on Metabolism and Aerobic Scope<br />

Restoring acid-base equilibrium after a hypercapnic disturbance represents a short term additional<br />

metabolic costs until a new steady state is reached which may be evident by an initial increase in <strong>the</strong><br />

resting oxygen consumption (Cruz-Neto and Steffensen 1997; Diegweiher et al., 2008; Baker et al.,<br />

2012). Maintaining a new steady state toge<strong>the</strong>r with hyperventilation would also represent an<br />

additional cost, but fish exposed to chronic hypercapnia do not generally have increased oxygen<br />

consumption at rest (McKenzie et al., 2003; Ishimatsu et al., 2008). Any increase in activity above<br />

standard metabolism (SMR) represents a fur<strong>the</strong>r increase in blood PCO 2 from respiring tissues ,<br />

proton load, and an increased use <strong>of</strong> <strong>the</strong> buffering capacity. The CO 2 excreted from cellular<br />

respiration is transported as bicarbonate (HCO - 3 ) inside <strong>the</strong> red blood cells. At <strong>the</strong> gills, <strong>the</strong> release<br />

<strong>of</strong> bicarbonate depends on <strong>the</strong> anion exchange with Cl - , and this step represents a potential<br />

limitation in <strong>the</strong> CO 2 excretion. At increased activity levels, e.g. during swimming, an increase in<br />

cardiac output will also reduce <strong>the</strong> time available for this exchange to take place (Randall 1982),<br />

which will fur<strong>the</strong>r limit CO 2 excretion. Swimming activity that involves <strong>the</strong> recruitment <strong>of</strong><br />

anaerobic muscle fibres will fur<strong>the</strong>r increase <strong>the</strong> proton load by <strong>the</strong> production <strong>of</strong> lactic acid. At a<br />

certain point <strong>the</strong> buffering capacity becomes exhausted and it is no longer possible to reduce <strong>the</strong><br />

changes in pH, which will <strong>the</strong>n decrease rapidly. As H + reduces <strong>the</strong> O 2 -affinity <strong>of</strong> <strong>the</strong><br />

haemoglobins, oxygen uptake will also eventually become limited. The overall result is that<br />

hypercapnia reduces <strong>the</strong> oxygen content <strong>of</strong> <strong>the</strong> blood, reduces <strong>the</strong> capacity for aerobic performance<br />

and <strong>the</strong>reby reduces <strong>the</strong> aerobic scope (Pörtner et al., 2008). Again, aerobic scope may not become<br />

significantly limited during exposure to mild hypercapnia and <strong>the</strong> limiting threshold is also species<br />

specific as is <strong>the</strong> bicarbonate and non-bicarbonate buffering capacity. For example, aerobic scope is<br />

not compromised in Atlantic cod (G. morhua) after long term acclimation to 4.5 mmHg (Melzner et<br />

39


al., 2009b). <strong>European</strong> eel (A. anguilla) is exceptionally tolerant to hypercapnia and aerobic scope is<br />

not reduced after long term acclimation to 45mmHg (McKenzie et al., 2003). In Paper III, A.<br />

anguilla was acclimated to a PCO 2 <strong>of</strong> 60mmHg for several weeks and this caused a reduction in<br />

maximum metabolic rate and consequently aerobic scope was reduced by about 25%.<br />

7.4.2 Feeding metabolism<br />

The effect <strong>of</strong> hypercapnia on postprandial (feeding) metabolism is relatively unstudied in fish. A<br />

few studies have demonstrated reduced growth rates in Atlantic salmon (Salmo salar) (Fivelstad et<br />

al. 2007), in white sturgeon (Acipenser transmontanus (Crocker and Cech 1996; Crocker et al.,<br />

2000) and Atlantic cod (G.morhua), (Moran and Støttrup 2011). This may be <strong>the</strong> result <strong>of</strong> an<br />

reduced feed intake, which have been observed in Atlantic salmon (Hosfeld et al., 2008) and in<br />

o<strong>the</strong>r species e.g. sea bass (Dicentrarchus labrax) (Cecchini et al. 2001) and spotted wolfish<br />

(Anarhichas minor) (Foss et al. 2003). Besides a decreased feed intake, hypercapnia may also affect<br />

o<strong>the</strong>r parameters <strong>of</strong> <strong>the</strong> energy balance equation (see chapter 2). These parameters include <strong>the</strong><br />

excretion <strong>of</strong> waste products (i.e. faeces, urea and ammonia) and protein syn<strong>the</strong>sis. How <strong>the</strong>se<br />

parameters are affected by hypercapnia may be evaluated in fish that have ingested a fixed ration<br />

size. In Paper III, <strong>European</strong> eel (A. anguilla) was chronically exposed to hypercapnia fluctuating<br />

between 20-60 mmHg, and fed a meal corresponding to 0.5% <strong>of</strong> <strong>the</strong> body weight. Hypercapnia did<br />

not affect <strong>the</strong> postprandial oxygen consumption, but <strong>the</strong> ammonia excretion was significantly<br />

reduced. This suggests that eels exposed to hypercapnia absorbed less protein from <strong>the</strong>ir diet and<br />

fur<strong>the</strong>r suggests that <strong>the</strong>re was a greater faecal loss. Thus <strong>the</strong> reduced growth observed in fish<br />

exposed to hypercapnia could also be <strong>the</strong> result <strong>of</strong> reduced digestibility <strong>of</strong> feed items.<br />

Although not evident from basal oxygen consumption rates, hypercapnia might also have a negative<br />

effect on <strong>the</strong> protein metabolism in non-feeding fish. Ammonia is <strong>the</strong> main nitrogenous waste<br />

product in fish, and <strong>the</strong> production and excretion <strong>of</strong> ammonia was increased in carp (Cyprinus<br />

carpio) exposed to 37mmHg (Claiborne and Heisler, 1986). Also in <strong>European</strong> eel, <strong>the</strong> basal nonfeeding<br />

ammonia excretion was increased in a low pH environment (Paper III). In two Antarctic<br />

species, Pachycara brachycephalum and Lepidonoto<strong>the</strong>n kempi, <strong>the</strong> protein syn<strong>the</strong>sis rate <strong>of</strong> liver<br />

cells was decreased by 80% in response to a hypercapnia-induced acidosis (Langenbuch and<br />

Pörtner, 2003). Toge<strong>the</strong>r, <strong>the</strong>se observations suggest that hypercapnia can cause a disturbance in <strong>the</strong><br />

protein metabolism <strong>of</strong> fish towards an increased catabolism (breakdown) and a decreased anabolism<br />

(syn<strong>the</strong>sis).<br />

40


CONCLUSIONS AND PERSPECTIVES<br />

This dissertation represents a series <strong>of</strong> studies which share <strong>the</strong> common element <strong>of</strong> cardio<strong>respiratory</strong><br />

capabilities <strong>of</strong> <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla) under extreme conditions. The<br />

majority <strong>of</strong> conditions studied are environmental; such as temperature, dissolved oxygen and carbon<br />

dioxide, while a fourth condition is physiological, focusing on <strong>the</strong> impressive spawning migration<br />

<strong>of</strong> A. anguilla.<br />

Temperature has been dubbed <strong>the</strong> `ecological master factor´ due to its influence on <strong>the</strong> rate <strong>of</strong> most<br />

biological functions. One <strong>of</strong> <strong>the</strong> objectives <strong>of</strong> this PhD <strong>the</strong>sis was to examine <strong>the</strong> influence <strong>of</strong><br />

temperature on basal and maximum rates <strong>of</strong> oxygen consumption, including those considered to be<br />

extremes, in order to quantify <strong>the</strong> aerobic metabolic capacity <strong>of</strong> A. anguilla over <strong>the</strong> entire range <strong>of</strong><br />

temperatures that this species might encounter in its natural environment. We found A. anguilla to<br />

have a wide <strong>the</strong>rmal optimum as aerobic scope was constant between 10°C and 30°C, and eels were<br />

able to maintain a high oxygen uptake, even at <strong>the</strong> highest temperature studied (30°C). Still, <strong>the</strong><br />

scope for o<strong>the</strong>r activities was considerably reduced as aerobic metabolism could only be increased<br />

about 3 fold at <strong>the</strong> highest temperature, a condition that would limit for instance <strong>the</strong> rate <strong>of</strong><br />

postprandial processes. Extrapolation from <strong>the</strong> results <strong>of</strong> <strong>the</strong>se studies suggests that <strong>the</strong> upper<br />

critical temperature, where aerobic capacity is reduced to zero, occurs at 42.8C. In an ecological<br />

context, quantifying metabolic scope and <strong>the</strong>rmal tolerance windows <strong>of</strong> fishes and o<strong>the</strong>r aquatic<br />

animals contributes to <strong>the</strong> understanding <strong>of</strong> how temperature influences <strong>the</strong> bio-geographical<br />

distribution <strong>of</strong> aquatic species.<br />

Ambient temperature has direct effects on physiological functions for instance <strong>the</strong> heart rate, while<br />

some effects occur gradually through <strong>the</strong> process <strong>of</strong> acclimation. After long term acclimation to 0°C<br />

and 10°C, we observed in A. anguilla that <strong>the</strong> size <strong>of</strong> <strong>the</strong> ventricle relative to body mass was<br />

increased compared to individuals acclimated to 20°C. A ventricular enlargement can be viewed as<br />

a compensatory mechanism for <strong>the</strong> increase in blood viscosity at low temperatures, as it may serve<br />

to increase <strong>the</strong> ability to generate pressure. It may also serve to compensate for <strong>the</strong> considerably<br />

prolonged duration <strong>of</strong> contraction that was observed at low temperature (0°C) in vitro, as this<br />

suggests a limit in maximum attainable heart rate in vivo. Besides changes in gross morphology,<br />

seasonal changes in temperature may also elicit ultra-structural changes e.g. in mitochondrial<br />

densities or <strong>the</strong> densities <strong>of</strong> different Ca 2+ channels and moreover, <strong>the</strong> expression <strong>of</strong> different<br />

functional protein is<strong>of</strong>orms within <strong>the</strong> contractile apparatus. Fur<strong>the</strong>r knowledge <strong>of</strong> this may come<br />

41


from future studies. A direct effect <strong>of</strong> temperature was observed after an acute decrease in ambient<br />

temperature from 20°C to 10°C, as <strong>the</strong> time-course <strong>of</strong> contraction was prolonged. At <strong>the</strong> same time<br />

myocardial contractility was increased which may serve to <strong>of</strong>fset <strong>the</strong> depressant effect on heart rate<br />

and thus ensure adequate cardiac performance when diving to cooler depths.<br />

The critical O 2 partial pressure (P CRIT ) was positively correlated with temperature in A. anguilla,<br />

indicating that adequate transfer <strong>of</strong> oxygen from <strong>the</strong> environment became progressively limited at<br />

higher temperatures. From this it can be concluded that hypoxia tolerance is temperature dependent<br />

in A. anguilla and <strong>the</strong> determinations <strong>of</strong> P CRIT across <strong>the</strong> broad temperature spectrum may prove<br />

useful in <strong>the</strong> assessments <strong>of</strong> suitable environments for A. anguilla. Exposure to low levels <strong>of</strong><br />

dissolved oxygen, where basal energy requirements cannot be sustained aerobically, led to excess<br />

oxygen consumption when normoxic conditions were restored demonstrating a switch to anaerobic<br />

ATP production. Again, temperature was observed to have an influence on different aspects <strong>of</strong> this<br />

excess post-hypoxic oxygen consumption (EPHOC). The total amount <strong>of</strong> EPHOC increased with<br />

temperature, reflective <strong>of</strong> <strong>the</strong> influence <strong>of</strong> temperature on metabolic rate. Recovery time at 0°C was<br />

reduced to about a third compared to 10, 20, and 30°C, which may have been a consequence <strong>of</strong> <strong>the</strong><br />

hypo-metabolic state at this temperature, but may it may also indicate a difference in <strong>the</strong> use <strong>of</strong><br />

metabolic substrates during anaerobiosis. A follow-up study on this subject could include<br />

determinations <strong>of</strong> metabolic substrate levels in plasma and in different tissues (e.g. white muscle<br />

and liver), as well as cellular energy status and enzyme activities. The total amount <strong>of</strong> O 2 consumed<br />

during recovery was modest compared to <strong>the</strong> few o<strong>the</strong>r species that have been examined, which<br />

may be related to an ability <strong>of</strong> A. anguilla to reduce cellular ATP demands during anoxia. The<br />

influence <strong>of</strong> temperature on this ability is unknown and also warrants future attention. The fraction<br />

<strong>of</strong> <strong>the</strong> aerobic scope utilized for recovery from severe hypoxic exposure was greatest at <strong>the</strong> extreme<br />

temperatures. The implications <strong>of</strong> this would be that hypoxic events narrow <strong>the</strong> <strong>the</strong>rmal tolerance<br />

windows and thus represents a `habitat squeeze´ for <strong>the</strong> <strong>European</strong> eel.<br />

Hypercapnia is a common problem in recirculation aquaculture systems especially in those that<br />

practise high stocking densities. Removal <strong>of</strong> excess CO 2 can be problematic and represents an<br />

added expense that must be balanced by an increased yield. Fish differ in <strong>the</strong>ir tolerance to elevated<br />

CO 2 as assessed by studying various physiological parameters including cardio-<strong>respiratory</strong> function<br />

and acid-base homeostasis. For a few cultured species, it has now been demonstrated that<br />

hypercapnia can affect fish health and growth. Although past studies on <strong>the</strong> A. anguilla have<br />

demonstrated a high degree <strong>of</strong> tolerance for elevated CO 2 with no limitations in aerobic capacity,<br />

42


swimming performance and cardiac function, we found that hypercapnia affected <strong>the</strong> postprandial<br />

processes in this species adversely. We observed that eels exposed to hypercapnia took longer time<br />

digesting a meal and that <strong>the</strong> excretion <strong>of</strong> nitrogen was reduced. From <strong>the</strong>se observations we<br />

propose that hypercapnia may limit <strong>the</strong> appetite and feed intake as it takes longer time to process a<br />

meal. In addition, a smaller amount <strong>of</strong> dietary energy and nitrogen content will be allocated to<br />

growth due to a reduced absorption /assimilation efficiency. Fish feed is expensive especially feed<br />

for carnivorous species like A. anguilla that require a high protein content in <strong>the</strong>ir diet, and<br />

<strong>the</strong>refore it must be an overall objective to reduce <strong>the</strong> waste <strong>of</strong> resources. We propose that fur<strong>the</strong>r<br />

studies should be made looking into <strong>the</strong> effect <strong>of</strong> hypercapnia on nitrogen metabolism and growth<br />

in A. anguilla as well as in o<strong>the</strong>r cultured fish species.<br />

The <strong>European</strong> eel undertakes <strong>the</strong> longest spawning migration <strong>of</strong> any known fish species (5-<br />

6000km), a feat that has and continues to attract, considerable scientific interest. The use <strong>of</strong> satellite<br />

tracking technology has provided a wealth <strong>of</strong> information on <strong>the</strong> migration routes <strong>of</strong> many species<br />

<strong>of</strong> fish, including eels, but is currently restricted to larger species due to <strong>the</strong> physical size <strong>of</strong> <strong>the</strong><br />

tags. The objective <strong>of</strong> <strong>the</strong> fourth study was to assess <strong>the</strong> feasibility <strong>of</strong> using satellite tags, by<br />

assessing how swimming with a tag affects <strong>the</strong> swimming efficiency <strong>of</strong> migrating eel. The results <strong>of</strong><br />

<strong>the</strong> study showed that swimming with an externally attached tag was associated with an increase in<br />

energy expenditure at <strong>the</strong> optimal cruising speed, a reduced swimming efficiency and a poorer<br />

swimming performance. These effects were in all likelihood a direct consequence <strong>of</strong> an additional<br />

hydraulic drag from <strong>the</strong> tag. The increase in energy expenditure will have implications for a<br />

successful spawning migration as lipid energy reserves may not <strong>the</strong>n be sufficient to fuel both <strong>the</strong><br />

journey and <strong>the</strong> development <strong>of</strong> gonads that occurs en route. Alternatively, if cruising speed was<br />

lowered to reduce energy expenditure, <strong>the</strong> consequence could be an untimely arrival at <strong>the</strong><br />

spawning site. Uncovering <strong>the</strong> oceanic journey <strong>of</strong> <strong>the</strong> <strong>European</strong> eel, must be considered a high<br />

priority and an important step in fully understanding this species for future management and<br />

conservation purposes, and this is likely to occur through advances in tracking technology.<br />

43


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56


PAPER I


The influence <strong>of</strong> temperature and hypoxia on oxygen consumption in <strong>the</strong><br />

<strong>European</strong> eel (Anguilla anguilla) over a wide range <strong>of</strong> temperatures<br />

C. METHLING a *, P.V. SKOV b AND J.F. STEFFENSEN a<br />

a Marine Biological Section, University <strong>of</strong> Copenhagen Strandpromenaden 5, DK-3000, Helsingør<br />

b National <strong>Institut</strong>e <strong>of</strong> Aquatic Resources, Section for Aquaculture, Technical University <strong>of</strong><br />

Denmark, North Sea Science Park DK-9850, Hirtshals<br />

Abstract<br />

Resting and maximum oxygen consumption rates (MO 2 ) were measured in <strong>the</strong> <strong>European</strong> eel<br />

(Anguilla anguilla) acclimated to 0, 10, 20 and 30°C in order to quantify <strong>the</strong> effect <strong>of</strong> ambient<br />

temperature on metabolic rate and on aerobic metabolic scope. Standard metabolic rate (SMR),<br />

maximum metabolic rate (MMR) and absolute metabolic scope (MS ABS ) was significantly<br />

suppressed at 0°C compared to 10C as indicated by values <strong>of</strong> Q 10 (3.5, 4.1 and 4.2 for SMR,<br />

MMR and MS ABS respectively). SMR and MMR increased significantly with increasing acclimation<br />

temperature, and thus MS ABS was maintained between 10° and 30°C. The factorial scope (MS FAC ),<br />

on <strong>the</strong> o<strong>the</strong>r hand was significantly suppressed at temperatures above 10°C. The ability to regulate<br />

MO 2 during hypoxia in relation to ambient temperature was assessed by determination <strong>of</strong> <strong>the</strong><br />

critical O 2 partial pressure (P CRIT ) at 0, 10, 20 and 30°C. P CRIT increased significantly with<br />

temperature, and this was best described by an exponential function according to: P CRIT = 0.97*exp<br />

(0.061*TEMP) , (R 2 = 0.99). Excess post-hypoxic oxygen consumption (EPHOC) was quantified after 2<br />

hours <strong>of</strong> severe hypoxia exposure over <strong>the</strong> same range <strong>of</strong> temperatures. MO 2 remained elevated<br />

above SMR for 54 minutes at 0°C, while at 10, 20 and 30°C, recovery time was significantly longer<br />

(152-163 minutes). EPHOC significantly increased with temperature between 0°C and 20°C. The<br />

fraction <strong>of</strong> MS ABS occupied during recovery from hypoxia exposure was elevated at <strong>the</strong> temperature<br />

extremes, most pronounced at 30°C, with ~60% <strong>of</strong> MS ABS occupied. The present study confirms <strong>the</strong><br />

metabolic depression observed in Anguilla spp. at low seasonal temperatures, and for <strong>the</strong> first time<br />

provides an estimate <strong>of</strong> metabolic scope at <strong>the</strong> temperature extremes for A. anguilla. A wide<br />

<strong>the</strong>rmal tolerance was demonstrated at normoxia, however this would be narrowed following<br />

hypoxic events as EPHOC occupied a larger a larger proportion <strong>of</strong> MS ABS at <strong>the</strong> extreme<br />

temperatures.<br />

Key words: Anguilla anguilla, temperature, aerobic scope, hypoxia, critical O 2 partial pressure,<br />

excess post-hypoxic O 2 consumption.<br />

1


Introduction<br />

Ambient temperature is unequivocally <strong>the</strong> most important physical factor in aquatic environments,<br />

as it influences most biochemical and physiological processes <strong>of</strong> ecto<strong>the</strong>rmic animals (Brett and<br />

Groves, 1979). Ano<strong>the</strong>r important factor is dissolved oxygen. The oxygen content <strong>of</strong> water bodies<br />

can be influenced by several natural factors, both temporal and spatial, including; patterns <strong>of</strong> algal<br />

respiration, stratification, flooding, ice covers, in addition to a range <strong>of</strong> anthropogenic activities<br />

(Diaz and Breitburg, 2009). Hypoxia can be defined as shortage <strong>of</strong> oxygen and can occur at any<br />

level <strong>of</strong> DO, where an O 2 -requiring process becomes compromised. Temperature is defined as a<br />

controlling factor, as it determines <strong>the</strong> metabolic rate, while dissolved oxygen is considered a<br />

limiting factor as it sets an upper limit to aerobic metabolism (Fry, 1971). Thus both temperature<br />

and dissolved oxygen place constraints on <strong>the</strong> energy metabolism <strong>of</strong> aquatic animals, constraints<br />

that reduce <strong>the</strong> aerobic scope, scope for activity and <strong>the</strong>reby influence <strong>the</strong> Darwinian fitness <strong>of</strong> <strong>the</strong><br />

organism (Fry, 1971; Priede, 1985; Portner et al., 2008). The metabolic scope <strong>of</strong> an organism can be<br />

considered an integral measure <strong>of</strong> <strong>the</strong> quality <strong>of</strong> <strong>the</strong> environment in which it resides (Claireaux et<br />

al., 2007). Thus it was recently demonstrated, that <strong>the</strong> scope for activities like swimming, feeding<br />

etc. is proportional to dissolved oxygen in ano<strong>the</strong>r temperate species, <strong>the</strong> Atlantic cod (Gadus<br />

morhua), (Chabot et al., 2008). Also in Pacific salmon (Oncorhynchus nerka), successful spawning<br />

migration was recently linked to aerobic scope (Farrell et al., 2008).<br />

If confined to environments where <strong>the</strong> oxygen content becomes sparse, <strong>the</strong> vast majority <strong>of</strong> fish<br />

species can regulate <strong>the</strong>ir oxygen uptake by making behavioural, <strong>respiratory</strong> and circulatory<br />

adjustments. However, a constant O 2 uptake rate, sustaining <strong>the</strong> basal metabolic demands, can only<br />

be maintained down to a certain point that is species specific. This critical point (P CRIT ) represents a<br />

measure <strong>of</strong> <strong>the</strong> animal´s capacity to extract O 2 from <strong>the</strong> environment and can be used as an indicator<br />

<strong>of</strong> hypoxia tolerance (Chapman et al., 2002). However, this value <strong>of</strong> P CRIT refers to <strong>the</strong> minimum<br />

metabolic requirements, and <strong>the</strong> partial pressures at which swimming, feeding and reproduction are<br />

compromised will be higher (Richards, 2009). Once O 2 partial pressure falls below P CRIT , <strong>the</strong><br />

maximum amount <strong>of</strong> O 2 that can be extracted from <strong>the</strong> water conforms to <strong>the</strong> ambient PO 2 . Survival<br />

below P CRIT depends on <strong>the</strong> ability to suppress cellular ATP demands and <strong>the</strong> amount <strong>of</strong> fuels<br />

available for anaerobic ATP production (Hochachka et al., 1996). The <strong>European</strong> eel (Anguilla<br />

anguilla) is notoriously tolerant to hypoxic conditions, and is able to overwinter in muddy hypoxic<br />

sediments (Bertin, 1956; Nyman, 1972; Andersen et al., 1985), survive prolonged aerial exposure<br />

(Hyde et al., 1987; Hyde et al., 1987) and even several hours (5.7 hrs at 15°C) <strong>of</strong> anoxia (van<br />

2


Waarde et al., 1983). When introduced to hypoxic environments, anguillid eels display a typical<br />

piscine response i.e. hyperventilation (Chan1986; Peyraud-Waitzenegger et al., 1989; CruzNeto et<br />

al., 1997) and lowering <strong>of</strong> <strong>the</strong> heart rate (reflex bradycardia), (Peyraud-Waitzenegger et al., 1989;<br />

Iversen et al., 2010), but a compensatory increase in stroke volume has not been observed (Chan,<br />

1986; Peyraud-Waitzenegger et al., 1989; Iversen et al., 2010). Also, anguillid eels possess<br />

functionally heterogenous hemoglobin components, with one component being less sensitive to pH<br />

changes and having a high O 2 -affinity and cooperativity (Fago et al., 1995; Brauner et al., 1998;<br />

Weber et al., 2004), which confer protection against hypoxia and acidosis (Fago et al., 1995;<br />

Brauner et al., 1998; Weber et al., 2004). During complete anoxia, eels are able to suppress whole<br />

animal metabolic rate by 70% (van Ginneken et al., 2001), reduce hepatic ATP production by 85%<br />

and stabilize cellular ATP levels to prolong survival during this extreme condition (Busk et al.,<br />

2005). However, <strong>the</strong> ability to survive short-term anoxic conditions is not correlated with an ability<br />

to ferment glucose to ethanol as observed in some Cyprinid species (van Waarde et al., 1983; van<br />

Ginneken et al., 2001).<br />

Recovery from environmental hypoxia (or anoxia) has received comparatively little attention<br />

compared to <strong>the</strong> oxygen debt following exhaustive exercise, but is also characterized by an increase<br />

in oxygen consumption (van den Thillart et al., 1991; Nonnotte et al., 1993; Maxime et al., 2000;<br />

Virani et al., 2000; Mandic et al., 2008; Svendsen et al., 2012). The excess <strong>of</strong> oxygen consumed<br />

during recovery, can be viewed as an reflection <strong>the</strong> anaerobic capacity as it is used for restoring<br />

cellular levels <strong>of</strong> ATP and phosphocreatine (CrP), re-syn<strong>the</strong>sises <strong>of</strong> glycogen and for clearing<br />

anaerobic end products (Scarabello et al., 1992; Mandic et al., 2008). Recovery from hypoxia has<br />

recently been referred to as excess post-hypoxic oxygen consumption (EPHOC) as to distinguish<br />

this from excess post-exercise oxygen consumption (EPOC) (Svendsen et al., 2012).<br />

The primary objective <strong>of</strong> <strong>the</strong> present study was to quantify <strong>the</strong> aerobic metabolic scope <strong>of</strong> <strong>the</strong><br />

<strong>European</strong> eel (Anguilla anguilla) over <strong>the</strong> wide range <strong>of</strong> temperatures (0-30°C) that occur in <strong>the</strong>ir<br />

natural environment. A second objective was to determine <strong>the</strong> critical oxygen tension (P CRIT ) for <strong>the</strong><br />

standard metabolic rate (SMR) over <strong>the</strong> same range <strong>of</strong> temperatures to assess <strong>the</strong> influence <strong>of</strong><br />

temperature on hypoxia tolerance. A final objective was to quantify excess post-hypoxic oxygen<br />

consumption (EPHOC) after 2 hours exposure to severe hypoxia and examine how this was related<br />

to ambient temperature. A thorough understanding <strong>of</strong> when environmental conditions become<br />

3


adverse is critical for future management and conservation <strong>of</strong> this species that is now considered<br />

critically endangered (Freyh<strong>of</strong> and Kottelat, 2010).<br />

Materials and methods<br />

Experimental animals<br />

<strong>European</strong> eel, Anguilla anguilla, were caught in <strong>the</strong> Øresund with traps and transported to <strong>the</strong><br />

Marine Biological Laboratory in Helsingør, Denmark in <strong>the</strong> fall <strong>of</strong> 2010. Fish were kept in a large<br />

circular tank with a volume <strong>of</strong> 2500 litres and supplied with 10°C re-circulated 35‰ aerated<br />

seawater. The light regime was 16h light /8h dark. The fish did not eat, although <strong>of</strong>fered a variety <strong>of</strong><br />

food items. Fish were acclimated to laboratory conditions for one month prior to temperature<br />

acclimation. For temperature acclimation to 0°C, eels were transferred to a separate insulated 450L<br />

tank supplied with 10°C seawater at a reduced flow. Cooling was achieved by pumping water from<br />

<strong>the</strong> tank through <strong>the</strong> heat exchanger on a custom built industrial-grade cooler. Flow <strong>of</strong> coolant<br />

through <strong>the</strong> heat exchanger was controlled by a motorized 3-way valve connected to a <strong>the</strong>rmostat.<br />

For acclimation to 20°C and 30°C eels where transferred to two insulated 250L aquaria Heating was<br />

achieved by submerging a set <strong>of</strong> 500W titanium aquarium heaters into <strong>the</strong> tank, and was regulated<br />

by a <strong>the</strong>rmostat. Cooling/heating to desired acclimation temperature was done at a rate <strong>of</strong> 1°C per<br />

day. Fish were kept at <strong>the</strong> final acclimation temperature for at least 4 weeks. The study was carried<br />

out in accordance with <strong>the</strong> EC Directive 86/609/EEC.<br />

Respirometry setup:<br />

Mass specific oxygen consumption (MO 2 ) was measured by <strong>the</strong> principle <strong>of</strong> computerized<br />

intermittent flow through respirometry (Steffensen, 1989). Two 2.1L Plexiglas respirometers where<br />

submerged in a 50L outer tank supplied with water from a reservoir. To maintain good quality,<br />

water in <strong>the</strong> holding tank was continuously aerated, filtered and circulated through an aquarium UV<br />

sterilizer to minimize bacterial growth. Temperature was kept constant at ± 0.1°C by controlling <strong>the</strong><br />

flow <strong>of</strong> water through a cooler (CBN 8-30; Heto, Denmark), equipped with <strong>the</strong>rmostatted heaters<br />

(HMT 200; Heto, Denmark). The respirometers were periodically flushed for 4 minutes with water<br />

from <strong>the</strong> outer tank, followed by a closed 1 minute waiting period to obtain steady state and a 5<br />

minute measuring period. Oxygen partial pressure (PO 2 ) was continuously measured by a set <strong>of</strong><br />

fibre optic oxygen transmitters (Fibox 3 from PreSens GmbH, Germany) and recorded by <strong>the</strong><br />

AutoResp4 s<strong>of</strong>tware (Loligo Systems, Denmark). MO 2 was derived from <strong>the</strong> decrease in PO 2<br />

during <strong>the</strong> 5 minute measuring period according to: MO 2 = V((pO 2 )/t) αM -1 , where V is <strong>the</strong><br />

4


volume <strong>of</strong> <strong>the</strong> respirometer, α is <strong>the</strong> specific oxygen solubility and M is <strong>the</strong> mass <strong>of</strong> <strong>the</strong> fish.<br />

Desired PO 2 levels were achieved by injecting pure N 2 or O 2 gas into a 1 meter water column<br />

connected to <strong>the</strong> outer tank. Flow <strong>of</strong> gas was controlled via a solenoid valve, by a programmable<br />

LED instrument (PR electronics, Rønde, Denmark) that received signal from a OxyGuard mini<br />

probe (OxyGuard International A/S, Birkerød, Denmark).<br />

Protocol:<br />

Experimental eels were removed from <strong>the</strong> holding tank, subjected to a chasing protocol <strong>of</strong> no more<br />

than 10 minutes duration and <strong>the</strong>n introduced to <strong>the</strong> respirometer. The first measurement was taken<br />

to be representative <strong>of</strong> <strong>the</strong> maximum metabolic rate (MMR). Measurements recorded <strong>the</strong> following<br />

48 hours were used to determine a value <strong>of</strong> <strong>the</strong> standard metabolic rate (SMR). Oxygen tension<br />

(PO 2 ) was <strong>the</strong>n reduced in a stepwise manner and maintained at each set point for 3 consecutive<br />

measurement periods (total 30min). Once P CRIT was reached eels were subjected to a 2 hour severe<br />

hypoxia challenge. A severe hypoxia challenge was chosen instead <strong>of</strong> exposing eel to full anoxia to<br />

minimize <strong>the</strong> risk <strong>of</strong> fatalities. In order to make comparisons between temperatures, it was decided<br />

that <strong>the</strong> hypoxia challenge should correspond to 0.5 times <strong>the</strong> critical oxygen tension (P CRIT ) for<br />

each experimental temperature. The temperature specific P CRIT was determined preliminarily in a<br />

pilot study (N=2) that was performed on eels from <strong>the</strong> same acclimation groups as in <strong>the</strong> main<br />

study. Determination After 2 hours PO 2 was returned to normoxic levels and MO 2 recorded until<br />

values had reached SMR.<br />

Calculations and statistics:<br />

Standard metabolic rate (SMR) was estimated by fitting data to a bimodal normal distribution<br />

(Steffensen 1989). Absolute metabolic scope (MS ABS ) was quantified as MS ABS = MMR-SMR and<br />

factorial metabolic scope (MS FAC ) as MS FAC = MMR/SMR. A temperature curve for MS ABS was<br />

produced by subtracting <strong>the</strong> regression curve for SMR from <strong>the</strong> regression curve for MMR. The<br />

upper and lower critical temperatures were estimated as <strong>the</strong> points were <strong>the</strong> SMR and MMR curves<br />

intercepted, i.e. where MS ABS is zero. The critical partial pressure <strong>of</strong> oxygen (P CRIT ) was quantified<br />

by linear regression <strong>of</strong> MO 2 values below SMR during hypoxia versus PO 2 . P CRIT was determined as<br />

<strong>the</strong> PO 2 where <strong>the</strong> regression line intercepted with <strong>the</strong> estimated SMR. To quantify excess post<br />

hypoxic oxygen consumption (EPHOC), a double exponential equation: f(x) = a*exp (b*x) +<br />

c*exp(d*x) + SMR was fitted to <strong>the</strong> post-hypoxic MO 2 measurements (Scarabello et al., 1991).<br />

Individual equations were integrated to determine <strong>the</strong> area under <strong>the</strong> curve and <strong>the</strong> area occupied by<br />

5


SMR was subtracted to quantify EPHOC. (TableCurve 2D v. 4, Systat s<strong>of</strong>tware Inc. USA). The<br />

recovery pr<strong>of</strong>ile was fur<strong>the</strong>r analyzed by quantifying: <strong>the</strong> duration <strong>of</strong> EPHOC, <strong>the</strong> post-hypoxic<br />

peak in MO 2 , (MO 2,Peak ,), <strong>the</strong> % utilization <strong>of</strong> absolute scope during <strong>the</strong> peak and % utilization <strong>of</strong><br />

absolute scope during <strong>the</strong> entire duration <strong>of</strong> EPHOC. The effect <strong>of</strong> temperature (Q 10 ) on MO 2 was<br />

quantified according to <strong>the</strong> van’t H<strong>of</strong>f equation: Q 10 = (MO 2, (2) / MO 2, (1) ) ^(10/t 2 -t 1 ). To determine if<br />

observed difference between temperatures was statistically significant, a one-way ANOVA was<br />

performed on all variables (SigmaPlot v.11, Systat s<strong>of</strong>tware Inc. USA) accepting a P


At 0°C, MMR was significantly decreased and in <strong>the</strong> low temperature interval (0-10°C), MMR was<br />

strongly influenced by temperature with a Q 10 <strong>of</strong> 4.08. At 30°C, MMR was significantly higher than<br />

at 20°C. In <strong>the</strong> range between 10°C and 30°C, temperature had no significant effect on <strong>the</strong> absolute<br />

metabolic scope with Q 10 values <strong>of</strong> 0.95 and 1.09 between 10-20°C and 20-30°C respectively. A<br />

large and significant temperature effect on absolute scope was observed between 0°C and 10°C, Q 10<br />

=4.17 (Table 1). The factorial scope however, was not significantly different between 0°C and<br />

10°C, with a Q 10 =0.94. MS FAC was significantly decreased at 20°C compared to 10°C and again at<br />

30°C compared to 20°C (Table 1).<br />

The temperature curve for MS ABS is shown in Figure 1. SMR as a function <strong>of</strong> temperature (T) was<br />

fitted to an exponential equation: SMR = 7.46*exp 0.075*T with R 2 = 0.995 (Figure 1). MMR as a<br />

function <strong>of</strong> temperature was fitted to a polynomial equation: MMR = 47.69 +10.93*T -0.181*T 2 ,<br />

with R 2 = 0.929 (Figure 1). The upper and lower critical temperatures were estimated to be 42.67°C<br />

and -3.63°C respectively. The maximum value <strong>of</strong> MS ABS was estimated to be 161.38 mgO 2 kg -1 h -1<br />

and occur at <strong>the</strong> optimal temperature <strong>of</strong> 22.04°C. The upper and lower temperature, where MS ABS is<br />

80% <strong>of</strong> maximum was estimated to be 32.01°C and 11.04°C respectively.<br />

Figure 1. Temperature curve for aerobic metabolic scope curve in A. anguilla.<br />

MO 2<br />

(mgO 2<br />

kg -1 h -1 )<br />

200<br />

150<br />

100<br />

50<br />

0<br />

SMR<br />

MS ABS<br />

MMR<br />

0 10 20 30 40<br />

Temperature (°C)<br />

Solid lines represent regression lines for SMR<br />

(=7.46*exp ( 0.075*T ), R 2 = 0.995) and MMR (= 47.69<br />

+10.93*T -0.181*T 2 , R 2 = 0.929), where T is<br />

temperature (°C). Dashed line represents <strong>the</strong><br />

metabolic scope (MS ABS ) curve as constructed by<br />

subtracting <strong>the</strong> regression line for SMR from <strong>the</strong><br />

regression line <strong>of</strong> MMR. Refer to Table 1 for data<br />

points. Vertical lines represent <strong>the</strong> temperature<br />

where MS ABS is maximized, and <strong>the</strong> upper and<br />

lower temperature corresponding to 80% <strong>of</strong><br />

maximum MS ABS . The upper and lower critical<br />

temperatures correspond to <strong>the</strong> intercepts between<br />

<strong>the</strong> SMR and MMR curves. See text for fur<strong>the</strong>r<br />

details.<br />

Hypoxia and EPHOC<br />

The critical oxygen saturation (P CRIT ) increased significantly with temperature (Figure 2); a<br />

relationship best described by <strong>the</strong> equation: P CRIT = 0.99*exp 0.060*T , (R 2 = 0.999), where T is<br />

temperature (°C). When normoxic conditions were restored after <strong>the</strong> 2 hours exposure to severe<br />

hypoxia, MO 2 increased relative to <strong>the</strong> pre-hypoxic levels <strong>of</strong> SMR (Table 2). The post-hypoxic peak<br />

in MO 2 increased significantly with temperature and MO 2 remained elevated for 54 minutes at 0°C,<br />

7


Figure 2. Determinations <strong>of</strong> <strong>the</strong> critical O 2 tension (P CRIT ) in A. anguilla at a range <strong>of</strong> temperatures.<br />

P CRIT<br />

(kpa)<br />

12<br />

10<br />

8<br />

6<br />

4<br />

2<br />

a<br />

b<br />

A<br />

0 10 20 30<br />

Temperature (°C)<br />

B<br />

c<br />

C C<br />

D<br />

E<br />

d<br />

<strong>Eel</strong>s were subjected to a progressive decline in ambient PO 2 ,<br />

while measuring <strong>the</strong> MO 2 . P CRIT was quantified as <strong>the</strong> PO 2<br />

MO 2 fell below <strong>the</strong> standard metabolic rate (SMR). Symbols<br />

and error bars represent group means ± s.e.m. (N=7-10). Line<br />

is <strong>the</strong> regression line described by: P CRIT = 0.99*exp<br />

(0.060*T), with R 2 = 0.999 and T is temperature. Lower case<br />

letters above symbols indicate significant differences between<br />

temperatures (One-way ANOVA, p


anguilla under a broad range <strong>of</strong> temperatures including <strong>the</strong> extremes (0°C and 30°C). In <strong>the</strong> closely<br />

related American eel (A. rostrata), temperature has a strong influence on standard metabolic rate<br />

between 5 and 10°C with a Q 10 <strong>of</strong> 4.10, while at higher ranges <strong>of</strong> temperature (i.e. 10-15°C and 15-<br />

20°C) <strong>the</strong> influence is lessened with Q 10 <strong>of</strong> 1.12 and 1.65, respectively (Walsh et al., 1983). The<br />

slight difference in Q 10 values between <strong>the</strong> present study and <strong>the</strong> study by Walsh and co-workers<br />

(1983) could be attributed to a difference in size (~55g vs ~285g) as <strong>the</strong> temperature effect on MO 2<br />

decreases with body size in A. rostrata (Degani et al., 1989).The high Q 10 values for SMR and<br />

MMR in <strong>the</strong> low temperature range corresponds well with <strong>the</strong> low activity (torpor) displayed by<br />

both Anguilla species eels under <strong>the</strong>se conditions. Also in A. rostrata <strong>the</strong> pH <strong>of</strong> white muscle is not<br />

regulated according to <strong>the</strong> alpha-stat model at decreasing temperatures, which effectively causes a<br />

tissue acidification affecting enzyme activities (Walsh et al., 1982). Considering that white muscle<br />

comprises ~50% <strong>of</strong> body mass in fish (Goolish, 1991) significant metabolic savings can be accrued<br />

by suppressing metabolic rate in this tissue. Several <strong>respiratory</strong> adjustments to low ambient<br />

temperature have been observed in A. anguilla, which could influence MMR. For one, acclimation<br />

to low temperature (5°C) causes a left shift <strong>of</strong> <strong>the</strong> O 2 -dissociation curve, which increases <strong>the</strong><br />

haemoglobin O 2 -affinity, but at <strong>the</strong> same time reduces <strong>the</strong> O 2 -unloading potential at <strong>the</strong> tissues<br />

(Andersen et al., 1985). Secondly, acclimation to low temperature (5°C) causes a thickening <strong>of</strong> <strong>the</strong><br />

branchial water-blood barrier by a factor 2.5 compared to 25°C (Tuurala et al., 1998). These<br />

changes may impair <strong>the</strong> O 2 -uptake from <strong>the</strong> environment and <strong>of</strong>fers a plausible explanation as to<br />

why <strong>the</strong> MMR was strongly depressed at 0°C. The low MMR at 0°C could also reflect a limitation<br />

in cardiac performance, as <strong>the</strong> maximal attainable in vitro heart rate in A. anguilla at 0°C is between<br />

6-12 beats per minute (Methling et al., 2012b). In <strong>the</strong> o<strong>the</strong>r end <strong>of</strong> <strong>the</strong> <strong>the</strong>rmal spectrum, MMR was<br />

not limited at 30°C, which suggests that nei<strong>the</strong>r <strong>the</strong> O 2 -extraction nor <strong>the</strong> O 2 -delivery had become<br />

limited even at this high temperature.<br />

According to <strong>the</strong> concept <strong>of</strong> oxygen and capacity limited <strong>the</strong>rmal tolerance (OCLTT), aerobic scope<br />

decreases towards <strong>the</strong> temperature extremes due to limitations in <strong>the</strong> oxygen cascade. Aerobic scope<br />

has a window <strong>of</strong> optimal temperatures that is defined by an upper and lower threshold temperature<br />

(termed pejus temperatures) beyond which, aerobic scope decreases and <strong>the</strong>re is a loss <strong>of</strong><br />

performance and fitness (Portner et al., 2007). At fur<strong>the</strong>r upper and lower temperature extremes<br />

(termed critical temperatures), where aerobic scope is minimal, <strong>the</strong>re is an onset <strong>of</strong> anaerobic<br />

metabolism and protective mechanisms such as metabolic depression are activated (Portner et al.,<br />

2007). The width <strong>of</strong> <strong>the</strong> <strong>the</strong>rmal optimum (window) reflects <strong>the</strong> amplitude <strong>of</strong> temperature<br />

9


fluctuations in natural environment <strong>of</strong> a given species, with temperate species having a wide<br />

<strong>the</strong>rmal window and polar and tropical species having a narrow window (Portner, 2002; Nilsson et<br />

al., 2009). In light <strong>of</strong> this, A. anguilla would be expected to have a wide <strong>the</strong>rmal optimum, which<br />

was also presently confirmed as <strong>the</strong> absolute aerobic scope was not significantly different between<br />

10°C and 30°C. If setting <strong>the</strong> 80% boundaries <strong>of</strong> MS ABS to represent <strong>the</strong> pejus temperatures, A.<br />

anguilla has an estimated optimal <strong>the</strong>rmal window between 11.0°C and 32.0°C. A reduced aerobic<br />

capacity implies that less energy can be expended on routine activities like feeding and swimming,<br />

<strong>the</strong>refore it can be expected that <strong>the</strong>se activities are reduced beyond <strong>the</strong> pejus temperatures. To this<br />

end, cessation <strong>of</strong> feeding occurs around 8-10°C in yellow stage eels (Bruun1963), and below 8°C<br />

eels remain completely inactive (Nyman, 1972). The optimal temperature was presently estimated<br />

to be 22.0°C. This corresponds well with <strong>the</strong> optimal temperature for growth, which have been<br />

reported to be around 22-23°C (Sadler, 1979). Towards <strong>the</strong> extremes, <strong>the</strong> present MS ABS curve was<br />

a less accurate assessment and <strong>the</strong> lower critical temperature was in all likelihood overestimated (-<br />

3.6°C). There are no reported values <strong>of</strong> <strong>the</strong> lower lethal temperature in A. anguilla, however no<br />

mortalities were observed after acclimation to -1.8°C during winter (P.V. Skov, pers. obs.). The<br />

upper lethal temperature has been reported to be 38°C in A. anguilla (Sadler, 1979) and this<br />

suggests that <strong>the</strong> upper critical temperature (42.8°C) was also overestimated in <strong>the</strong> present study.<br />

Thus, <strong>the</strong> actual temperature curve for MS ABS in A. anguilla most likely have steeper slopes towards<br />

<strong>the</strong> extremes, which remains to be verified by quantifying MS ABS at closer intervals .<br />

Even though maximum MS ABS was maintained at 30°C, eels would only be able to increase<br />

metabolism by approximately 3 times that <strong>of</strong> <strong>the</strong> minimum maintenance requirements. This leaves<br />

little room for e.g. feeding activities. For instance, digesting a small meal (0.5% body mass)<br />

occupies approximately 33% <strong>of</strong> <strong>the</strong> available scope during peak SDA at 23°C in A. anguilla<br />

(Methling et al., 2012a). As <strong>the</strong> post-prandial peak in MO 2 increases with temperature (McCue,<br />

2006), digesting a larger meal at 30°C could potentially occupy <strong>the</strong> entire scope thus compromising<br />

o<strong>the</strong>r activities like predator avoidance or increase <strong>the</strong> vulnerability to hypoxia.<br />

Several estimates <strong>of</strong> <strong>the</strong> P CRIT at temperatures between 18-25°C have been reported for A. anguilla<br />

(summarized in Figure 2), but from <strong>the</strong>se earlier reports a clear correlation between temperature and<br />

P CRIT is not apparent. The present study provides evidence for a significant influence <strong>of</strong> temperature<br />

on P CRIT over a broad range <strong>of</strong> temperatures in A. anguilla. The P CRIT observed at 20°C and 30°C,<br />

was in <strong>the</strong> low range <strong>of</strong> previously reported observations, however <strong>the</strong>re is considerable variation<br />

10


among those previous estimates, which may reflect differences in methodology and determination<br />

<strong>of</strong> SMR, or o<strong>the</strong>r factors not controlled for, such as <strong>the</strong>rmal history, nutritional status, season and<br />

size. Considering that oxygen solubility decreases with temperature while O 2 requirements<br />

increases with temperature, fish have to ventilate larger volumes <strong>of</strong> increasingly hypoxic water<br />

across <strong>the</strong>ir gills to maintain a constant MO 2 during hypoxia. Also considering that <strong>the</strong> amount <strong>of</strong><br />

water that can be ventilated across <strong>the</strong> gills is limited by <strong>the</strong> capacity <strong>of</strong> <strong>the</strong> branchial pump (Farrell<br />

et al., 1987), it can rationally be expected that if no o<strong>the</strong>r compensatory mechanisms are brought<br />

into play, P CRIT will increase with temperature. However, some controversy exists with regards to<br />

<strong>the</strong> influence <strong>of</strong> temperature on P CRIT . A positive correlation has been described for several species<br />

<strong>of</strong> fish (Fernandes et al., 1989; Schurmann et al., 1997; Valverde et al., 2006; Corkum et al., 2009).<br />

In some species however, P CRIT is only positively correlated with temperature within a certain range<br />

(Ott et al., 1980; Barnes et al., 2011), while in o<strong>the</strong>rs P CRIT is independent <strong>of</strong> temperature (Ott et al.,<br />

1980; Cerezo et al., 2004). The present results support <strong>the</strong> expected positive correlation between<br />

temperature and P CRIT suggesting that <strong>the</strong> capacity <strong>of</strong> <strong>the</strong> branchial pump may limit <strong>the</strong> maximum<br />

O 2 -uptake in A. anguilla during hypoxia.<br />

Increased rates <strong>of</strong> oxygen consumption have previously been reported in o<strong>the</strong>r species <strong>of</strong> fish<br />

recovering from exposure to severe hypoxia or anoxia (van den Thillart et al., 1991; Nonnotte et al.,<br />

1993; Maxime et al., 2000; Virani et al., 2000; Mandic et al., 2008; Svendsen et al., 2012). The<br />

post-hypoxic increase in MO 2 demonstrated that anaerobic ATP production was activated to<br />

compensate for <strong>the</strong> reduced oxygen availability. EPHOC after exposure to severe hypoxia have only<br />

quantified for a few species. For example, in rainbow trout (Oncorhynchus mykiss) recovering from<br />

approx. 1 hour at a PO 2 corresponding to 1/4 times P CRIT , EPHOC was reported to be 212.6 mgO 2<br />

kg -1 at 10°C (Svendsen et al., 2012). A similar observation was made in turbot (Scophthalmus<br />

maximus) recovering from 1 hour hypoxia corresponding to 1/3 <strong>of</strong> P CRIT , where EPHOC amounted<br />

to 213.1 mgO 2 kg -1 at 14-18°C (Maxime et al., 2000). The values <strong>of</strong> EPHOC observed in eels in <strong>the</strong><br />

present study were considerably lower even at <strong>the</strong> highest temperature than what was observed in<br />

trout and turbot. Also taking into account that <strong>the</strong> hypoxia challenge was more severe in <strong>the</strong> present<br />

study, this suggests that eels instead <strong>of</strong> maintaining a certain rate <strong>of</strong> metabolism via substrate<br />

phosphorylation, suppressed metabolic demands i.e. ATP production as observed during complete<br />

anoxia (van Ginneken et al., 2001; Busk et al., 2005).<br />

11


The increase in EPHOC with temperature most likely reflected <strong>the</strong> increase in basic metabolic rate<br />

as <strong>the</strong> temperature effect was similar to <strong>the</strong> effect on resting oxygen consumption (SMR) i.e. a large<br />

effect in <strong>the</strong> low range <strong>of</strong> temperatures and a less strong effect at higher temperatures. The recovery<br />

process from exhaustive exercise has been divided into two components i.e. an initial fast<br />

component, where internal O 2 , ATP and phosphocreatine (CrP) levels are restored, and a slower<br />

component where o<strong>the</strong>r O 2 consuming processes e.g. gluconeogenesis or oxidation <strong>of</strong> lactate occur<br />

(Scarabello et al., 1991; Gleeson1996). In <strong>the</strong> American eel (A. rostrata), CrP is primarily utilized<br />

as substrate for ATP production during severe hypoxia (at 15°C), while glucogenolysis and lactate<br />

formation becomes increasingly significant during anoxia (van Waarde et al., 1983). The shorter<br />

recovery time at 0°C might suggest that <strong>the</strong> CrP pool was adequate to support ATP demands during<br />

hypoxia at this temperature and that (muscle) glycogen breakdown was <strong>of</strong> minor significance.<br />

Recovering from hypoxia exposure occupied <strong>the</strong> largest fraction <strong>of</strong> <strong>the</strong> available metabolic scope at<br />

<strong>the</strong> temperature extremes especially at 30°C. In accordance with <strong>the</strong> concept <strong>of</strong> OCLLT, this<br />

suggests that hypoxic events at <strong>the</strong> temperature extremes constitute a squeeze in fitness for A.<br />

anguilla by a narrowing <strong>of</strong> <strong>the</strong>rmal tolerance windows (Portner et al., 2008).<br />

Acknowledgements<br />

This study was funded by <strong>the</strong> Danish Agency for Science and Innovation, The Elisabeth and Knud<br />

Petersen Foundation and <strong>the</strong> Faculty <strong>of</strong> Science, University <strong>of</strong> Copenhagen.<br />

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15


PAPER II<br />

Reprinted with permission from Elsevier


Comparative Biochemistry and <strong>Physiology</strong>, Part A 163 (2012) 66–73<br />

Contents lists available at SciVerse ScienceDirect<br />

Comparative Biochemistry and <strong>Physiology</strong>, Part A<br />

journal homepage: www.elsevier.com/locate/cbpa<br />

The temperature challenges on cardiac performance in winter-quiescent and<br />

migration-stage eels Anguilla anguilla<br />

C. Methling a, ⁎, J.F. Steffensen a , P.V. Skov b<br />

a Marine Biological Section, University <strong>of</strong> Copenhagen Strandpromenaden 5, DK-3000, Helsingør, Denmark<br />

b National <strong>Institut</strong>e <strong>of</strong> Aquatic Resources, Section for Aquaculture, Technical University <strong>of</strong> Denmark, North Sea Science Park DK-9850, Hirtshals, Denmark<br />

article<br />

info<br />

abstract<br />

Article history:<br />

Received 8 February 2012<br />

Received in revised form 8 May 2012<br />

Accepted 8 May 2012<br />

Available online 12 May 2012<br />

Keywords:<br />

A. anguilla<br />

Adrenaline<br />

Contractility<br />

Relative ventricular mass<br />

Temperature<br />

Spawning migration<br />

Winter-quiescence<br />

The present study was undertaken to examine cardiac responses to some <strong>of</strong> <strong>the</strong> temperature challenges that<br />

eels encounter in <strong>the</strong>ir natural environment. The contractile properties <strong>of</strong> ventricular muscle was studied on<br />

electrically paced tissue strips after long term acclimation at 0 °C, 10 °C, or 20 °C, and following acute ±10 °C<br />

temperature changes. The time-course <strong>of</strong> contraction, and thus maximal attainable heart rates, was greatly<br />

influenced by working temperature, but was independent <strong>of</strong> acclimation history. The absolute force <strong>of</strong><br />

contraction and power production (i.e. <strong>the</strong> product <strong>of</strong> force and stimulation frequency) was significantly<br />

influenced by acute temperature decrease from 20 °C to 10 °C. The role <strong>of</strong> adrenaline as a modulator <strong>of</strong><br />

contraction force, power production, rates <strong>of</strong> contraction and relaxation, and minimum time in contraction<br />

was assessed. Increased adrenergic tonus elicited a positive inotropic, temperature-dependent response,<br />

but did not influence twitch duration. This suggests that adrenaline acts as an agent in maintaining an adequate<br />

contractile force following temperature challenges. A significant increased relative ventricular mass<br />

was observed in 0 °C and 10 °C-acclimated eels compared to 20 °C-acclimated, which suggests that at<br />

low temperatures, eels secure cardiac output by heart enlargement. Inhibition <strong>of</strong> specific sarcolemmal Ca 2+<br />

channels by selective drug treatment revealed that, depending on temperature, L-type channels is <strong>the</strong> major<br />

entry site, but also that reverse-mode Na + /Ca 2+ -exchange and store operated calcium entry contribute to<br />

<strong>the</strong> pool <strong>of</strong> activator Ca 2+ .<br />

© 2012 Elsevier Inc. All rights reserved.<br />

1. Introduction<br />

The <strong>European</strong> eel (Anguilla anguilla) is an eury<strong>the</strong>rmal fish which<br />

tolerates temperatures ranging from sub-zero to above 30 °C. For<br />

instance, during <strong>the</strong>ir spawning-migration to <strong>the</strong> Sargasso Sea, eels<br />

are faced with a gradual increase in temperature (McCleave, 2003;<br />

Aarestrup et al., 2009). Recently, it was reported that migrating eels<br />

perform diel vertical migration <strong>of</strong> up to 400 m, diving into colder<br />

waters (~6–8 °C) during <strong>the</strong> day and ascending to shallow warmer<br />

waters (12–14 °C) at night (Aarestrup et al., 2009). Meanwhile, nonmigrating<br />

individuals are faced with decreased temperatures during<br />

fall and winter. When <strong>the</strong> temperature starts to decrease in <strong>the</strong> fall,<br />

eels cease activity, eventually become torpid and bury <strong>the</strong>mselves<br />

in <strong>the</strong> sediment until spring where <strong>the</strong>ir activity increases along with<br />

<strong>the</strong> rise in temperature (Bertin, 1956; Nyman, 1972; Walsh et al.,<br />

1983). Under <strong>the</strong>se conditions eels are able to survive to temperatures<br />

down to, or below 0 °C.<br />

At all temperatures faced by <strong>the</strong> eel, a match must exist between activity<br />

levels and cardiac performance that may need to be modulated<br />

⁎ Corresponding author. Tel.: +45 35321977.<br />

E-mail address: cmethling@bio.ku.dk (C. Methling).<br />

accordingly. A common, but not universal response to low seasonal<br />

temperatures is an increase in relative ventricular mass (RVM) or cardiac<br />

somatic index, but no clear correlation between cardiac enlargement<br />

and (in)activity during winter has been established (Driedzic<br />

et al., 1996). Thus cardiac growth has been observed in several species<br />

e.g. rainbow trout (Tsukuda et al., 1985; Graham and Farrell, 1989),<br />

goldfish (Tsukuda et al., 1985), common carp (Goolish, 1987), and sea<br />

raven (Graham and Farrell, 1985), while little or no change in RVM<br />

has been observed in o<strong>the</strong>r species like <strong>the</strong> crucian carp (Matikainen<br />

and Vornanen, 1992), white perch and yellow perch (Sephton and<br />

Driedzic, 1991). Intracellular responses to cold temperatures in fish include<br />

ultrastructural changes e.g. in mitochondrial volume (Rodnick<br />

and Sidell, 1997), expression <strong>of</strong> different myosin heavy chain is<strong>of</strong>orms<br />

(Vornanen, 1994), my<strong>of</strong>ibrillar ATPase activity (Aho and Vornanen,<br />

1999; Tiitu and Vornanen, 2001; Shiels et al., 2002), increased expression<br />

<strong>of</strong> β-adrenoreceptors (Keen et al., 1993) and increased SERCA2<br />

activity and protein expression (Landeira-Fernandez et al., 2004). The<br />

effect <strong>of</strong> low temperature on excitation–contraction coupling in <strong>the</strong><br />

fish myocardium includes prolonged action potential duration (APD)<br />

and slow deactivation <strong>of</strong> <strong>the</strong> SL L-type Ca 2+ channels (Shiels et al.,<br />

2002). Adrenaline (AD) has long been recognized as a modulator <strong>of</strong><br />

cardiac performance in fish (Nilsson, 1983; Farrell and Jones, 1992). In<br />

addition to regulating heart rate, cardiac contractility is also supported<br />

1095-6433/$ – see front matter © 2012 Elsevier Inc. All rights reserved.<br />

doi:10.1016/j.cbpa.2012.05.183


C. Methling et al. / Comparative Biochemistry and <strong>Physiology</strong>, Part A 163 (2012) 66–73<br />

67<br />

by AD, this by increasing <strong>the</strong> sarcolemmal (SL) Ca 2+ influx through <strong>the</strong><br />

L-type Ca 2+ channels (Vornanen, 1998; Bers, 2002). In eels, AD has previously<br />

been demonstrated to have a positive inotropic effect on contractility<br />

under hypoxic and acidotic conditions (Gesser et al., 1982),<br />

but not during winter (Pennec and Peyraud, 1983). Contractility is ultimately<br />

determined by <strong>the</strong> flux <strong>of</strong> Ca 2+ across <strong>the</strong> myocyte membrane<br />

and within <strong>the</strong> cell. Generally, <strong>the</strong> SL L-type channels are <strong>the</strong> major<br />

entry-ways for activator Ca 2+ , although <strong>the</strong> Na + /Ca 2+ -exchanger<br />

(NCX), operating in reverse mode, has also been recognized as Ca 2+<br />

entry in teleosts (Shiels et al., 2002). In <strong>the</strong> crucian carp, as much as<br />

one third to one half <strong>of</strong> <strong>the</strong> SL Ca 2+ influx can be ascribed to reverse<br />

mode NCX (Vornanen, 1999). In mammalian skeletal myocytes<br />

(Kurebayashi and Ogawa, 2001) and cardiocytes (Huang et al., 2006),<br />

ano<strong>the</strong>r SL Ca 2+ entry, Store Operated Calcium Entry (SOCE) has<br />

been described. SOCE increases <strong>the</strong> Ca 2+ loading <strong>of</strong> <strong>the</strong> sarcoplasmatic<br />

reticulum (SR), and is suggested to play a role when <strong>the</strong> intracellular<br />

stores are low or depleted (Putney, 1986).<br />

Cardiac function and performance at very low temperatures has<br />

only been studied in a limited number <strong>of</strong> species and it is unknown<br />

how eels, that are characterized as cold dormant, modulate cardiac<br />

function to meet <strong>the</strong> challenges imposed by low temperature. Moreover,<br />

because <strong>of</strong> <strong>the</strong> migrating life-stage, that imposes completely<br />

different challenges on cardiac performance, <strong>the</strong> <strong>European</strong> eel is an<br />

interesting species for studying cardiac function in fish.<br />

With <strong>the</strong> following, we aimed partly to study <strong>the</strong> cardiac responses<br />

<strong>of</strong> winter-quiescent eels after long‐term acclimation to 0 °C.<br />

We expected that at this very low temperature, some means <strong>of</strong> compensation<br />

might be necessary to ensure adequate cardiac output and<br />

hence survival. We investigated this by examining RVM and cardiac<br />

contractility — and how AD might support this. Secondly we aimed<br />

to study cardiac performance <strong>of</strong> migration-stage eels faced with<br />

acute temperatures changes, expecting that contractility would be<br />

modified as observed in o<strong>the</strong>r diving species (Shiels et al., 2002),<br />

and that AD would support contractility — especially during a temperature<br />

decrease. Finally, we wanted to investigate <strong>the</strong> individual<br />

contributions <strong>of</strong> three different SL Ca 2+ entry-ways (L-type channels,<br />

NCX and SOCE) to force development, and how this might be affected<br />

by temperature.<br />

2. Materials and methods<br />

2.1. Fish origin and care<br />

Silver stage female eels where caught in <strong>the</strong> Øresund by local<br />

fishermen and transported to <strong>the</strong> Marine Biological Laboratory in<br />

Helsingør, Denmark in <strong>the</strong> fall <strong>of</strong> 2007. Fish were kept in a large circular<br />

tank with a volume <strong>of</strong> 2500 l and supplied with 10 °C re-circulated<br />

35‰ aerated seawater. The light–dark period was 16 h–8 h. The<br />

fish did not eat, although <strong>of</strong>fered a variety <strong>of</strong> food items. Fish were<br />

acclimated to laboratory conditions for a minimum <strong>of</strong> 2 months<br />

prior to <strong>the</strong> experiments. During <strong>the</strong> acclimation period, <strong>the</strong> fish did<br />

not eat although a variety <strong>of</strong> food items was <strong>of</strong>fered. The mass and<br />

length <strong>the</strong> experimental animals was 577.27±151.58 g and 68.48±<br />

6.26 cm respectively. For temperature acclimation to 0 and 20 °C,<br />

eels were transferred to separate insulated 450 l tanks supplied<br />

with 10 °C seawater at a reduced flow. Cooling was achieved by<br />

pumping water from <strong>the</strong> tank through <strong>the</strong> heat exchanger on a<br />

custom-built industrial-grade cooler. Flow <strong>of</strong> coolant through <strong>the</strong><br />

heat exchanger was controlled by a motorized 3-way valve connected<br />

to a <strong>the</strong>rmostat. Heating was achieved by submerging a set <strong>of</strong> 1000 W<br />

titanium aquarium heaters into <strong>the</strong> tank, and was regulated by a<br />

<strong>the</strong>rmostat. Cooling/heating to desired acclimation temperature was<br />

done at a rate <strong>of</strong> 1 °C per day (Ta). Fish were kept at <strong>the</strong> final acclimation<br />

temperature for at least 4 weeks. The study was carried out in<br />

accordance with <strong>the</strong> Danish Animal Experimentation Act and <strong>the</strong><br />

protocol was approved by <strong>the</strong> Danish Animal Experimentation Board<br />

(license number: 2004/561-894).<br />

2.2. Tissue preparation<br />

<strong>Eel</strong>s were stunned by a blow to <strong>the</strong> head and killed by pithing <strong>of</strong><br />

<strong>the</strong> spinal cord and brain. The heart was quickly and carefully excised<br />

and placed in cold ringer's solution. Four tissue strips (app. 1 mm<br />

thick) were cut longitudinally from <strong>the</strong> ventricle and <strong>the</strong> compact<br />

layer was trimmed. Strips were fixed to tissue holders in organ<br />

baths (Myobath, WPI) and connected to force transducers (Fort 10,<br />

WPI, Florida, USA). Signals were amplified using a Transbridge 4 M<br />

(TBM4M, WPI, USA) amplifier and recorded with <strong>the</strong> AcqKnowledge<br />

s<strong>of</strong>tware (v.3.8.1 Biopac Systems Inc., California, USA) using a BioPac<br />

MP100 system. The bathing medium consisted <strong>of</strong> (in mM): 150<br />

NaCl, 5.0 KCL, 1.5 CaCl 2 , 0.17 MgSO 4 , 0.17 NaHPO 4 , 2.33 Na 2 HPO 4 ,<br />

11.0 NaHCO 3 , 5.0 D-glucose, 10.0 HEPES. pH was adjusted to 7.80 at<br />

10 °C and was allowed to change with <strong>the</strong> experimental temperature<br />

(T t ), (7.85 at 0 °C and 7.76 at 20 °C). The medium was continuously<br />

bubbled with pure oxygen throughout <strong>the</strong> whole <strong>of</strong> <strong>the</strong> experiment.<br />

A tonic level <strong>of</strong> adrenaline (1 nM) was added initially to <strong>the</strong> medium<br />

(standard condition). Experimental temperature (0 °C, 10 °C or 20 °C)<br />

was controlled by a set <strong>of</strong> coolers (CBN 8–30; Heto, Denmark),<br />

equipped with <strong>the</strong>rmostated heaters (HMT 200; Heto) circulating a<br />

coolant. In each experiment, three <strong>of</strong> four preparations were working<br />

at <strong>the</strong> acclimation temperature while one was working at ±10 °C.<br />

The preparations were allowed to hang in <strong>the</strong> medium for 30 min<br />

before being stimulated with a square wave pulse (10 ms duration)<br />

at 0.1 Hz with a Grass SD9 stimulator (Grass Product Group, Rhode<br />

Island, USA). The stimulus voltage was slowly increased until contractions<br />

became apparent, after which <strong>the</strong> voltage was increased by 50%.<br />

To achieve maximum force <strong>of</strong> contraction, tension was increased by<br />

stretching <strong>the</strong> tissue strips and under <strong>the</strong>se conditions <strong>the</strong>y were<br />

allowed to stabilize for ano<strong>the</strong>r 30 min.<br />

2.3. Experimental protocol<br />

Contraction <strong>of</strong> <strong>the</strong> ventricular muscle was studied at four different<br />

adrenaline concentrations [AD], (1 nM, 10 nM, 100 nM and 1 mM).<br />

Stimulation frequency was increased in steps until contractions<br />

became erratic. Data was collected for 5 min at each frequency.<br />

Stimulation frequency was <strong>the</strong>n returned to 0.1 Hz and <strong>the</strong> bathing<br />

media was replaced with fresh ringer containing <strong>the</strong> next [AD].<br />

Tissues were allowed to stabilize for 30 min and <strong>the</strong> above was<br />

repeated for all [AD]. Finally <strong>the</strong> stimulation frequency was returned<br />

to 0.1 Hz and <strong>the</strong> [AD] in <strong>the</strong> bathing media was returned to 1 nM.<br />

Fig. 1. Trace <strong>of</strong> isometric tension (PT) development in eel (A. anguilla) ventricle tissue<br />

strips paced at 0.1 Hz. The trace depicts a single contraction performed at ei<strong>the</strong>r 0 °C or<br />

10 °C with tissue taken from <strong>the</strong> same individual (acclimated to 0 °C). Values <strong>of</strong> peak<br />

tension normalized to percent <strong>of</strong> maximal tension (3.25 and 3.84 mN mm − 1 ,at0°C<br />

and 10 °C respectively) (see Materials and methods section).


68 C. Methling et al. / Comparative Biochemistry and <strong>Physiology</strong>, Part A 163 (2012) 66–73<br />

Tissues were allowed 15 min to stabilize upon which, one <strong>of</strong> three<br />

different Ca 2+ channel blocking agents was added to each separate<br />

preparation: Nifedipine, a drug that inhibits <strong>the</strong> SL L-type Ca 2+<br />

channels; KB-R7943, that inhibits <strong>the</strong> reverse mode NCX (both<br />

from Calbiochem); and SKF-96365, an inhibitor <strong>of</strong> SOCE (Cayman<br />

Chemical, MI, USA). All agents were added in concentrations <strong>of</strong> 10 μM<br />

according to <strong>the</strong> supplier's recommendations and previous studies<br />

(Vornanen, 1998; Woo and Morad, 2001; Huang et al., 2006). After<br />

15 min 1 mM AD was added to each preparation to examine if<br />

this high [AD] could ameliorate <strong>the</strong> negative effect <strong>of</strong> <strong>the</strong> drug. As<br />

<strong>the</strong> blocking protocol was performed at <strong>the</strong> end <strong>of</strong> <strong>the</strong> experiment,<br />

some preparations performed poorly and were excluded from fur<strong>the</strong>r<br />

analysis.<br />

2.4. Data analysis<br />

For each 5 min recording, only <strong>the</strong> last 10 consecutive peaks were<br />

used for fur<strong>the</strong>r analysis. Peak tension (PT) (g), time to peak tension<br />

(TPT) (s) and time to half relaxation (THR) (s), was quantified using a<br />

custom made Matlab script (MathWorks, Inc., USA). Peak tension was<br />

standardized to mN mm − 2 , where <strong>the</strong> mean cross-sectional area (A)<br />

<strong>of</strong> <strong>the</strong> tissues was calculated as A=M/L×1.06, where M is tissue<br />

mass in mg, L is <strong>the</strong> length <strong>of</strong> <strong>the</strong> strip in mm when positioned in<br />

<strong>the</strong> organ bath and 1.06 is <strong>the</strong> assumed density <strong>of</strong> muscle.<br />

2.5. Calculations and statistics<br />

Power production (PP) was calculated as <strong>the</strong> product <strong>of</strong> peak tension<br />

(mN mm −2 ) and contractions per minute according to (Matikainen<br />

and Vornanen, 1992). Contraction rate (TR) and relaxation rate (RR)<br />

was calculated as PT/TPT and PT/THR respectively. The product <strong>of</strong><br />

heart rate (min −1 ) and <strong>the</strong> sum <strong>of</strong> TPT and THR was expressed as <strong>the</strong><br />

variable minimum time in contraction (MTIC). The effects <strong>of</strong> temperature<br />

change and AD were assessed by a two-way repeated measures<br />

ANOVA, with pacing frequency as <strong>the</strong> second variable followed by <strong>the</strong><br />

Holm–Sidak multi comparisons procedure (SigmaPlot v. 11, Systat systems<br />

inc. USA). For all statistics, a pb0.05 was considered significant. All<br />

data are presented as mean values±SEM. Data points with Nb3 were<br />

included in tables and graphs supplementary, but were not included<br />

in <strong>the</strong> statistical analysis.<br />

Table 1<br />

Kinematic variables in eel (A. anguilla) ventricle tissue strips at 0 °C or 10 °C. <strong>Eel</strong>s were<br />

acclimated to 0 °C and paced to contract at tonic (1 nM) or high AD concentration<br />

(1000 nM). Rates <strong>of</strong> contraction and relaxation expressed as mN mm − 2 s − 1 , and<br />

minimum time in contraction as s min − 1 . An asterisk denotes significant difference<br />

(pb0.05) from <strong>the</strong> lowest adrenaline concentration (1 nM). A dagger denotes a significant<br />

difference (pb0.05) between working temperatures. Values are mean±s.e.m.<br />

(see Materials and methods section).<br />

T a /T t [AD] F STIM (Hz)<br />

(°C) (nM)<br />

0.1 0.2<br />

0/0 1 TR (mN mm − 2 s − 1 ) 2.28 ±0.57 2.06 ±0.87<br />

RR (mN mm − 2 s − 1 ) 5.40 ±1.14 4.81 ±1.71<br />

MTIC (s min − 1 ) 22.99 ±1.75 43.00±0.36<br />

N 6 2<br />

1000 TR (mN mm − 2 s − 1 ) 3.14 ±1.01* 2.45 ±1.02<br />

RR (mN mm − 2 s − 1 ) 8.44 ±2.21* 5.78 ±2.40<br />

MTIC (s min − 1 ) 25.28 ±1.83 43.97±1.59<br />

N 6 4<br />

0/10 1 TR (mN mm − 2 s − 1 ) 5.86 ±1.30† 6.91 ±1.38†<br />

RR (mN mm − 2 s − 1 ) 11.85 ±2.64† 13.82±2.30†<br />

MTIC (s min − 1 ) 10.89 ±0.74† 19.51±0.84†<br />

N 5 3<br />

1000 TR (mN mm − 2 s − 1 ) 6.45 ±1.63 8.06 ±1.71<br />

RR (mN mm − 2 s − 1 ) 13.20± 16.73±3.49<br />

MTIC (s min − 1 ) 11.17 ±0.79 19.33±0.67<br />

N 4 4<br />

whereas blocking <strong>the</strong> reverse mode NCX Ca 2+ entry with KB-R7943<br />

had no effect on peak tension.<br />

3.2. Acclimated to 10 °C or 20 °C<br />

Twitch duration was affected by <strong>the</strong> acute change in temperature,<br />

i.e. prolonged after a decrease and shortened after an increase, but<br />

<strong>the</strong>re were no differences in twitch duration between tissues working<br />

at <strong>the</strong> same temperature (Fig. 4). Preparations from 10 °C-acclimated<br />

3. Results<br />

3.1. Acclimated to 0 °C<br />

RVM was significantly higher (one-way ANOVA and Tukey post<br />

hoc test) in eels acclimated to 0 °C and 10 °C, being 0.073±0.015<br />

and 0.075±0.016 respectively, compared to eels acclimated to 20 °C<br />

with a RVM <strong>of</strong> 0.049±0.01. Twitch duration at 0 °C lasted approx.<br />

8s(Fig. 1), allowing a maximal stimulation frequency (F STIM ) <strong>of</strong> less<br />

than 0.2 Hz, as only few preparations could contract at 0.2 Hz<br />

(Table 1). Following an acute temperature increase to 10 °C, <strong>the</strong><br />

twitch duration was significantly shortened and lasted ~3 s (Fig. 1).<br />

Rates <strong>of</strong> contraction and relaxation increased (Table 1), with Q 10<br />

values [(R 1 /R 2 )^(10/(T 1 −T 2 ))] <strong>of</strong> 2.4±0.2 and 2.0±0.2 respectively<br />

(at 0.1 Hz), whereas peak tension and power production were not<br />

significantly affected by an increase in test temperature (Fig. 2A, B).<br />

AD elicited an inotropic response at 0 °C, with an increase in peak tension<br />

and power production <strong>of</strong> ~50% at <strong>the</strong> highest [AD] (Fig. 2C, D).<br />

Contraction and relaxation rates increased significantly at <strong>the</strong> high<br />

[AD] (Table 1) while MTIC was unaffected by adrenergic stimulation.<br />

A similar responsiveness to AD was not observed when tested at<br />

10 °C. Blocking <strong>the</strong> SL Ca 2+ influx via L-type channels with Nifedipine<br />

caused a significant reduction in peak tension, which was not abolished<br />

by addition <strong>of</strong> 1 mM AD (Fig. 3). Treatment with SKF-96365 a blocker <strong>of</strong><br />

SOCE also caused a significant, albeit smaller reduction in peak tension,<br />

Fig. 2. Peak tension and power production in eel (A. anguilla) ventricle tissue strips at<br />

0 °C or 10 °C. <strong>Eel</strong>s were acclimated to 0 °C and <strong>the</strong> working temperature was ei<strong>the</strong>r 0 °C<br />

or 10 °C. Left side: A) <strong>the</strong> force–frequency response and B) <strong>the</strong> power production as a<br />

function <strong>of</strong> pacing frequency. Right side: <strong>the</strong> effect <strong>of</strong> increased adrenergic tonus on<br />

C) peak tension and D) power production in strips paced at 0.1 Hz. Values presented<br />

are mean ±s.e.m. (N=2–6). An asterisk signifies significant difference (pb0.05)<br />

from <strong>the</strong> lowest adrenaline concentration (1 nM). Brackets denotes data points with<br />

Nb3 (see Materials and methods section).


C. Methling et al. / Comparative Biochemistry and <strong>Physiology</strong>, Part A 163 (2012) 66–73<br />

69<br />

Fig. 3. Peak tension in eel (A. anguilla) ventricle tissue strips treated with Ca 2+ -channel<br />

blocking agents. The acclimation and working temperature was 0 °C, and strips were<br />

paced at 0.1 Hz. Height <strong>of</strong> bars represents means and error bars s.e.m. (N=4–5). An<br />

asterisk signifies significant difference (pb0.05) from control conditions (see Materials<br />

and methods section).<br />

animals did not display any changes in <strong>the</strong> force <strong>of</strong> contraction when<br />

working at 20 °C compared to 10 °C (Fig. 5A), consequently <strong>the</strong>re was<br />

no difference in power production between <strong>the</strong> two working temperatures<br />

(Fig. 5B). The shortened twitch duration at <strong>the</strong> increased temperature<br />

significantly increased both contraction and relaxation rates<br />

with Q 10 values <strong>of</strong> 2.2±0.2 and 1.8±0.2 (at 0.1 Hz) respectively<br />

(Table 2). A significant inotropic response to AD was observed<br />

at <strong>the</strong> high concentrations (100 and 1000 nM) only in tissues performing<br />

contractions at 10 °C (when compared at 0.1 Hz) (Fig. 5C,<br />

D). A decrease in temperature, from 20 °C to10 °C, increased contraction<br />

force with a resultant significant increase in power production<br />

(Fig. 6A, B) and decreased rates <strong>of</strong> contraction and relaxation with<br />

Q 10 <strong>of</strong> 1.5±0.4 and 1.4±0.2 respectively at 0.1 Hz (Table 3). Similarly,<br />

a significant inotropic response to AD was observed at 100 and<br />

1000 nM (Fig. 6C, D) in tissues performing contractions at 10 °C<br />

Fig. 5. Peak tension and power production in eel (A. anguilla) ventricle tissue strips at<br />

10 °C or 20 °C. <strong>Eel</strong>s were acclimated to 10 °C and <strong>the</strong> working temperature was ei<strong>the</strong>r<br />

10 °C or 20 °C. Left side: A) <strong>the</strong> force–frequency response and B) <strong>the</strong> power production<br />

as a function <strong>of</strong> pacing frequency. Right side: <strong>the</strong> effect <strong>of</strong> increased adrenergic tonus<br />

on C) peak tension and D) power production in strips paced at 0.1 Hz. Values presented<br />

are mean ±s.e.m. (N=2–7). An asterisk signifies significant difference (pb0.05) from<br />

<strong>the</strong> lowest adrenaline concentration (1 nM). Brackets denote data points with Nb3<br />

(see Materials and methods section).<br />

only (paced at 0.1 Hz). At higher pacing frequencies, however, a significant<br />

inotropic response was also observed in tissues working<br />

at 20 °C (Fig. 7). Moreover, at both test temperatures, <strong>the</strong> magnitude<br />

<strong>of</strong> <strong>the</strong> inotropic response was positively correlated with pacing<br />

frequency. The time-course <strong>of</strong> contraction was unaffected by adrenergic<br />

stimulation so as a consequence <strong>of</strong> <strong>the</strong> increased peak tension,<br />

rates <strong>of</strong> contraction and relaxation were also increased (Table 3, 4).<br />

Treatment with Nifedipine (L-type channel blocker) caused a considerable<br />

and significant reduction in peak tension at both 10 °C and<br />

20 °C, which was not abolished by <strong>the</strong> addition <strong>of</strong> AD. Inhibition <strong>of</strong> reverse<br />

mode NCX as well as SOCE caused a negative inotropic response<br />

at both temperatures. This effect, unlike that <strong>of</strong> Nifedipine, could be<br />

reversed by adrenergic stimulation (Fig. 8).<br />

4. Discussion<br />

4.1. Acclimated to 0 °C<br />

Fig. 4. Traces <strong>of</strong> isometric tension development in eel (A. anguilla) ventricle tissue<br />

strips paced at 0.1 Hz. The traces depict single contractions at ei<strong>the</strong>r 10 °C or 20 °C by<br />

tissue taken from <strong>the</strong> same individual. A) Acclimated to 10 °C. B) Acclimated to 20 °C.<br />

Values <strong>of</strong> peak tension normalized to percent <strong>of</strong> maximal tension (in A, 7.53 and<br />

4.4156 mN mm − 1 at 10 °C and 20 °C respectively, and in B, 5.05 and 5.56 mN mm − 1<br />

at 10 °C and 20 °C respectively) (see Materials and methods section).<br />

Acclimation to cold temperatures (4 °C) reduces <strong>the</strong> heart rate,<br />

increases <strong>the</strong> duration <strong>of</strong> contraction and <strong>the</strong> refractoriness <strong>of</strong><br />

<strong>the</strong> heart in <strong>the</strong> cold dormant crucian carp, but does not stimulate<br />

cardiac growth. These responses are considered an adaptive strategy<br />

to minimize energy expenditure during extreme winter conditions<br />

(Matikainen and Vornanen, 1992; Tiitu and Vornanen, 2001). The<br />

present observations on eels after cold acclimation partly resemble<br />

those <strong>of</strong> <strong>the</strong> crucian carp in that <strong>the</strong> time-course <strong>of</strong> contraction was<br />

noticeably prolonged thus allowing a maximal heart rate between<br />

6 and 12 beats per minute. No in vivo heart rates, at 0 °C, have so<br />

far been reported for A. anguilla, however, Seibert (1979) recorded a<br />

resting heart rate <strong>of</strong> ~12 bpm at 5 °C in unrestrained eels. This value<br />

corresponds well with <strong>the</strong> present results, and with a study on <strong>the</strong><br />

closely related American eel (Anguilla rostrata), where electrically<br />

paced ventricle strips and in situ perfused hearts could not be stimulated<br />

to contract above a frequency 18 bpm when tested at 5 °C<br />

(Bailey et al., 1991). A significantly increased RVM was presently


70 C. Methling et al. / Comparative Biochemistry and <strong>Physiology</strong>, Part A 163 (2012) 66–73<br />

Table 2<br />

Kinematic variables in eel (A. anguilla) ventricle tissue strips at 10 °C or 20 °C. <strong>Eel</strong>s were acclimated to 10 °C and paced to contract at tonic (1 nM) or high AD concentration<br />

(1000 nM). Rates <strong>of</strong> contraction and relaxation expressed as mN mm − 2 s − 1 , and minimum time in contraction as s min − 1 . An asterisk denotes significant difference (pb0.05)<br />

from <strong>the</strong> lowest adrenaline concentration (1 nM). A dagger denotes a significant difference (pb0.05) between working temperatures. Values are mean±s.e.m. (see Materials<br />

and methods section).<br />

T a/ T t<br />

(°C)<br />

[AD]<br />

(nM)<br />

F STIM (Hz)<br />

0.1 0.2 0.4 0.6<br />

10/10 1 TR (mN mm − 2 s − 1 ) 3.67 ±0.84 3.79±0.78 3.37 ±0.94 2.07 ±0.10<br />

RR (mN mm − 2 s − 1 ) 9.16 ±1.99 9.98±2.21 8.67 ±2.41 5.85 ±0.12<br />

MTIC (s min − 1 ) 11.04 ±0.39 19.97±0.55 34.84±0.69 46.19±1.08<br />

N 7 7 5 3<br />

1000 TR (mN mm − 2 s − 1 ) 5.10 ±1.28 6.31±1.61* 7.82 ±1.86* 4.77 ±0.31*<br />

RR (mN mm − 2 s − 1 ) 11.67 ±2.56* 13.66±2.87* 18.10±3.91* 13.08±0.95*<br />

MTIC (s min − 1 ) 11.26 ±0.33 20.28±0.44 33.57±0.57 43.16±1.21<br />

N 7 7 7 3<br />

10/20 1TR (mN mm − 2 s − 1 ) 8.90 ±2.06† 9.32±2.25† 9.75 ±2.46† 8.39 ±0.74†<br />

RR (mN mm − 2 s − 1 ) 18.89 ±3.08† 19.92±3.86† 21.25±4.51† 19.02±2.50†<br />

MTIC (s min − 1 ) 4.34 ±0.11† 8.48±0.23† 16.580.39† 24.30±1.78†<br />

N 6 6 6 2<br />

1000 TR (mN mm − 2 s − 1 ) 8.50 ±2.18 10.41±2.42 12.48±2.71 15.48±3.71<br />

RR (mN mm − 2 s − 1 ) 21.18 ±503 24.06±5.14 27.76±5.32 35.56±7.35<br />

MTIC (s min − 1 ) 4.70 ±4.70 9.21±0.26 17.25±0.41 24.45±1.24<br />

N 6 6 6 4<br />

observed after cold acclimation demonstrating that eels, unlike <strong>the</strong><br />

crucian carp, compensate for <strong>the</strong> depressing effect <strong>of</strong> low seasonal<br />

temperature by increasing <strong>the</strong> size <strong>of</strong> <strong>the</strong> ventricle. Possibly o<strong>the</strong>r<br />

conditions, like anoxia, have necessitated a different survival strategy<br />

in <strong>the</strong> crucian carp. The specific temperature conditions (e.g. threshold<br />

value and/or rate <strong>of</strong> decrease), at which this response is initiated,<br />

is unknown, but evidently it occurs at 10 °C. At this temperature eels<br />

also reduce feed intake activity levels (Nyman, 1972; Walsh et al.,<br />

1983). The increase in RVM may be viewed as an early response in preparing<br />

for winter and possibly makes additional changes (increased<br />

contractility) redundant.<br />

Adrenergic stimulation did not affect <strong>the</strong> twitch duration, but it<br />

increased <strong>the</strong> force <strong>of</strong> contraction. This suggests that eels increase<br />

<strong>the</strong> adrenergic tone as a compensatory mechanism at low temperatures,<br />

and that cardiac performance is modulated via inotropic ra<strong>the</strong>r<br />

than chronotropic adjustments. A similar observation was recently<br />

made in two species <strong>of</strong> Antarctic fishes, Chaenocephalus aceratus<br />

and Noto<strong>the</strong>nia coriiceps, where ventricle tissues working at 0 °C,<br />

responded to AD solely by increases in contraction force (Skov et al.,<br />

2009). Also, previous studies on A. anguilla reported that AD does<br />

not have a positive chronotropic effect, instead it prolongs <strong>the</strong> ventricular<br />

action potential duration (APD) and can even have a negative<br />

chronotropic effect (Peyraud-Waitzenegger et al., 1980; Pennec and<br />

Peyraud, 1983). Fur<strong>the</strong>rmore, <strong>the</strong> reactivity to catecholamines in eels<br />

was observed to be seasonally conditioned in that bradycardia and prolonged<br />

APD was mediated by a predominance <strong>of</strong> α-adrenoreceptors<br />

in winter-acclimated (8 °C) eels, while tachycardia, mediated by a predominance<br />

<strong>of</strong> β-adrenoreceptors, was observed in summer-acclimated<br />

(16 °C) eels (Peyraud-Waitzenegger et al., 1980; Pennec and Peyraud,<br />

1983). More recently, it was observed that a β3-adrenoreceptor agonist<br />

caused a negative inotropic response in <strong>the</strong> eel myocardium (at 20 °C),<br />

higher temperatures (Imbrogno et al., 2006). Possibly, <strong>the</strong> expression <strong>of</strong><br />

β3-ARs is also influenced by seasonal factors i.e. ambient temperature<br />

and <strong>the</strong> seasonal expression pattern <strong>of</strong> α‐ and β-ARs is an interesting<br />

subject for future studies.<br />

4.2. Acclimated to 10 °C or 20 °C<br />

Fig. 6. Peak tension and power production in eel (A. anguilla) ventricle tissue strips at<br />

20 °C or 10 °C. <strong>Eel</strong>s were acclimated to 20 °C and <strong>the</strong> working temperature was ei<strong>the</strong>r<br />

20 °C or 10 °C Left side: A) <strong>the</strong> force–frequency response and B) <strong>the</strong> power production<br />

as a function <strong>of</strong> pacing frequency. An asterisk signifies significant difference (pb0.05)<br />

between test temperatures. Right side: <strong>the</strong> effect <strong>of</strong> increased adrenergic tonus on<br />

C) peak tension and D) power production in strips paced at 0.1 Hz. An asterisk signifies<br />

significant difference (pb0.05) from <strong>the</strong> lowest adrenaline concentration (1 nM).<br />

Values presented are mean ±s.e.m. (N=2–7). Brackets denotes data points with<br />

Nb3. (See materials and methods section).<br />

In most species examined, an acute temperature decrease, increases<br />

<strong>the</strong> contractile force <strong>of</strong> <strong>the</strong> ventricular myocardium thus<br />

safeguarding cardiac output when e.g. diving to low ambient temperatures<br />

(Shiels et al., 2002). Conversely, increased temperatures<br />

have been demonstrated to decrease contractile force or cause a<br />

downward shift in <strong>the</strong> force–frequency curve in e.g. rainbow trout<br />

(Shiels and Farrell, 1997; Shiels et al., 2002). In accordance with this<br />

general observation, an increase in power production was also observed<br />

in eels when <strong>the</strong> working temperature was acutely decreased<br />

from 20 °C to 10 °C and it seems reasonable to interpret this as<br />

a mechanism to maintain cardiac output when eels make diurnal<br />

excursions to deeper cooler waters (Aarestrup et al., 2009). Unlike<br />

<strong>the</strong> response observed in rainbow trout, increasing <strong>the</strong> working<br />

temperature had no effect on isometric tension and did not cause a<br />

downward shift in <strong>the</strong> force–frequency response when working ei<strong>the</strong>r<br />

at 10 °C or 20 °C (compared to 0 °C or 10 °C). From <strong>the</strong> present results,<br />

it appears that in eels, cardiac output is uncompromised during acute


C. Methling et al. / Comparative Biochemistry and <strong>Physiology</strong>, Part A 163 (2012) 66–73<br />

71<br />

Table 3<br />

Kinematic variables in eel (A. anguilla) ventricle tissue strips at 20 °C or 10 °C. <strong>Eel</strong>s were acclimated to 20 °C and paced to contract at tonic (1 nM) or high AD concentration<br />

(1000 nM). Rates <strong>of</strong> contraction and relaxation expressed as mN mm − 2 s − 1 , and minimum time in contraction as s min − 1 . An asterisk denotes significant difference (pb0.05)<br />

from <strong>the</strong> lowest adrenaline concentration (1 nM). A dagger denotes a significant difference (pb0.05) between working temperatures. Values are mean±s.e.m. (see Materials<br />

and methods section).<br />

T a/ T t<br />

(°C)<br />

[AD]<br />

(nM)<br />

F STIM (Hz)<br />

0.1 0.2 0.4 0.6 1.4<br />

20/20 1 TR (mN mm − 2 s − 1 ) 9.33 ±1.34 8.92 ±1.20 8.33 ±0.97 8.09 ±0.92 … 5.99 ±0.90<br />

RR (mN mm − 2 s − 1 ) 20.34±2.64 20.38 ±2.49 20.41±2.28 20.68 ±2.28 … 17.05±2.54<br />

MTIC (s min − 1 ) 4.78 ±0.25 9.26 ±0.47 17.42±0.85 24.54 ±1.14 … 44.89±1.36<br />

N 7 7 7 7 … 7<br />

1000 TR (mN mm − 2 s − 1 ) 7.44 ±1.06 9.53 ±1.27* 12.46±1.69* 13.96 ±1.88* … 13.59±1.84<br />

RR (mN mm − 2 s − 1 ) 23.43±3.69 27.54 ±4.34 33.75±5.19 38.40 ±6.05* … 41.68±6.24*<br />

MTIC (s min − 1 ) 5.01 ±0.13 9.45 ±0.22 17.37±0.42 24.20 ±0.61 … 44.50±0.65<br />

N 7 7 7 7 … 7<br />

20/10 1TR (mN mm − 2 s − 1 ) 5.05 ±0.67† 5.43 ±0.61† 5.82 ±0.71 6.95 ±0.62<br />

RR (mN mm − 2 s − 1 ) 12.86±1.99 14.03 ±2.02 15.49±2.31 21.83 ±0.95<br />

MTIC (s min − 1 ) 13.21±1.64† 21.20 ±0.62† 35.83±0.90† 45.99 ±0.36†<br />

N 7 7 7 2<br />

1000TR (mN mm − 2 s − 1 ) 7.37 ±0.93* 8.69 ±1.22* 10.07±1.27* 9.63 ±1.54<br />

RR (mN mm − 2 s − 1 ) 17.13±2.95* 18.65 ±3.32* 23.73±3.69* 25.00 ±4.42<br />

MTIC (s min − 1 ) 11.38±0.39 20.90 ±0.74 35.24±1.07 44.78 ±0.83<br />

N 6 6 7 4<br />

increases in temperature. This is in support <strong>of</strong> observations from<br />

A. rostrata where even an increase in isometric force was observed<br />

following an acute increase in temperature from 10 °C to 15 °C (Bailey<br />

et al., 1991).<br />

Increasing <strong>the</strong> adrenergic tone as a regulatory mechanism is more<br />

efficient at low temperature in e.g. rainbow trout (Graham and<br />

Farrell, 1989; Keen et al., 1993; Farrell et al., 1996; Shiels and<br />

Farrell, 1997; Aho and Vornanen, 2001) and pacific mackerel (Shiels<br />

and Farrell, 2000). In rainbow trout <strong>the</strong> β-AR expression is increased<br />

at low ambient temperature (Keen et al., 1993) and <strong>the</strong> SL L-type<br />

Ca 2+ -channel current, I Ca , is more sensitive to AD after an acute<br />

temperature decrease (Shiels et al., 2003). On <strong>the</strong> o<strong>the</strong>r hand, <strong>the</strong> cardiac<br />

performance is less sensitive to adrenergic stimulation at warm<br />

temperatures (Graham and Farrell, 1989), and in trout atrial cells, I Ca<br />

AD sensitivity is decreased when temperature is increased (Shiels<br />

et al., 2003). A similar temperature-dependence was observed in <strong>the</strong><br />

present study, as isometric tension increased following <strong>the</strong> AD treatment<br />

when contracting at 10 °C, but not at 20 °C. However, this was<br />

also frequency-dependent as <strong>the</strong>re was a positive inotropic response<br />

at both test temperatures when paced at higher frequencies. In<br />

perspective <strong>of</strong> diel vertical migrations, an increased adrenergic tone<br />

would act as compensation for <strong>the</strong> decrease in heart rate while<br />

descending. When ascending on <strong>the</strong> o<strong>the</strong>r hand, <strong>the</strong> increase in<br />

heart rate might not be sufficient to meet <strong>the</strong> concurrently increasing<br />

metabolic demands and during this period AD would <strong>the</strong>n support<br />

cardiac performance. Besides AD, o<strong>the</strong>r factors have been shown to<br />

Fig. 7. Force–frequency response in eel (A. anguilla) ventricle tissue strips at tonic<br />

and high adrenaline concentrations. <strong>Eel</strong>s were ei<strong>the</strong>r acclimated to 10 °C or 20 °C and<br />

performed contractions ei<strong>the</strong>r 10 °C (A, D) or 20 °C (B, C). An asterisk denotes a significant<br />

difference (pb0.05) from <strong>the</strong> lowest adrenaline concentration (1 nM). Values<br />

presented are mean ±s.e.m. (N=2–7). Brackets denotes data points with Nb3. (See<br />

materials and methods section).<br />

Fig. 8. Peak tension in eel (A. anguilla) ventricle tissue strips treated with Ca 2+ -channel<br />

blocking agents. A) Acclimation and working temperature: 10 °C. B) Acclimation and<br />

working temperature: 20 °C. Strips were paced at 0.1 Hz. Height <strong>of</strong> bars represent<br />

means and error bars s.e.m. (N=4–7). An asterisk signifies significant difference<br />

(pb0.05) from control conditions (see Materials and methods section).


72 C. Methling et al. / Comparative Biochemistry and <strong>Physiology</strong>, Part A 163 (2012) 66–73<br />

modulate cardiac performance in A. anguilla. Endogenous release<br />

<strong>of</strong> nitric oxide (NO) increases <strong>the</strong> sensitivity <strong>of</strong> <strong>the</strong> Frank–Starling<br />

response (Imbrogno et al., 2001) and NO can influence myocardial<br />

relaxation on a beat-to-beat basis by regulating <strong>the</strong> Ca 2+ reuptake by<br />

<strong>the</strong> SR‐Ca 2+ ATPase (SERCA2a) (Gar<strong>of</strong>alo et al., 2009).<br />

Out <strong>of</strong> <strong>the</strong> 3 SL Ca 2+ -blockers, Nifedipine caused <strong>the</strong> largest reduction<br />

in peak tension. This is in accordance with general observations<br />

on fish cardiocytes; that L-type Ca 2+ channels serve as <strong>the</strong> main activator<br />

Ca 2+ entry site (Driedzic and Gesser, 1988; Hovemadsen and<br />

Gesser, 1989; Vornanen, 1989). Also in line with previous studies<br />

using Nifedipine, <strong>the</strong> negative effect could not be abolished by AD as<br />

this agent binds irreversibly to <strong>the</strong> L-type Ca 2+ channels. The reverse<br />

operating mode <strong>of</strong> NCX, i.e. bringing Ca 2+ into <strong>the</strong> cell, is conditioned<br />

by <strong>the</strong> properties <strong>of</strong> <strong>the</strong> AP (Vornanen, 1999), and <strong>the</strong>oretically, low<br />

ambient temperature favors Ca 2+ -influx via NCX due to a prolongation<br />

<strong>of</strong> <strong>the</strong> APD (Vornanen et al., 2002). But contrary to this assumption, <strong>the</strong><br />

effect <strong>of</strong> blocking <strong>the</strong> reverse mode NCX was more pronounced at high<br />

temperature, while <strong>the</strong>re was no effect at 0 °C. Possibly, <strong>the</strong> conditions<br />

favoring Ca 2+ -influx via NCX are modified after <strong>the</strong>rmal acclimation,<br />

but fur<strong>the</strong>r insight must come from <strong>the</strong> study <strong>of</strong> AP characteristics<br />

at different temperatures.<br />

As a novelty in fish cardiocytes, a decrease in isometric force could<br />

be attributed to <strong>the</strong> blockade <strong>of</strong> SOCE. The response was lower at 0 °C<br />

than at 10 °C and 20 °C, indicating also a temperature sensitivity <strong>of</strong><br />

this Ca 2+ entry. In mammalian myocytes, SOCE increases <strong>the</strong> Ca 2+<br />

loading <strong>of</strong> <strong>the</strong> SR, and is suggested to play a role when <strong>the</strong> intracellular<br />

stores are low or depleted (Putney, 1986). Assuming that <strong>the</strong> function<br />

<strong>of</strong> SOCE is homologous in mammals and teleosts, <strong>the</strong> current<br />

results suggest that <strong>the</strong> SR could be involved in force development<br />

in eel ventricular myocytes. To this end, it was demonstrated that treatment<br />

with ryanodine, a blocker <strong>of</strong> SR function, decreased isometric force<br />

at 20 °C and high Ca 2+ loads in <strong>the</strong> American eel (A. rostrata) (Bailey<br />

et al., 2000). The contributing role <strong>of</strong> SR to force development is more<br />

significant in active species e.g. scombrids and in rainbow trout at high<br />

temperatures (Hovemadsen, 1992; Shiels and Farrell, 1997; Shiels<br />

et al., 2002), and possibly this could be true for migrating eels as well.<br />

In summary we found that, in <strong>the</strong> <strong>European</strong> eel acclimatory<br />

responses to low temperature includes an increase in RVM that<br />

may compensate for <strong>the</strong> prolonged twitch duration and low heart<br />

rate. An acute temperature decrease, as experienced by migrating<br />

eels, increased <strong>the</strong> ventricular power production, which may serve as<br />

compensation for <strong>the</strong> upper limit placed on heart rate by acute cooling.<br />

Adrenergic stimulation generally improved contractility, suggesting a<br />

role for AD in supporting cardiac performance both during winterquiescence<br />

and spawning migration. At all temperatures, contractions<br />

were mainly supported by L-type channel influx, but at higher temperatures<br />

also by reverse mode NCX and SOCE.<br />

Acknowledgments<br />

This study was funded by <strong>the</strong> Danish Agency for Science and Innovation,<br />

The Elisabeth and Knud Petersen Foundation and <strong>the</strong> Faculty<br />

<strong>of</strong> Science, University <strong>of</strong> Copenhagen.<br />

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PAPER III


Tolerance towards hypercapnia does not preclude a negative impact on<br />

metabolism and postprandial processes in <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla)<br />

C. METHLING a *, P.B. PEDERSEN b , J.F. STEFFENSEN a AND P.V. SKOV b<br />

a Marine Biological Section, University <strong>of</strong> Copenhagen Strandpromenaden 5, DK-3000, Helsingør<br />

b National <strong>Institut</strong>e <strong>of</strong> Aquatic Resources, Section for Aquaculture, Technical University <strong>of</strong><br />

Denmark, North Sea Science Park DK-9850, Hirtshals<br />

Abstract<br />

The effect <strong>of</strong> elevated CO 2 partial pressure on <strong>the</strong> specific dynamic action (SDA) and ammonia<br />

excretion in <strong>European</strong> eel (Anguilla anguilla) fed a 0.5% body weight-ration was assessed. Two<br />

different hypercapnic scenarios were investigated; one PCO2 oscillating between 20-60mmHg in a<br />

24 hour period, and one PCO2 constant at 60mmHg. To asses to what extent any observations<br />

might be an effect <strong>of</strong> pH, a low pH but normocapnic environment (pCO 2 ≈ 3mmHg, pH=6.5) was<br />

also investigated. Both constant hypercapnia (60mmHg) and low pH (pH=6.5) significantly<br />

prolonged <strong>the</strong> postprandial rise in oxygen consumption (MO 2 ) by approximately 22% and 29%<br />

respectively. Hypercapnia had no effect on standard metabolic rate (SMR), but eels kept in constant<br />

or oscillating hypercapnia had a significantly lower maximum metabolic rate (MMR) compared to<br />

controls to an extent that caused a significant reduction <strong>of</strong> <strong>the</strong> aerobic scope during constant<br />

hypercapnia. Under conditions <strong>of</strong> oscillating pCO 2 , <strong>the</strong> postprandial increase in ammonia excretion<br />

was significantly reduced and returned earlier to pre-feeding levels. This group also respired<br />

significantly less <strong>of</strong> <strong>the</strong> consumed nitrogen. No significant effects on <strong>the</strong> post-prandial ammonia<br />

excretion were observed in eels at 60mmHg or low pH/normocapnia. On <strong>the</strong> o<strong>the</strong>r hand, <strong>the</strong> fasting<br />

ammonia excretion rate, ammonia quotient (AQ) and protein respiration during rest was increased<br />

at low pH/normocapnia during rest, while ammonia excretion was reduced at MMR in eels at<br />

60mmHg. The results demonstrate that in spite <strong>of</strong> <strong>the</strong> exceptional tolerance eels have towards<br />

elevated pCO 2 and acidosis, <strong>the</strong> post-prandial metabolic processes are adversely affected by<br />

hypercapnia and low pH.<br />

Key words: Anguilla anguilla, aquaculture, hypercapnia, specific dynamic action, ammonia<br />

excretion<br />

1


Introduction<br />

Elevated partial pressure <strong>of</strong> CO 2 (pCO 2 ) frequently occurs in recirculating aquaculture systems<br />

(RAS), particularly under high degrees <strong>of</strong> water re-use and high rearing densities, due to an<br />

accumulation <strong>of</strong> expired CO 2 (Steffensen and Lomholt, 1990; Crocker et al., 2000). CO 2 readily<br />

diffuses across <strong>the</strong> gill epi<strong>the</strong>lia with a resultant decline in plasma pH and blood oxygen carrying<br />

capacity (Heisler, 1984; Heisler, 1993). The resulting extra- and intracellular acidosis can be<br />

compensated for by an accumulation <strong>of</strong> bicarbonate (HCO - 3 ) ions in exchange for Cl - ions (Heisler,<br />

1984; Heisler, 1993). Physiological effects <strong>of</strong> hypercapnia in fishes may be acute in <strong>the</strong> form <strong>of</strong><br />

perturbations in acid-base regulation, respiration and cardiac function, while chronic effects may be<br />

manifested as reduced growth or survival (for review see (Portner et al., 2004; Ishimatsu et al.,<br />

2005). <strong>Eel</strong>s have been shown to be exceptionally tolerant to both acute and chronic exposure to<br />

elevated ambient pCO 2 with no changes <strong>the</strong>ir metabolic rate and no elevations in <strong>the</strong> traditional<br />

indicators <strong>of</strong> stress, such as plasma catecholamine or cortisol (McKenzie et al., 2002; McKenzie et<br />

al., 2003). The tolerance to hypercapnia has been explained by <strong>the</strong> ability <strong>of</strong> eels to regulate<br />

intracellular pH despite severe extracellular acidosis (McKenzie et al., 2003), which is made<br />

possible by a tolerance to very low plasma Cl - levels (Farrell and Lutz, 1975; McKenzie et al.,<br />

2003), which again enables a maintained cardiac output despite acidosis and hypoxaemia<br />

(McKenzie et al., 2002). After an initial hypercapnic disturbance it takes hours to days before a<br />

steady state in acid-base status is reached (Hyde and Perry, 1989) and <strong>the</strong> adjustments in gene<br />

expression <strong>of</strong> acid-base regulatory ion exchangers differs between short term and long term<br />

exposure to hypercapnia in fish gills (Deigweiher et al., 2008). This suggests that negative effects <strong>of</strong><br />

hypercapnia may be more likely to occur under acute or unstable pCO 2 conditions. An example <strong>of</strong><br />

<strong>the</strong> former was shown by (CruzNeto and Steffensen, 1997), where acute exposure to 25mmHg<br />

pCO 2 affected <strong>the</strong> ability to regulate oxygen uptake during hypoxia in A. anguilla.<br />

In Europe, aquaculture production <strong>of</strong> A. anguilla occurs under extensive conditions, and are in some<br />

places characterized by high rearing densities and an accumulation <strong>of</strong> expired CO 2 in recirculating<br />

systems (Steffensen and Lomholt, 1990). The extent <strong>of</strong> hypercapnia will also depend on feeding<br />

schedules because general activity and respiration increases during feeding events (Owen et al.,<br />

1998). Feeding once or twice daily will cause pCO 2 levels to fluctuate while shorter intervals<br />

between meals or continuous feeding will cause constantly elevated pCO 2 . The effect <strong>of</strong> elevated<br />

pCO 2 on feeding and growth in fish has only been studied in a few species, and <strong>the</strong> results indicate<br />

that <strong>the</strong> effect depends on species, size, salinity and temperature (Ishimatsu et al., 2008). Severe<br />

2


chronic hypercapnia ultimately reduces growth in Atlantic salmon parr (Salmo salar) ((Fivelstad et<br />

al., 2007) and in juvenile white sturgeon (Acipenser transmontanus(Crocker and Cech, 1996;<br />

Crocker et al., 2000). Reduced feed intake have been reported in Sea bass (Dicentrarchus labrax)<br />

(Cecchini et al., 2001) and in spotted wolfish (Anarhichas minor) (Foss et al., 2003). Fur<strong>the</strong>rmore,<br />

in a preliminary growth study performed in this lab under <strong>the</strong> same conditions as <strong>the</strong> present, a<br />

reduction in growth rate <strong>of</strong> 42% and 56% was observed in A. anguilla when exposed to a pCO 2 <strong>of</strong><br />

60mmHg and oscillating between 20-60mmHg respectively (P.B. Pedersen unpubl. obs.).<br />

The post-prandial increase in metabolic rate that occurs after feeding (specific dynamic action;<br />

SDA) represents <strong>the</strong> cumulative energy spent on ingesting and digesting a meal, and <strong>the</strong> subsequent<br />

absorption, assimilation and deposition <strong>of</strong> nutrients. This also includes <strong>the</strong> biochemical processes <strong>of</strong><br />

amino acid deamination, protein anabolism and syn<strong>the</strong>sis <strong>of</strong> excretory products (Jobling, 1981;<br />

Beamish and Trippel, 1990). Protein syn<strong>the</strong>sis represents <strong>the</strong> largest fraction <strong>of</strong> <strong>the</strong> SDA response<br />

(Brown and Cameron, 1991; Owen, 2001; Mccue, 2006a; Secor, 2009). The majority <strong>of</strong> SDA<br />

studies have focused on factors like meal size, feeding frequency and diet composition. With regard<br />

to environmental factors affecting <strong>the</strong> SDA response, most studies have focused on temperature<br />

(Mccue, 2006b; Secor, 2009), and some attention has been given to ambient O 2 saturation (Owen,<br />

2001; Jordan and Steffensen, 2007; Jourdan-Pineau et al., 2010; Zhang et al., 2010), but to our<br />

knowledge <strong>the</strong> effect <strong>of</strong> hypercapnia on SDA has so far not been studied in a teleost. The postprandial<br />

rise in oxygen consumption is accompanied by an increase in ammonia excretion, which is<br />

<strong>the</strong> main dissolved nitrogenous waste product in freshwater fishes (Wood, 2001). Post-prandial<br />

ammonia excretion rates depend on several factors including species, temperature, ration size and<br />

protein intake (Owen et al., 1998; Leung et al., 1999; Engin and Carter, 2001). A few studies have<br />

documented that environmental hypercapnia has an effect on protein metabolism in fish. An<br />

increased endogenous ammonia production and excretion was observed in carp (Cyprinus<br />

carpio)(Claiborne and Heisler, 1986). More recently, (Langenbuch and Portner, 2003) reported an<br />

80% decrease in hepatic protein syn<strong>the</strong>sis rate in two Antarctic teleosts (Pachycara<br />

brachycephalum and Lepidonoto<strong>the</strong>n kempi) in response to a hypercapnia-induced acidosis.<br />

Toge<strong>the</strong>r, <strong>the</strong>se observations suggest that environmental hypercapnia can cause a shift in<br />

intermediary metabolism towards increased protein catabolism and decreased anabolism. In an eel<br />

farming context, this is a highly undesirable effect because it reduces protein retention but also<br />

because <strong>the</strong> increased ammonia excretion will fur<strong>the</strong>r deteriorate <strong>the</strong> water quality.<br />

3


The aim <strong>of</strong> this work was to study oxygen consumption and ammonia excretion in <strong>the</strong> <strong>European</strong> eel<br />

(Anguilla anguilla) following feeding, and under conditions typical for intensive recirculating<br />

aquaculture environments i.e. elevated pCO 2 . Two scenarios <strong>of</strong> hypercapnia were chosen to mimic<br />

different feeding schedules. In one treatment (Osc·CO 2 ), oscillating pCO 2 levels (20-60mmHg)<br />

were chosen to mimic a single daily feeding event, and a second treatment (Hi·CO 2 ) with a high but<br />

constant pCO 2 (60mmHg) mimicked a 24 hours continuous feeding schedule. To determine whe<strong>the</strong>r<br />

any observed effects were caused by <strong>the</strong> elevation in pCO 2 levels or <strong>the</strong> concomitant reduction in<br />

pH, a third treatment (Lo·pH) was included, in which a normocapnic environment was maintained,<br />

but pH was lowered to <strong>the</strong> same level as <strong>the</strong> high pCO 2 treatment by addition <strong>of</strong> diluted HCl. The a<br />

priory hypo<strong>the</strong>sis was that hypercapnia would suppress <strong>the</strong> post-prandial peak in oxygen<br />

consumption rate (MO 2 ), reflective <strong>of</strong> a decreased protein syn<strong>the</strong>sis rate, and possibly prolong <strong>the</strong><br />

duration <strong>of</strong> <strong>the</strong> post-prandial state as observed in hypoxic cod (Jordan and Steffensen, 2007). The<br />

negative effect <strong>of</strong> hypercapnia was hypo<strong>the</strong>sized to be exacerbated at oscillating pCO 2 levels, owing<br />

to <strong>the</strong> added stress <strong>of</strong> <strong>the</strong> disequilibria in acid-base status and <strong>the</strong> preliminary observation <strong>of</strong><br />

reduced growth.<br />

Materials and methods<br />

Animals and holding conditions<br />

<strong>European</strong> eel, Anguilla anguilla were optained from a commercial eel farm (Stensgaard Åleopdræt,<br />

Randbøl, Denmark) and transported to <strong>the</strong> holding facility at <strong>the</strong> Technical University <strong>of</strong> Denmark,<br />

National <strong>Institut</strong>e <strong>of</strong> Aquatic Resources, Section for Aquaculture, Hirtshals, Denmark. Fish were<br />

evenly distributed into 4 separate 330L tanks at a density <strong>of</strong> approx. 1.2kg m -3 and received a 24<br />

hours bath treatment with 1mg L -1 mebendazole (Vermox, Janssen Pharmaceuticals Inc., Belgium)<br />

to avoid infestations with Pseudodactylogyrus spp. (Buchmann and Bjerregaard, 1990). Water was<br />

continuously recirculated (40l min -1 , Eheim 1260) through a submerged bi<strong>of</strong>ilter (25m 2 filter media)<br />

connected to each tank and 20% <strong>of</strong> <strong>the</strong> volume was exchanged daily with fresh tap water (pH 7.76 ±<br />

0.11, alkalinity 3.8 mEq L -1 ). Temperature was maintained at 23±1°C by aquarium heaters<br />

controlled by <strong>the</strong>rmostats (T Controller 2001C, Aqua Medic, GmbH, Bissendorf, Germany). Light<br />

conditions were 24h dimmed lighting. Proper oxygenation was maintained by injecting pure O 2 into<br />

<strong>the</strong> outlet from <strong>the</strong> bi<strong>of</strong>ilters. Fish were fed a daily ration (0.5% body mass) <strong>of</strong> commercial feed<br />

pellets (DAN-EX, 2mm, BioMar Group, Brande, Denmark). The composition <strong>of</strong> <strong>the</strong> feed was<br />

47.3% protein, 29.6% fat, 7.0% ash, with a total <strong>of</strong> 94.2% dry matter. The acclimation to<br />

hypercapnia or low pH (see below) was done at a rate corresponding to 0.2 pH units day -1 . When<br />

4


<strong>the</strong> desired conditions were reached, <strong>the</strong>se were maintained for minimum three weeks prior to<br />

experimentation. Water quality parameters (NH + 4 , NO - 3 and NO - 2 ) were monitored regularly, and<br />

always remained below 0.5, 1.0 and 50 mgL -1 respectively.<br />

Carbon dioxide and pH control<br />

A schematic <strong>of</strong> <strong>the</strong> experimental setup is presented in Figure 1. Water pCO 2 was controlled<br />

indirectly by monitoring water pH. The relationship between pH and pCO 2 was established with<br />

CO 2 equilibrated water at different pCO 2 tensions using a gas mixing pump (Radiometer GMA 2<br />

Precision Gas Supply). Alkalinity was monitored regularly and remained at 3.82 ± 0.15 mEq l -1 .<br />

Fluctuating pCO 2 levels (20-60mmHg) were achieved using <strong>the</strong> input <strong>of</strong> a pH meter (WTW340i,<br />

WTW GmbH, Weilheim, Germany) interfaced with a pc running a custom made Python script<br />

Figure 1. A schematic representation <strong>of</strong> <strong>the</strong> experimental setup.<br />

Holding conditions. 1a: Holding tank, 1b: Bi<strong>of</strong>ilter pump, 1c: Bi<strong>of</strong>ilter, 1d: Oxygen tank. CO 2 control. 2a:<br />

pH probe, 2b: pH meter, 2c: Galvanic isolation amplifier, 2d: Programmable instrument, 2e: Solenoid valve,<br />

2f: CO 2 gas, 2g: CO 2 mixing column with pump. Respirometry. 3a: Respirometer holding tank, 3b: Water<br />

supply to respirometer tank via UV sterilizer, 3c: Respirometer with recirculation loop (NB only 1 <strong>of</strong> 4<br />

depicted), 3d: AD converter, 3e: Flush pump, 3f: Fiber optic O 2 sensor, 3g: Laptop PC, 3h: Circulation<br />

pump. Arrows indicate flow <strong>of</strong> water. This schematic illustrates <strong>the</strong> setup for <strong>the</strong> Hi·pCO 2 experiment and a<br />

few modifications were applied to <strong>the</strong> Osc·pCO 2 and Lo·pH setups. See materials and methods section for<br />

fur<strong>the</strong>r details.<br />

5


controlling a solenoid valve and flow <strong>of</strong> pure CO 2 gas. pCO 2 levels oscillated within 24 hour<br />

periods with a daily high occurring between 18.00-20.00 hrs and a daily low between 08.00-10.00<br />

hrs. Constant high (60mmHg) pCO 2 was controlled via a programmable instrument (5714, PR<br />

electronics, Rønde, Denmark) that received input (optically isolated) from a pH meter (PHM 210,<br />

Radiometer, Denmark) controlling a solenoid valve and flow <strong>of</strong> CO 2 (<strong>the</strong> hysteresis was set to 0.1<br />

pH unit). To ensure that CO 2 and water was mixed and distributed into <strong>the</strong> tank thoroughly, CO 2<br />

was dispersed by wooden air stones (Aqua Medic GmbH, Bissendorf, Germany) into a tall acrylic<br />

cylinder (h= 1m, Ø = 50mm), that was continuously flushed with water from <strong>the</strong> tank (10L min -1 ,<br />

Eheim 1048). Low pH (6.5) and normocapnia was controlled exactly as above except in this setup,<br />

<strong>the</strong> instrument controlled <strong>the</strong> flow <strong>of</strong> a weak solution <strong>of</strong> HCl in tap water (~1.8mM) directly into <strong>the</strong><br />

tank.<br />

Respirometry<br />

In order to eliminate differences between holding conditions and experimental conditions, <strong>the</strong> water<br />

supply to respirometry setup was derived from <strong>the</strong> holding tank. Water was circulated through an<br />

aquarium UV sterilizer into a 160L tank holding 4 acrylic respirometers (2.1L) and returned to <strong>the</strong><br />

holding tank. The water in <strong>the</strong> respirometry tank was kept well mixed by a pump. Mass specific<br />

oxygen consumption MO 2 was measured by <strong>the</strong> principle <strong>of</strong> computerized intermittent flow through<br />

respirometry (Steffensen, 1989). The respirometers were periodically flushed for 4 minutes with<br />

water from <strong>the</strong> outer tank, followed by a closed 1 minute waiting period to obtain steady state and a<br />

5 minute measuring period. Oxygen partial pressure pO 2 was measured by a 4-channel fiber optic<br />

oxygen transmitter (OXY-4mini from PreSens GmbH, Germany) and recorded by <strong>the</strong> AutoResp4<br />

s<strong>of</strong>tware (Loligo Systems, Denmark). MO 2 was derived from <strong>the</strong> decrease in pO 2 during <strong>the</strong> 5<br />

minute measuring period according to: MO 2 = V(d(pO 2 )/dt) αM -1 , where V is <strong>the</strong> volume <strong>of</strong> <strong>the</strong><br />

respirometer, α is <strong>the</strong> specific oxygen solubility and M is <strong>the</strong> wet weight.<br />

Protocol<br />

Experimental eels were removed from <strong>the</strong> holding tank, subjected to a chasing protocol <strong>of</strong> no more<br />

than 10 minutes duration and transferred to <strong>the</strong> respirometer. The first measurement was taken to be<br />

representative <strong>of</strong> <strong>the</strong> maximum metabolic rate (MMR). Oxygen consumption measurements<br />

recorded <strong>the</strong> following 24 hours were used to calculate <strong>the</strong> standard metabolic rate (SMR). <strong>Eel</strong>s<br />

were <strong>the</strong>n removed from <strong>the</strong> respiromter, anaes<strong>the</strong>tized in 2-phenoxyethanol (400µl l -1 ) and force<br />

fed a s<strong>of</strong>tened mixture <strong>of</strong> feed pellets. This was deposited in <strong>the</strong> stomach by inserting a modified<br />

6


1mL syringe into <strong>the</strong> esophagus. The procedure lasted less than 1 minute after which eels were<br />

immediately returned to <strong>the</strong> respirometer. The ration size was 0.5% <strong>of</strong> body weight in dry pellets.<br />

MO 2 was <strong>the</strong>n measured <strong>the</strong> following 48 hours. To quantify <strong>the</strong> fraction <strong>of</strong> MO 2 that could be<br />

attributed to <strong>the</strong> handling stress, each eel was subjected to a sham feeding were <strong>the</strong> exact same<br />

procedure was followed only <strong>the</strong> feed was replaced with <strong>the</strong> same volume <strong>of</strong> water and MO 2 was<br />

monitored for following 24 hours. The sham feeding was ei<strong>the</strong>r done before or after <strong>the</strong> force<br />

feeding in a randomized order, but <strong>the</strong> order was <strong>the</strong> same for all 4 respirometers.<br />

Ammonia excretion<br />

To determine ammonia excretion, a 15ml water sample was retrieved from <strong>the</strong> respirometer<br />

immediately before <strong>the</strong> waiting period and again at <strong>the</strong> end <strong>of</strong> <strong>the</strong> measurement period. Samples<br />

were frozen at -20°C for later analysis. This was done during measurement <strong>of</strong> MMR, at SMR (after<br />

<strong>the</strong> first 24 hours) and at 0, 2, 4, 6, 12, 24, 36 and 48 hours after feeding. Determination <strong>of</strong> ammonia<br />

was performed spectrophotometrically at 680nm using a modification <strong>of</strong> <strong>the</strong> method described by<br />

(Bower and Holm-Hansen, 1980).<br />

Calculations and Statistics<br />

The SDA response was analyzed by method <strong>of</strong> non-linear quantile regression (Chabot and<br />

Claireaux, 2008) using <strong>the</strong> R Studio s<strong>of</strong>tware (http://www.r-project.org/). The following variables<br />

were quantified: SMR, Peak SDA -peak metabolic rate during <strong>the</strong> SDA response minus SMR, t peak -<br />

time to Peak SDA , SDA duration and SDA. The oxygen consumed during <strong>the</strong> sham feeding was<br />

analyzed as above and subtracted from SDA. Absolute metabolic scope (MS) was calculated as<br />

MMR-SMR, and factorial scope as MMR/SMR. The post-prandial metabolic scope was calculated<br />

as Peak net /SMR. The SDA coefficient was expressed as <strong>the</strong> total amount <strong>of</strong> energy used as a<br />

percentage <strong>of</strong> <strong>the</strong> digestible energy content <strong>of</strong> <strong>the</strong> meal using an oxycalorific coefficient <strong>of</strong> 14.06<br />

kJ/gO 2 (Gnaiger, 1983). Ammonia excretion rate was expressed as total ammonia-N excretion<br />

(TAN, mmol N kg -1 h -1 ) and was calculated as: [TAN= ΔcN*V resp /BM*Δt], where ΔcN is <strong>the</strong><br />

difference in ammonia-N concentration (mM) from <strong>the</strong> start <strong>of</strong> <strong>the</strong> waiting period to <strong>the</strong> end <strong>of</strong> <strong>the</strong><br />

measurement period, V resp is <strong>the</strong> volume <strong>of</strong> <strong>the</strong> respirometer less <strong>the</strong> volume <strong>of</strong> <strong>the</strong> fish, BM is <strong>the</strong><br />

body mass (kg) and Δt is <strong>the</strong> measurement period. Post-prandial TAN was analyzed as MO 2 ,<br />

quantifying duration, peak excretion rate TAN peak (mmol kg -1 h -1 ), TAN magnitude i.e. <strong>the</strong><br />

integrated excretion (mmol kg -1 ) and time to TAN peak (tTAN peak ). Ammonia quotients (AQ) were<br />

calculated as <strong>the</strong> ratio <strong>of</strong> <strong>the</strong> amount ammonia-N expired (mmol) to <strong>the</strong> amount <strong>of</strong> O 2 (mmol)<br />

7


consumed. The percentage <strong>of</strong> metabolism fueled by protein oxidation was calculated as AQ /<br />

0.248*100, where 0.248 is <strong>the</strong> <strong>the</strong>oretical value <strong>of</strong> AQ if 100% protein respiration (Gnaiger and<br />

Bitterlich, 1984). The percentage <strong>of</strong> <strong>the</strong> meal N content excreted, was calculated as TAN/ N<br />

intake*100, assuming a protein/nitrogen ratio <strong>of</strong> 5.8 (Gnaiger and Bitterlich, 1984). For all<br />

variables, Student´s t-tests were used to test if observed differences were statistically significant,<br />

accepting a P


metabolic rates (SMR) ± s.e.m as determined before feeding. Dotted line in B represents <strong>the</strong> actual CO 2<br />

partial pressures during <strong>the</strong> post-prandial phase. See materials and method section for details.<br />

The duration <strong>of</strong> <strong>the</strong> SDA response was significantly longer in <strong>the</strong> Hi·CO 2 and <strong>the</strong> Lo·pH group<br />

compared to <strong>the</strong> control group (Table 1) lasting approximately 44 and 47 hours respectively, but<br />

were not significantly different from each o<strong>the</strong>r. In <strong>the</strong> Osc·CO 2 group, <strong>the</strong> duration <strong>of</strong> <strong>the</strong> SDA<br />

response was not prolonged (Table 1). The differences in SDA between groups were not<br />

significantly different, and on average eels utilized between 34-40% <strong>of</strong> <strong>the</strong> aerobic metabolic scope<br />

with <strong>the</strong> Hi·CO 2 group using <strong>the</strong> highest fraction <strong>of</strong> <strong>the</strong>ir scope. The energetic cost <strong>of</strong> processing a<br />

meal, <strong>the</strong> SDA coefficient, expressed as <strong>the</strong> percentage <strong>of</strong> <strong>the</strong> energy content <strong>of</strong> <strong>the</strong> meal was not<br />

significantly affected by ei<strong>the</strong>r hypercapnia or low pH (Table 1).<br />

The fasting oxygen consumption rates at rest (SMR) were not affected by hypercapnia and/or low<br />

pH, but maximal metabolic rates (MMR) where significantly depressed in both hypercapnic groups<br />

(Table 1). The actual metabolic scope was also reduced in both hypercapnic groups but only<br />

significantly so in <strong>the</strong> Hi·CO 2 group, this group also had significantly reduced factorial scope<br />

(Table 1). It should be noted that MMR in <strong>the</strong> Osc·CO 2 group was measured at <strong>the</strong> lowest pCO 2<br />

(20mmHg) while SMR was measured at oscillating pCO 2 (20-60mmHg) which may have been a<br />

confounding factor.<br />

Table 1. Oxygen consumption in A. anguilla pre- and post feeding and SDA pr<strong>of</strong>ile after long-term<br />

exposure to hypercapnia and/or low pH<br />

Osc·pCO 2 Hi·pCO 2 Lo·pH Control<br />

Post-prandial<br />

Duration (hrs) 36.64 ± 1.81 43.94 ± 2.52* 46.70 ± 1.30* 36.14 ± 1.98<br />

t peak (hrs) 16.86 (7-23) 8.13 (6.5-15) 11.70 (6.5-26.5) 8.57 (7-10.5)<br />

Peak net (mgO 2 kg -1 h -1 ) 30.88 ± 4.10 25.81 ± 1.52 29.40 ± 3.01 33.55 ± 4.23<br />

SDA (mgO 2 kg -1 ) 610.37 ± 106.41 679.25 ± 99.96 781.61 ± 116.47 728.68 ± 90.05<br />

Post-prandial scope 1.87 ± 0.13 1.58 ± 0.05 1.68 ± 0.10 1.82 ± 0.11<br />

% aerobic scope 34.74 ± 3.50 40.07 ± 3.12 33.97 ± 1.62 33.80 ± 2.65<br />

SDA (kJ) 8.58 ± 1.50 10.22 ± 1.65 11.16 ± 1.38 11.51 ± 1.59<br />

SDA Coefficient 7.91 ± 1.38 9.42 ± 1.52 10.28 ± 1.27 10.61 ± 1.46<br />

Fasting<br />

SMR (mgO 2 kg -1 h -1 ) 36.87 ± 2.49 44.91 ± 1.35 43.92 ± 2.29 41.35 ± 1.52<br />

MMR (mgO 2 kg -1 h -1 ) 195.24 ± 14.70* 181.97 ± 10.54* 206.90 ± 10.41 229.76 ± 11.43<br />

Factorial scope 5.41 ± 0.34 4.10 ± 0.29* 4.94 ± 0.29 5.52 ± 0.37<br />

Absolute scope 159.06 ± 13.44 137.06 ± 11.24* 171.29 ± 10.99 184.18 ± 12.19<br />

<strong>Eel</strong>s were fed commercial feed pellets corresponding to 0.5% <strong>of</strong> <strong>the</strong>ir body weight. Osc·pCO 2 denotes<br />

hypercapnia at oscillating CO 2 partial pressures (20-60mmHg). Hi·pCO 2 denotes hypercapnia at constant<br />

high CO 2 partial pressure (60mmHg). Lo·pH denotes pH=6.5 and normocapnia. Values presented are mean ±<br />

9


s.e.m. except t peak , where numbers in brackets refer to <strong>the</strong> range. An asterisk signifies a value significantly<br />

different from control (Student´s t-test, p


protein respiration during MMR (fasting) were not significantly affected in any <strong>of</strong> <strong>the</strong> groups,<br />

except ammonia excretion was significantly decreased in <strong>the</strong> Hi·CO 2 group (Table 2).<br />

Table 2. Ammonia excretion pre- and post feeding in A. anguilla at hypercapnia and/or low pH<br />

O·pCO 2 H·pCO 2 L·pH Control<br />

Post-prandial<br />

Duration (hrs) 25.50 ± 3.45* 37.75 ± 3.18 36.70 ± 4.35 37.50 ± 3.43<br />

t peak (hrs) 10.57 (4-24) 12.00 (6-24) 16.80 (6-36) 12.75 (6-24)<br />

Peak net (mmol N kg -1 h -1 ) 0.19 ± 0.03 0.23 ± 0.03 0.18 ± 0.05 0.23 ± 0.04<br />

TAN net (mmol N kg -1 ) 2.78 ± 0.43* 3.85 ± 0.41 3.85 ± 0.90 4.47 ± 0.48<br />

AQ 0.16 ± 0.04 0.19 ± 0.02 0.13 ± 0.03 0.20 ± 0.03<br />

Protein respiration % 63.12 ± 14.73 76.39 ± 9.42 51.39 ± 10.90 80.54 ± 11.06<br />

N intake excreted % 9.08 ± 1.65 13.21 ± 1.43 11.72 ± 3.76 15.81 ± 1.82<br />

Fasting<br />

TAN SMR (mmol N kg -1 h -1 ) 0.05 ± 0.01 0.04 ± 0.01 0.09 ± 0.03* 0.02 ± 0.01<br />

TAN MMR (mmol N kg -1 h -1 ) 0.20 ± 0.05 0.11 ± 0.02* 0.20 ± 0.04 0.18 ± 0.01<br />

AQ SMR 0.03 ± 0.01 0.04 ± 0.01 0.05 ± 0.01* 0.01 ± 0.01<br />

AQ MMR 0.03 ± 0.01 0.02 ± 0.00 0.03 ± 0.01 0.03 ± 0.00<br />

Protein resp. SMR % 10.88 ± 1.36 15.78 ± 5.23 19.65 ± 6.71 5.63 ± 2.20<br />

Protein resp. MMR % 13.76 ± 2.25 9.06 ± 4.99 12.39 ± 6.83 10.26 ± 0.65<br />

<strong>Eel</strong>s were fed commercial feed pellets corresponding to 0.5% <strong>of</strong> <strong>the</strong>ir body weight. Osc·pCO 2 denotes<br />

hypercapnia at oscillating CO 2 partial pressures (20-60mmHg). Hi·pCO 2 denotes hypercapnia at constant<br />

high CO 2 partial pressure (60mmHg). Lo·pH denotes pH=6.5 and normocapnia. TAN is total ammonia<br />

nitrogen. AQ is ammonia Quotient. Values presented are mean ± s.e.m. except t peak , where numbers in<br />

brackets refer to <strong>the</strong> range. An asterisk signifies a value significantly different from control (Student´s t-test,<br />

p


suggest an arterial blood O 2 content reduced by 70% compared to normocapnic conditions. Limited<br />

O 2 availability has been reported to affect <strong>the</strong> SDA pr<strong>of</strong>ile in Atlantic cod (G. morhua) in that <strong>the</strong><br />

peak in MO 2 was significantly suppressed during hypoxia (6.8kPa) and that <strong>the</strong> duration <strong>of</strong> <strong>the</strong><br />

post-prandial increase in MO 2 was more than twice that in normoxia (Jordan and Steffensen, 2007).<br />

As observed in hypoxic cod, severe hypercapnia at a constant level prolonged <strong>the</strong> post-prandial<br />

state and although <strong>the</strong> peak increase in oxygen consumption was not significantly limited, <strong>the</strong><br />

observation still suggests that <strong>the</strong> energetically costly processes (i.e. protein syn<strong>the</strong>sis) was impaired<br />

in some way during chronic exposure to 60mmHg leading to <strong>the</strong> prolonged post-prandial state. A<br />

prolonged SDA duration was also observed in <strong>the</strong> Lo·pH group and was <strong>of</strong> comparative length to<br />

<strong>the</strong> Hi·CO 2 group, suggesting that <strong>the</strong> observed effect was caused by <strong>the</strong> acidosis and not by <strong>the</strong><br />

elevation in pCO 2 per se. A corroborating observation was made in a study on two Antarctic species<br />

(P. brachycephalum and L. kempi), where <strong>the</strong> in vitro hepatic protein syn<strong>the</strong>sis rate decreased by<br />

80% during extracellular acidosis regardless <strong>of</strong> <strong>the</strong> pCO 2 level (Langenbuch and Portner, 2003).<br />

However, <strong>the</strong> decrease in protein syn<strong>the</strong>sis was proposed to be <strong>the</strong> effect <strong>of</strong> a low intracellular pH<br />

(pHi), as this decreased in parallel with <strong>the</strong> decrease in extracellular pH (Langenbuch and Portner,<br />

2003). This leads to <strong>the</strong> question <strong>of</strong> whe<strong>the</strong>r or not A. anguilla regulates hepatic pHi. (McKenzie et<br />

al., 2003) observed that <strong>the</strong> pHi <strong>of</strong> heart and white muscle remained at normocapnic levels in spite<br />

<strong>of</strong> a severe arterial acidosis (at 45mmHg pCO 2 ). Regulation <strong>of</strong> pHi (in in heart, brain, liver and<br />

white muscle) during extracellular acidosis have also been observed in o<strong>the</strong>r hypercapnia tolerant<br />

species e.g. swamp eel (S. marmoratus, (Heisler, 1982), armored catfish (P. pardalis, (Brauner et<br />

al., 2004) and white sturgeon (A. transmontanus, (Baker et al., 2009). Supposing <strong>the</strong> prolonged<br />

post-prandial state was an effect <strong>of</strong> low pH and not pCO 2 per se, it could be an explanation as to<br />

why <strong>the</strong> same prolongation was not observed in <strong>the</strong> Osc·CO 2 . To this end, (McKenzie et al., 2002)<br />

observed that when subjected to a stepwise sequential increase in pCO 2 (from normocapnia to<br />

80mmHg in <strong>the</strong> course <strong>of</strong> two hours) arterial pCO 2 did not reach steady states within each step,<br />

leading to an increasing disequilibrium between water and blood pCO 2 levels. It is <strong>the</strong>refore<br />

possible, that although hypercapnia progressed at a much slower rate in <strong>the</strong> present study, <strong>the</strong><br />

arterial pCO 2 did not reach a steady state and that <strong>the</strong> acidosis was less pr<strong>of</strong>ound in this group.<br />

It follows that in consequence <strong>of</strong> <strong>the</strong> prolonged post-prandial increase in MO 2 (lasting almost two<br />

days), eels in aquaculture settings under those conditions (i.e. low pH) would be <strong>of</strong>fered a new meal<br />

before <strong>the</strong> previous one was fully digested. Studying <strong>the</strong> effect <strong>of</strong> different feeding schedules on<br />

metabolism in A. anguilla, (Heinsbroek et al., 2007) observed that feed intake was correlated to pre-<br />

12


feeding MO 2 and ammonia excretion rate and that meal size was adjusted according to <strong>the</strong><br />

metabolic scope available for processing <strong>the</strong> meal. Fur<strong>the</strong>rmore, it was observed that when eels fed<br />

once every second day, <strong>the</strong> postprandial phase was prolonged, and average feed intake and growth<br />

was reduced. Based on this, we believe that severe hypercapnia/ low pH have a negative impact on<br />

feed intake and growth in A. anguilla. In support <strong>of</strong> this, a preliminary growth study performed in<br />

this lab under <strong>the</strong> same conditions as <strong>the</strong> present, have demonstrated a 42% and 56% reduction in<br />

growth rates <strong>of</strong> ~40g eels at high (60mmHg) and oscillating (20-60mmHg) pCO 2 (P. Bovbjerg,<br />

pers. obs.). Data are presented in Table 3. In <strong>the</strong> present study, <strong>the</strong> duration <strong>of</strong> <strong>the</strong> SDA response in<br />

normocapnia was 36 hrs, which is longer than observed by (Owen et al., 1998) (~12hrs) in eels fed<br />

a similar ration once a day. This raises <strong>the</strong> question whe<strong>the</strong>r <strong>the</strong> stress <strong>of</strong> <strong>the</strong> protocol prolonged <strong>the</strong><br />

duration <strong>of</strong> <strong>the</strong> SDA response in <strong>the</strong> present study. However, <strong>the</strong> discrepancy could be explained by<br />

o<strong>the</strong>r factors. One, <strong>the</strong> difference in experimental approach i.e. force fed individuals vs bulk tank<br />

measurements. Two, by <strong>the</strong> fact that (Owen et al., 1998) made <strong>the</strong>ir observation on smaller eels<br />

(~45g) or three, that <strong>the</strong> minimum MO 2 observed did not represent fasting metabolic rates (Owen,<br />

2001). To this end, (Heinsbroek et al., 2007) observed that MO 2 only reached fasting levels (SMR)<br />

in eels (35-70g) fed once every 48hrs and that MO 2 remained elevated for approximately 37hrs after<br />

feeding.<br />

Table 3. Growth in A. anguilla under constant high or oscillating hypercapnia<br />

Control Control Osc·CO 2 Hi·CO 2<br />

W 0 (kg) 22.50 22.50 22.50 22.50<br />

W end (kg) 27.80 28.10 24.50 25.50<br />

Growth (kg) 5.30 5.60 2.00 3.00<br />

Dead (kg) 0.179 0.58 0.255 0.086<br />

time (days) 18 18 18 18<br />

SGR (% day -1 ) 1.22 1.25 0.53 0.72<br />

Osc·pCO 2 denotes oscillating CO 2 partial<br />

pressures (20-60mmHg), Hi·pCO 2 denotes<br />

constant high CO 2 partial pressure<br />

(60mmHg). Water pCO 2 was controlled<br />

indirectly by monitoring water pH. The<br />

relationship between water pH and pCO 2 was<br />

established by equilibrating water with CO 2<br />

using a gas mixing pump and assuming a<br />

constant alkalinity. (see CruzNeto and Steffensen, 1997) and materials and methods section for fur<strong>the</strong>r<br />

details). <strong>Eel</strong>s were acclimated to hypercapnic conditions for one month prior to <strong>the</strong> start <strong>of</strong> experiments. The<br />

temperature was held at 23°±1°C and light conditions were 24hrs dimmed lighting. The initial individual<br />

weight was approximately 40g. <strong>Eel</strong>s were fed commercial feed pellets ad libitum both during <strong>the</strong> acclimation<br />

and <strong>the</strong> growth phase using automated belt feeders.W 0 is <strong>the</strong> initial weight and W end is <strong>the</strong> final weight per<br />

tank. SGR is <strong>the</strong> specific growth rate according to: SGR = [(lnW end – nW 0 )/time)*100%].<br />

The loss <strong>of</strong> metabolic scope at chronically elevated pCO 2 (60mmHg) was <strong>the</strong> result <strong>of</strong> a reduced<br />

maximum metabolic rate (MMR). Also it should be viewed as a consequence <strong>of</strong> <strong>the</strong> severity <strong>of</strong> <strong>the</strong><br />

hypercapnic condition as (McKenzie et al., 2003) did not observe any reduction in metabolic scope<br />

or limitation in MMR at 45mmHg in similar sized eels at <strong>the</strong> same temperature. While some fish<br />

13


espond to hypercapnia by decreasing stroke volume (SV) leading to a reduced cardiac output (CO)<br />

(Ishimatsu et al., 2005), an increase in contractile force was observed A.anguilla ventricle strips<br />

exposed to 13% CO 2 (Gesser et al., 1982). This was later confirmed in vivo by McKenzie et al.<br />

(2002), who observed CO was maintained by an increase in SV when eels where acutely exposed to<br />

80mmHg at rest. At <strong>the</strong> same time, hypercapnia (10% CO 2 ) leads to a decrease in <strong>the</strong> blood O 2<br />

carrying capacity via combined Root and Bohr effects (Bridges et al., 1983). Toge<strong>the</strong>r <strong>the</strong>se<br />

observations demonstrate that despite suffering from pr<strong>of</strong>ound hypoxaemia, eels are able to meet<br />

<strong>the</strong> routine O 2 tissue demands, but <strong>the</strong>y may not be able to meet <strong>the</strong> increased demands <strong>of</strong><br />

exhaustive exercise. During <strong>the</strong> chasing protocol eels had to ei<strong>the</strong>r increase SV must or heart rate (f)<br />

to reach <strong>the</strong> required CO. It can be assumed that <strong>the</strong> scope left to increase CO via SV was<br />

diminished or exhausted, leaving only <strong>the</strong> possibility to increase f. As <strong>the</strong> increase in pumping<br />

capacity as measured in vitro at 20°C begins to level <strong>of</strong>f around 60 beats per minute (Methling et<br />

al., 2012), little was also left to gain from fur<strong>the</strong>r increases in f, and ultimately <strong>the</strong> capacity for<br />

aerobic exercise was reduced.<br />

Ammonia excretion<br />

The post-prandial ammonia excretion rates observed in this study during normocapnia were slightly<br />

lower than previously reported for A. anguilla fed <strong>the</strong> same ration <strong>of</strong> a diet with a similar nitrogen<br />

content at 25°C (Owen et al., 1998). This might be related to a mass specific (~45g vs ~170g)<br />

differences in metabolic rates between experimental animals, ra<strong>the</strong>r than a difference in N<br />

metabolism as such, since ammonia excretion rates are known to decrease with size in A. anguilla<br />

(Heinsbroek et al., 2007). Also, <strong>the</strong> AQ and <strong>the</strong> fraction <strong>of</strong> metabolism fuelled by protein<br />

respiration in <strong>the</strong> present study (0.20 and 80.5 for AQ and %protein respiration, respectively) were<br />

in range <strong>of</strong> <strong>the</strong> observed values reported by (Owen et al., 1998) (i.e. 0.148-0.331 and 59.7-133.5 for<br />

AQ and % protein respiration, respectively).<br />

Increased nitrogen excretion rates may indicate an increase in protein catabolism while an increase<br />

in nitrogen/oxygen (N/O) ratio indicates an increased oxidation <strong>of</strong> amino acids as metabolic<br />

substrates. In effect, this means that <strong>the</strong>re will be fewer amino acids available for protein syn<strong>the</strong>sis<br />

and growth. Environmental hypercapnia has been reported to cause an increase in nitrogen<br />

excretion and N/O ratios in carp (Cyprinus carpio) (Claiborne and Heisler, 1986) during fasting,<br />

which is in agreement with <strong>the</strong> observations on fasting eels in <strong>the</strong> present study. However, nei<strong>the</strong>r<br />

hypercapnia nor low pH affected <strong>the</strong> postprandial ammonia excretion rates or <strong>the</strong> protein<br />

14


espiration, and that eels exposed to oscillating pCO 2 actually excreted a smaller fraction <strong>of</strong> <strong>the</strong><br />

nitrogen intake during <strong>the</strong> post-prandial phase. This however, does not necessarily imply a higher N<br />

retention and biosyn<strong>the</strong>sis in this group on several accounts. For one, <strong>the</strong> peak MO 2 and <strong>the</strong> SDA<br />

were not increased, in fact SDA was lower although not significantly so. Secondly, an increase in<br />

protein catabolism and deamination would increase ammonia excretion, which was also not<br />

observed. Finally, it would be unprecedented and counterintuitive that hypercapnia should have<br />

positive effect on energy retention and growth as <strong>the</strong> body <strong>of</strong> literature points to no or negative<br />

effects (Crocker and Cech, 1996; Foss et al., 2003; Fivelstad et al., 2003; Danley et al., 2005;<br />

Fivelstad et al., 2007; Petochi et al., 2011; Moran and Stottrup, 2011). Although <strong>the</strong> majority <strong>of</strong><br />

nitrogenous waste is excreted as ammonia in A. anguilla (Owen et al., 1998), as much as 30-50%<br />

can be excreted as urea (Heinsbroek et al., 1991) and likewise in A. australis (Engin and Carter,<br />

2001). It is possible that in <strong>the</strong> present study, protein catabolism was unaffected by hypercapnia and<br />

a larger fraction <strong>of</strong> N was excreted as urea. However, urea syn<strong>the</strong>sis is more costly (requires more<br />

ATP) (Wood, 2001), so an increase in urea excretion (production) would be reflected in a higher<br />

MO 2 , which was not observed. Alternatively, <strong>the</strong> present observations could be explained by an<br />

increase in fecal loss, which would suggest a limited ability to absorb nutrients. A similar<br />

observation was made on carp (Cyprinus carpio) exposed to severe hypoxia, where fish had<br />

significantly decreased assimilation efficiencies as well as increased fecal losses (Zhou et al., 2001).<br />

Also, gastrointestinal blood flow can be reduced during hypoxia (Axelsson and Fritsche, 1991),<br />

which would limit nutrient absorption.<br />

The low pH (HCl) treatment caused a significant increase in fasting ammonia excretion rates and<br />

protein respiration at rest, while an effect <strong>of</strong> equal magnitude was not observed during hypercapnia.<br />

This must be related to <strong>the</strong> water alkalinity and suggests that <strong>the</strong> effect <strong>of</strong> low pH was aggravated in<br />

water low in bicarbonate content. Increased sensitivity to hypercapnic acidosis at reduced<br />

bicarbonate levels has also been observed in o<strong>the</strong>r aquatic animals (Portner, 2008). The increased<br />

ammonia excretion, AQ and protein respiration points to a shift in fasting metabolism towards<br />

breakdown <strong>of</strong> body protein and perhaps a preferential catabolism <strong>of</strong> alkalizing amino acids (e.g.<br />

asparagine and glutamine) as proposed by (Langenbuch and Portner, 2002).<br />

15


Acknowledgements<br />

This study was funded by <strong>the</strong> Danish Agency for Science and Innovation, The Elisabeth and Knud<br />

Petersen Foundation and <strong>the</strong> Faculty <strong>of</strong> Science, University <strong>of</strong> Copenhagen. A thank is due to B.Sc.<br />

Anders Jørgensen for creation <strong>of</strong> <strong>the</strong> Python script and technical assistance.<br />

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anguilla L.). Comparative Biochemistry and <strong>Physiology</strong> A-Molecular and Integrative <strong>Physiology</strong>,<br />

128, 631-644.<br />

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<strong>of</strong> <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla) fed at high and low ration levels. Canadian Journal <strong>of</strong><br />

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concentrations: Lessons from animal physiology and earth history. Journal <strong>of</strong> Oceanography, 60,<br />

705-718.<br />

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1-56.<br />

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Them. Fish <strong>Physiology</strong> and Biochemistry, 6, 49-59.<br />

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water. In R.C.Ryans (Ed.), Fish physiolohy, fish toxicology and fisheries management (pp. 157-161).<br />

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90/011.<br />

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In P.A.Wrigth and P. M. Anderson (Eds.), Nitrogen excretion (pp. 201-238). New York: Academic<br />

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Biochemical Systemic and Environmental <strong>Physiology</strong>, 171, 49-57.<br />

18


PAPER IV<br />

Printed from PlosOne


Pop Up Satellite Tags Impair Swimming Performance and<br />

Energetics <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla)<br />

Caroline Methling 1 *, Christian Tudorache 2 , Peter V. Skov 3 , John F. Steffensen 1,4<br />

1 Marine Biological Section, University <strong>of</strong> Copenhagen, Helsingør, Denmark, 2 <strong>Institut</strong>e <strong>of</strong> Biology, Leiden University, Leiden, <strong>the</strong> Ne<strong>the</strong>rlands, 3 National <strong>Institut</strong>e <strong>of</strong><br />

Aquatic Resources, Section for Aquaculture, Technical University <strong>of</strong> Denmark, Hirtshals, Denmark, 4 DTU Aqua, National <strong>Institut</strong>e <strong>of</strong> Aquatic Resources, Technical University<br />

<strong>of</strong> Denmark, Charlottenlund, Denmark<br />

Abstract<br />

Pop-up satellite archival tags (PSATs) have recently been applied in attempts to follow <strong>the</strong> oceanic spawning migration <strong>of</strong><br />

<strong>the</strong> <strong>European</strong> eel. PSATs are quite large, and in all likelihood <strong>the</strong>ir hydraulic drag constitutes an additional cost during<br />

swimming, which remains to be quantified, as does <strong>the</strong> potential implication for successful migration. Silver eels<br />

(L T = 598.6629 mm SD, N = 9) were subjected to swimming trials in a Steffensen-type swim tunnel at increasing speeds <strong>of</strong><br />

0.3–0.9 body lengths s 21 , first without and subsequently with, a scaled down PSAT dummy attached. The tag significantly<br />

increased oxygen consumption (MO 2 ) during swimming and elevated minimum cost <strong>of</strong> transport (COT min ) by 26%. Standard<br />

(SMR) and active metabolic rate (AMR) as well as metabolic scope remained unaffected, suggesting that <strong>the</strong> observed<br />

effects were caused by increased drag. Optimal swimming speed (U opt ) was unchanged, whereas critical swimming speed<br />

(U crit ) decreased significantly. Swimming with a PSAT altered swimming kinematics as verified by significant changes to tail<br />

beat frequency (f), body wave speed (v) and Strouhal number (St). The results demonstrate that energy expenditure,<br />

swimming performance and efficiency all are significantly affected in migrating eels with external tags.<br />

Citation: Methling C, Tudorache C, Skov PV, Steffensen JF (2011) Pop Up Satellite Tags Impair Swimming Performance and Energetics <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

(Anguilla anguilla). PLoS ONE 6(6): e20797. doi:10.1371/journal.pone.0020797<br />

Editor: Yan Ropert-Coudert, <strong>Institut</strong> Pluridisciplinaire Hubert Curien, France<br />

Received March 20, 2011; Accepted May 9, 2011; Published June 8, 2011<br />

Copyright: ß 2011 Methling et al. This is an open-access article distributed under <strong>the</strong> terms <strong>of</strong> <strong>the</strong> Creative Commons Attribution License, which permits<br />

unrestricted use, distribution, and reproduction in any medium, provided <strong>the</strong> original author and source are credited.<br />

Funding: This study was funded by <strong>the</strong> Danish Agency for Science Technology and Innovation, The Elisabeth and Knud Petersen Foundation, and The Faculty <strong>of</strong><br />

Science, University <strong>of</strong> Copenhagen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation <strong>of</strong> <strong>the</strong> manuscript.<br />

Competing Interests: The authors have declared that no competing interests exist.<br />

* E-mail: cmethling@bio.ku.dk<br />

Introduction<br />

The <strong>European</strong> eel (Anguilla anguilla) is common in waters <strong>of</strong><br />

Western Europe. The spawning site <strong>of</strong> this species is believed to be<br />

<strong>the</strong> Sargasso Sea since this is where <strong>the</strong> smallest eel larvae have been<br />

found [1–3], but so far nei<strong>the</strong>r spawning adults nor eggs have been<br />

found in <strong>the</strong> Sargasso Sea to confirm this. <strong>European</strong> eel stocks have<br />

seen a strong decline since <strong>the</strong> 1980’s and numbers are now believed<br />

to have declined by as much as 90 to 99% [4]. There are several<br />

hypo<strong>the</strong>ses as to <strong>the</strong> causative mechanisms, including overfishing,<br />

pollution, and mass infections with <strong>the</strong> swim bladder parasite<br />

Anguillicola crassus. In addition, <strong>the</strong> nutritional status <strong>of</strong> individuals at<br />

<strong>the</strong> onset <strong>of</strong> migration may also play a role, in that many eels<br />

apparently do not have <strong>the</strong> minimum fat content required to fuel <strong>the</strong><br />

journey [5–8]. Fur<strong>the</strong>r insights into this part <strong>of</strong> <strong>the</strong> <strong>European</strong> eels<br />

reproduction cycle, are very valuable for future management <strong>of</strong> <strong>the</strong><br />

species both with regards to conservation and successful breeding<br />

programs in aquaculture. Several attempts have been made to follow<br />

eels during <strong>the</strong>ir spawning migration to gain information on <strong>the</strong><br />

migration route and <strong>the</strong> direction. In many <strong>of</strong> <strong>the</strong>se studies eels were<br />

tracked with acoustic transmitters, and individuals were only<br />

followed for a limited time, up to 156 hours [9–13]. During <strong>the</strong><br />

past decades, externally attached pop-up satellite archival tags<br />

(PSATs) have frequently been used in tracking studies on a variety <strong>of</strong><br />

large pelagic fish species [14–19]. PSATs make it possible to recover<br />

information on a multitude <strong>of</strong> parameters including temperature,<br />

depth and geo-location, and thus provide valuable information on<br />

swimming velocity, direction and depth that may help obtain a better<br />

understanding <strong>of</strong> <strong>the</strong> migratory behavior <strong>of</strong> <strong>European</strong> eel as this<br />

information could provide <strong>the</strong> key to <strong>the</strong>ir reproduction success and<br />

recent decline in population strength. PSATs have also been used on<br />

one <strong>of</strong> <strong>the</strong> largest eel species, <strong>the</strong> New Zealand longfin eel (Anguilla<br />

dieffenbachii) by Jellyman and Tsukamoto [20], who tracked 7.6–<br />

11.4 kg eels for 2–3 months, and recently, Aarestrup and co-workers<br />

[21] used PSATs to track migrating <strong>European</strong> eels for distances up to<br />

1300 km. The time required to cover this distance was approximately<br />

2 months and corresponded to a swimming speed between<br />

0.06–0.3 body lengths per second. Despite energy spent on diel<br />

vertical migrations, <strong>the</strong> distance was shorter and <strong>the</strong> speed was<br />

slower than anticipated. Assuming a cruising speed <strong>of</strong> 0.8 to 1 body<br />

length per second, as suggested by Palstra and co-workers [22], a 1<br />

meter long eel should be able to swim <strong>the</strong> 5000–6000 km from<br />

continental Europe to <strong>the</strong> Sargasso Sea in 3 to 4 months. Migrating<br />

<strong>European</strong> eel are much smaller than o<strong>the</strong>r species traditionally used<br />

in PSAT studies, and it is possible that <strong>the</strong> hydrodynamic resistance<br />

<strong>of</strong> <strong>the</strong> tag is a barrier to successful migration.<br />

The objectives <strong>of</strong> <strong>the</strong> present study were to measure <strong>the</strong> energy<br />

expenditure at different swimming speeds, analyze <strong>the</strong> swimming<br />

capacity and <strong>the</strong> biomechanics <strong>of</strong> migratory silver eels equipped<br />

with a PSAT dummy, and to compare <strong>the</strong>m with those <strong>of</strong><br />

untagged eels.<br />

Materials and Methods<br />

This study was carried out in accordance with <strong>the</strong> Danish<br />

Animal Experimentation Act and <strong>the</strong> protocol was approved by<br />

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PSATs Impair Swimming in <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

<strong>the</strong> Danish Animal Experimentation Board (licence number:<br />

2004/561–894).<br />

Fish origin and husbandry<br />

<strong>Eel</strong>s were caught with traps in <strong>the</strong> vicinity <strong>of</strong> <strong>the</strong> Marine<br />

Biological Laboratory, University <strong>of</strong> Copenhagen, in October<br />

2009. Fish were kept in a circular 3000 L tank, supplied with<br />

recirculating aerated seawater with a salinity <strong>of</strong> .32%, at a<br />

constant temperature <strong>of</strong> 10uC. <strong>Eel</strong>s were kept under <strong>the</strong>se<br />

conditions to acclimatize for at least two months prior to<br />

experimentation. In accordance with <strong>the</strong> general observation <strong>of</strong><br />

migrating silver eels, <strong>the</strong>y did not feed, although <strong>of</strong>fered a variety<br />

<strong>of</strong> food items. All eels were determined to be females in <strong>the</strong>ir<br />

migrating phase (Stage IV) [23].<br />

Setup<br />

Tests were performed in a 90 l Steffensen-type swim tunnel,<br />

downsized to 55 l by inserting a solid section, blocking <strong>the</strong> lower<br />

half <strong>of</strong> most <strong>of</strong> <strong>the</strong> tunnel leaving a 70*20*10 cm (l*w*d)<br />

swimming section (Fig. 1A). Turbulence was minimized by<br />

directing <strong>the</strong> flow through two sets <strong>of</strong> baffles and a 10 cm<br />

honeycomb. The swim tunnel was submerged in an outer tank,<br />

supplied with aerated water from a reservoir. The water in <strong>the</strong><br />

outer tank was maintained at 1060.1uC by continuously pumping<br />

it through a <strong>the</strong>rmostat, a filter and an aquarium UV sterilizer. In<br />

addition, <strong>the</strong> water was kept well-mixed by a submerged Eheimpump.<br />

Water velocity was controlled by a motor-driven propeller<br />

and motor controller (WEG, Germany) and <strong>the</strong> output voltage<br />

calibrated against a TAD flow meter (Höntzsch, Germany).<br />

Velocities were corrected for solid blocking effect according to Bell<br />

and Terhune [24]. A CCDTV video camera (TSR481, ELMO<br />

CO, LTD, Japan) was mounted above <strong>the</strong> swimming section<br />

illuminated with a single white LED allowing filming <strong>of</strong> <strong>the</strong> entire<br />

swimming section. The entire setup was shielded from daylight<br />

and o<strong>the</strong>r disturbances by black curtains. Video sequences were<br />

recorded by <strong>the</strong> PCTV USB2 s<strong>of</strong>tware (Pinnacle systems Inc. CA,<br />

USA). Oxygen tension was continuously measured (1 Hz) with a<br />

Fibox 3 electrode by <strong>the</strong> Oxyview s<strong>of</strong>tware (version 5.31,<br />

PreSense, Germany) and recorded by <strong>the</strong> AutoResp TM 1 s<strong>of</strong>tware<br />

(version 1.6, Loligo systems). Intermittent flow through respirometry<br />

[25–27] was used to monitor <strong>the</strong> oxygen consumption at<br />

different swimming velocities. The swim tunnel was periodically<br />

flushed for 8 min with water from <strong>the</strong> outer tank, followed by a<br />

closed 2 min waiting period, to obtain steady state conditions, and<br />

a 20 min measuring period.<br />

Protocol<br />

Three swimming trials were completed on 9 individuals, with<br />

<strong>the</strong> first serving as control without a tag for <strong>the</strong> subsequent two<br />

trials with a tag attached. The third trial was performed in order to<br />

investigate if swimming performance was affected by repeated<br />

trials. Fur<strong>the</strong>r trials were not undertaken as preliminaries showed<br />

no difference between second and third trials. Before being<br />

introduced to <strong>the</strong> swim tunnel, eels were quickly (3–4 min)<br />

anaes<strong>the</strong>tized in a 40 mg L 21 benzocaine solution. Benzocaine is<br />

rapidly excreted across <strong>the</strong> gills with a half-life <strong>of</strong> ,20 min [28].<br />

Total length (L T = 598.6629 mm SD), mass (339.6651.5 g SD),<br />

maximum height and width, were recorded to adjust for solid<br />

blocking and in addition to <strong>the</strong>se, pectoral fin length, vertical and<br />

horizontal eye diameter were recorded to classify silver stage. <strong>Eel</strong>s<br />

were left to acclimatize in <strong>the</strong> swim tunnel for 24 hrs at a velocity<br />

<strong>of</strong> 0.3 body lengths per second (BL s 21 ), corresponding to <strong>the</strong><br />

lowest speed that incited swimming. After 24 hrs, <strong>the</strong> velocity was<br />

increased in increments <strong>of</strong> 0.1 Bl s 21 during <strong>the</strong> 2 min waiting<br />

period. <strong>Eel</strong>s swam at each new speed for 20 min (<strong>the</strong> measurement<br />

period) and <strong>the</strong> speed was increased until <strong>the</strong>y were unable to<br />

maintain swimming and keep <strong>of</strong>f <strong>the</strong> rear grid. <strong>Eel</strong>s were <strong>the</strong>n<br />

removed from <strong>the</strong> swim tunnel, anaes<strong>the</strong>tized as above and <strong>the</strong> tag<br />

was attached. The tag was a scaled-down replica <strong>of</strong> a PSAT (X-tag<br />

Archival) (Microwave Telemetry, Inc. DC, USA). A scaled-down<br />

tag was chosen to match <strong>the</strong> size <strong>of</strong> <strong>the</strong> eels, as <strong>the</strong>y were smaller<br />

than <strong>the</strong> migrating eels tagged with a PSAT in previous tracking<br />

studies [20,21]. The PSAT dummy was manufactured from a<br />

cylindrical piece <strong>of</strong> PVC (16 mm in diameter, 60 mm long and<br />

mass in air 5.6 g), compared to 32 mm, 130 mm and 42 g <strong>of</strong> <strong>the</strong><br />

original tag. The frontal cross-sectional area <strong>of</strong> <strong>the</strong> dummy tag was<br />

Figure 1. Schematic <strong>of</strong> swim tunnel and PSAT dummy. A. 1. Motor, 2. Propeller, 3. Flushpump (inlet), 4. Flush outlet, 5. Honeycomb, 6. Mixing<br />

pump, 7. Outlet from tank to water reservoir, 8. Inlet to tank from water reservoir. Arrows indicate water flow.B. PSAT dummy. C. PSAT dummy<br />

attached to eel. Refer to text for details.<br />

doi:10.1371/journal.pone.0020797.g001<br />

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PSATs Impair Swimming in <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

on average 24% <strong>of</strong> <strong>the</strong> cross-sectional area <strong>of</strong> <strong>the</strong> eel. As <strong>the</strong><br />

original tag, <strong>the</strong> dummy tag was positively buoyant. The drag (g)<br />

<strong>of</strong> <strong>the</strong> tag was measured separately in a flow chamber with a force<br />

transducer, converted to mN and expressed as function <strong>of</strong> water<br />

velocity (cm s 21 ) by y = 0.0136 1.79 (r 2 = 0.99). The tag was<br />

attached to <strong>the</strong> eels by a stainless steel wire from <strong>the</strong> tag to two<br />

plastic attachment plates (30*15*2 mm) positioned on ei<strong>the</strong>r side<br />

<strong>of</strong> <strong>the</strong> body, in order to evenly distribute <strong>the</strong> drag. The tag was<br />

positioned approximately J <strong>of</strong> a body length from <strong>the</strong> snout, so<br />

that <strong>the</strong> lift from <strong>the</strong> tag would be approximately centred. The<br />

attachment plates were rounded and equipped with silicone pads<br />

to minimize stress to <strong>the</strong> skin, and attached to <strong>the</strong> eel by two<br />

parallel surgical steel wires (0.3 mm Ø) transversing <strong>the</strong> dorsal<br />

body musculature. The position and placement <strong>of</strong> <strong>the</strong> tag was in<br />

close similarity to <strong>the</strong> study by Aarestrup and co-workers [21]. The<br />

attachment <strong>of</strong> <strong>the</strong> tag was completed within 2 min, during which<br />

<strong>the</strong> gills were flushed with aerated water containing a weak dose<br />

(20 mg L 21 ) <strong>of</strong> anaes<strong>the</strong>tic. <strong>Eel</strong>s were returned to <strong>the</strong> swim tunnel,<br />

Figure 2. Swimming energetics in (A. anguilla) with a PSAT dummy. A. Oxygen consumption (MO 2 , mgO 2 kg 21 h 21 ) and B. Cost <strong>of</strong> transport<br />

(COT, mgO 2 kg 21 km 21 ) as a function <strong>of</strong> swimming speed (U, Bls 21 ) swimming with and without a PSAT dummy. Swim trials without (control) and<br />

with a tag (trials 2, 3) were performed on <strong>the</strong> same individual (N = 9). Lines are regression lines (refer to Table 1 for regression values). An asterisk<br />

denotes significant difference between control and tagged condition (Repeated measures ANOVA, p,0.05). Data are presented as mean6SD. C.<br />

Additional cost <strong>of</strong> tag on COT as a function <strong>of</strong> swimming speed (U, Bls 21 ). Line is regression line <strong>of</strong> <strong>the</strong> average value <strong>of</strong> trials 2 and 3.<br />

doi:10.1371/journal.pone.0020797.g002<br />

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PSATs Impair Swimming in <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

where <strong>the</strong>y were left to recover swimming at 0.3 Bl s 21 . The swim<br />

trial was repeated as above 24 and 48 hrs after attachment <strong>of</strong> <strong>the</strong><br />

tag.<br />

Calculations and statistics<br />

Mass specific oxygen consumption (MO 2 )wasderivedfrom<strong>the</strong><br />

decrease in oxygen partial pressure (pO 2 ) during <strong>the</strong> 20 min<br />

measuring period according to: MO 2 =V(d(pO 2 )/dt) aM 21 ,where<br />

V is volume <strong>of</strong> <strong>the</strong> swim tunnel, a is oxygen solubility and M is <strong>the</strong> wet<br />

weight. Oxygen consumption as a function <strong>of</strong> swimming speed (U)<br />

was fitted to <strong>the</strong> equation: MO 2 =aU b +SMR, with SMR being <strong>the</strong><br />

standard metabolic rate at zero speed or at rest. The critical<br />

swimming speed (U crit ) was calculated according to Beamish [29] as<br />

U crit = U f +(t f t i 21 DU) whereU f is <strong>the</strong> highest velocity maintained for<br />

an entire 20 min interval, DU is <strong>the</strong> velocity increment (5 cm s 21 ), t f is<br />

<strong>the</strong> duration <strong>of</strong> <strong>the</strong> final (fatigue) velocity increment and t i is <strong>the</strong> time<br />

interval (20 min; [30]). Active metabolic rate at <strong>the</strong> critical swimming<br />

speed (AMR crit ), sustained for 20 min, was used to calculate <strong>the</strong><br />

factorial metabolic scope (AMR crit SMR 21 ). A polynomial equation<br />

(ax 2 +bx+c) was fitted to <strong>the</strong> relationship between fish swimming speed<br />

and oxygen consumption. The swimming speed with <strong>the</strong> lowest cost<br />

<strong>of</strong> transport (U opt ) and <strong>the</strong> corresponding oxygen consumption<br />

(COT min ) was calculated from <strong>the</strong> roots <strong>of</strong> <strong>the</strong> derivative as x = 2b/<br />

2a and y = 2(b 2 24ac)/4a, respectively.<br />

Video recordings from each swimming speed were analysed to<br />

calculate tail beat frequency (f), tail beat amplitude (a) and body<br />

wave velocity (V). Tail beat frequency was obtained by counting<br />

during a 20 second period at <strong>the</strong> beginning, middle and end <strong>of</strong><br />

each swimming velocity. a was calculated using Vernier Logger<br />

Pro (v3.6., Vernier S<strong>of</strong>tware & Technology, USA). Frames where<br />

<strong>the</strong> tail was in <strong>the</strong> outermost position were chosen and position <strong>of</strong><br />

<strong>the</strong> tail tip recorded. The amplitude was calculated as <strong>the</strong><br />

difference between <strong>the</strong> two outermost positions <strong>of</strong> <strong>the</strong> tail tip<br />

during one tail beat. This was repeated ten times for each <strong>of</strong> <strong>the</strong><br />

three periods, and used to calculate an average for each swimming<br />

speed. Body wave velocity (V) was calculated as <strong>the</strong> distance<br />

travelled by a wave crest over time from <strong>the</strong> digitized video<br />

sequences using Vernier Logger Pro. The Strouhal number (St)<br />

was calculated according to <strong>the</strong> formula St = af/U. The Strouhal<br />

number is dimensionless and has been shown to be strongly<br />

correlated to force production and efficiency <strong>of</strong> flapping foils [31]<br />

and <strong>the</strong> propulsive efficiency <strong>of</strong> swimming fish [32,33].<br />

Data <strong>of</strong> tagged and untagged eels were compared at each<br />

swimming speed using repeated measurements ANOVA followed<br />

by a Holm-Sidak multi comparison procedure (SigmaPlot v. 11,<br />

Systat systems inc. USA) when significant effects were found.<br />

Significance value was p,0.05.<br />

Results<br />

Respirometry<br />

Oxygen consumption during swimming was significantly<br />

higher for tagged compared to non-tagged eels (Trial 1 v. Trials<br />

2, 3) at speeds above 0.4 Bl s 21 (Fig. 2A). There was no<br />

significant difference between Trial 2 and Trial 3. There were no<br />

significant differences in standard metabolic rate (SMR), active<br />

metabolic rate (AMR crit ) or metabolic scope between trials. Cost<br />

<strong>of</strong> transport was significantly higher in tagged versus untagged<br />

eels at all but <strong>the</strong> lowest swimming speed (0.3 BL s 21 ), again with<br />

no difference between tagged trials (Fig. 2B). The critical<br />

swimming velocity U crit was significantly lower with a tag<br />

attached compared to control, but <strong>the</strong>re was no difference<br />

between tagged trials (Table 1). Optimal swimming speed (U opt )<br />

was not affected by tagging, but minimum cost <strong>of</strong> transport<br />

Table 1. Regression values and swimming energetic<br />

parameters.<br />

Parameter Control Trial 2 Trial 3<br />

a 96.62638.64 a 148.79656.28 a 129.58625.17 a<br />

b 2.5161.24 a 2.2960.67 a 2.5160.98 a<br />

SMR (mgO 2 kg 21 h 21 ) 26.28611.24 a 28.3667.42 a 29.71613.70 a<br />

(r 2 = 0.993) (r 2 = 0.995) (r 2 = 0.985)<br />

AMR (mgO 2 kg 21 h 21 ) 106.83629.39 a 118.09635.99 a 112.15634.91 a<br />

Scope 5.1563.77 a 4.4662.01 a 4.0461.11 a<br />

U opt (Bl s 21 ) 0.6060.12 a 0.5460.06 a 0.5260.07 a<br />

COT min (mgO 2 kg 21 km 21 ) 40.7062.27 a 50.8765.35 b 49.5867.47 b<br />

U crit (Bl s 21 ) 0.9060.14 a 0.7360.13 b 0.8060.13 b<br />

Oxygen consumption (MO 2 , mgO 2 kg 21 h 21 ) was expressed as a function <strong>of</strong><br />

swimming speed (U, Body lengths s 21 ) with <strong>the</strong> formula f =abU b +SMR. Swim<br />

trials without (control) and with a tag (trials 2, 3) were performed on <strong>the</strong> same<br />

individual. Abbreviations: SMR, standard metabolic rate; AMR, active metabolic<br />

rate; U opt , optimal swimming speed; COT min , minimum cost <strong>of</strong> transport; U crit ,<br />

critical swimming speed. Values are mean6SD. Different superscripts indicate<br />

significant differences per row (repeated measurements ANOVA, p,0.05, N = 9).<br />

doi:10.1371/journal.pone.0020797.t001<br />

(COT min ) was significantly higher when swimming with a tag<br />

(Table 1). The additional cost <strong>of</strong> swimming with <strong>the</strong> tag, on cost<br />

<strong>of</strong> transport, increased with swimming speed according to <strong>the</strong><br />

formula (average <strong>of</strong> trials 2 and 3) y = 10.14e 1.52x (r 2 = 0.92) and<br />

was estimated to be 26% higher at <strong>the</strong> optimal swimming speed<br />

U opt <strong>of</strong> 0.6 Bl s 21 (Fig. 2C).<br />

Kinematics<br />

Tail beat frequency f, plotted against swimming speed (U)<br />

revealed a linear relationship, f =a+bU, with a being <strong>the</strong> intercept<br />

and b being <strong>the</strong> slope <strong>of</strong> <strong>the</strong> curve (Table 2). At swimming speeds<br />

greater than 0.4 Bl s 21 f was significantly higher in tagged eels<br />

compared to non-tagged eels at <strong>the</strong> same swimming speed<br />

(Fig. 3A). Tail beat amplitude (a) was not affected by tagging<br />

and remained constant across all swimming speeds at<br />

10.5761.38 cm (Fig. 3B). The width <strong>of</strong> <strong>the</strong> swim tunnel was<br />

20 cm and thus <strong>the</strong> eels were allowed <strong>the</strong>ir full range <strong>of</strong> motion<br />

without any obstructions at all speeds. Body wave velocity (V) was<br />

positively correlated with U and could be described by V =a+bU<br />

Table 2. Regression values and swimming kinematics.<br />

Parameter Control Trial 2 Trial 3<br />

Tail beat frequency, f a (slope) 1.4160.21 a 2.1760.17 b 2.1160.17 b<br />

b (intercept) 0.3160.07 a 0.1560.08 b 0.1060.05 b<br />

(r 2 = 1.00) (r 2 = 0.99) (r 2 = 1.00)<br />

Wave speed, V a (slope) 1.0160.02 a 1.1960.03 b 1.1560.02 a<br />

b (intercept) 0.0160.01 a 0.0260.01 a 0.0160.01 a<br />

(r 2 = 1.00) (r 2 = 1.00) (r 2 = 1.00)<br />

Strouhal (St) 0.3160.02 a 0.4360.05 b 0.4260.05 b<br />

Tail-beat frequency (f) and body wave speed (V) was expressed as a function <strong>of</strong><br />

swimming speed (U, Body lengths s 21 ) with <strong>the</strong> formula f =a+bU. Swim trials<br />

without (control) and with a tag (trials 2, 3) were performed on <strong>the</strong> same<br />

individual. Values are mean6SD. Different superscripts indicate significant<br />

differences per row (repeated measurements ANOVA, p,0.05, N =9).<br />

doi:10.1371/journal.pone.0020797.t002<br />

PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20797


PSATs Impair Swimming in <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

Figure 3. Swimming kinematics in (A. anguilla) with a PSAT dummy. A. Tail beat frequency (Hz) B. Tail beat amplitude (body lengths) and C.<br />

Body wave speed (body lengths) as a function <strong>of</strong> swimming speed (U, body lengths s 21 ) swimming with and without a PSAT dummy. Swim trials<br />

without (control) and with a tag (trials 2, 3) were performed on <strong>the</strong> same individual (N = 9). Lines are regression lines (refer to Table 2 for regression<br />

values). An asterisk denotes significant difference between control and tagged condition (Repeated measures ANOVA, p,0.05). Data are presented as<br />

mean6SD.<br />

doi:10.1371/journal.pone.0020797.g003<br />

(Table 2). At swimming speeds above 0.3 Bl s 21 V was<br />

significantly higher in tagged eels compared to non-tagged eels<br />

(Fig. 3C). The Strouhal number (St) was significantly lower in <strong>the</strong><br />

control trial compared to <strong>the</strong> two tagged trials, with no difference<br />

between tagged trials.<br />

Discussion<br />

This study demonstrates that attaching a PSAT dummy to<br />

<strong>European</strong> eels, results in an increased oxygen uptake during<br />

swimming, an increased cost <strong>of</strong> transport, and a decreased<br />

swimming efficiency and performance.<br />

No changes to standard metabolic rate, active metabolic rate or<br />

metabolic scope were associated with fitting eels with a PSAT<br />

dummy. This implies that <strong>the</strong>re was no increased energy<br />

expenditure during rest, for example from maintaining buoyancy,<br />

and that aerobic capacity was uncompromised. The standard<br />

metabolic rate was similar and within range <strong>of</strong> what is reported in<br />

<strong>the</strong> literature for A. anguilla [34] and A. rostrata [35], and for resting<br />

PLoS ONE | www.plosone.org 5 June 2011 | Volume 6 | Issue 6 | e20797


PSATs Impair Swimming in <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

A. anguilla <strong>of</strong> similar size at <strong>the</strong> same temperature (unpubl. obs.).<br />

The increased oxygen uptake during swimming in <strong>the</strong> tagged trials<br />

was likely to be a result <strong>of</strong> <strong>the</strong> additional drag <strong>of</strong> <strong>the</strong> transmitter as<br />

SMR was not significantly increased. This has previously been<br />

suggested with regards to o<strong>the</strong>r externally attached transmitters<br />

[36–38] and recently, a similar observation <strong>of</strong> increased oxygen<br />

uptake during swimming was reported in a study on Atlantic cod<br />

(Gadus morhua) with an externally attached acoustic dummy<br />

transmitter [39]. They found no difference in standard or active<br />

metabolic rates, but a decrease in <strong>the</strong> optimal and critical<br />

swimming speed. In <strong>the</strong> present study, <strong>the</strong> maximum metabolic<br />

rate was reached at a lower swimming speed in tagged eel, which<br />

corroborates an additional metabolic cost <strong>of</strong> swimming with <strong>the</strong><br />

tag. The values for AMR and U crit were slightly lower than<br />

previously reported for silver eels <strong>of</strong> similar size [40]; Palstra and<br />

co workers reported an AMR <strong>of</strong> ,120 mgO 2 kg 21 h 21 and U crit<br />

<strong>of</strong> ,1.1Bl s 21 , and Quintella and co workers [41] a U crit <strong>of</strong><br />

,1.2 Bl s 21 . However, those studies were performed at 18uC<br />

compared to 10uC in <strong>the</strong> present study, which may explain <strong>the</strong><br />

discrepancy, as it is generally observed that performance depends<br />

on ambient temperature [42,43], and is at its maximum at <strong>the</strong><br />

preferred temperature, that has been reported to be approximately<br />

18uC in <strong>the</strong> closely related American eel (A. rostrata) [44]. In <strong>the</strong><br />

present experiment a temperature <strong>of</strong> 10uC was chosen since this<br />

represents what has been demonstrated to be <strong>the</strong> average<br />

temperature for a considerable part <strong>of</strong> <strong>the</strong> journey to <strong>the</strong> Sargasso<br />

Sea [21]. Additionally, differences in set up size can account for<br />

<strong>the</strong> differences in results, as it has been shown previously [45–47].<br />

<strong>Eel</strong>s migrating in <strong>the</strong> wild might swim at very different optimal<br />

and cruising speeds, than found in any <strong>of</strong> <strong>the</strong>se studies, due to <strong>the</strong><br />

experimental set up. However, as ours and <strong>the</strong> o<strong>the</strong>r studies<br />

mentioned are comparative studies, <strong>the</strong> findings are conclusive.<br />

The optimal swimming speed did not decrease significantly when<br />

<strong>the</strong> eels were tagged. Swimming at a reduced U opt would minimize<br />

<strong>the</strong> additional cost <strong>of</strong> swimming with a tag, but assuming that eel<br />

migrate at <strong>the</strong>ir optimal swimming speed, would also prolong <strong>the</strong><br />

journey for a tagged individual, who <strong>the</strong>n perhaps would not reach<br />

<strong>the</strong> spawning area in due time. Even though U opt did not change it<br />

has to be kept in mind that <strong>the</strong> energy consumption was 26%<br />

higher for <strong>the</strong> tagged animals swimming at U opt . Recently,<br />

Quintella and co workers [41] reported that male and female silver<br />

eels had <strong>the</strong> same absolute critical swimming speed, measured in<br />

meters per second, despite a 37% difference in length. The authors<br />

proposed that this, by way <strong>of</strong> natural selection, was to favour <strong>the</strong><br />

synchronized arrival at <strong>the</strong> spawning grounds. That study did not<br />

consider swimming energetics, but clearly a comparison <strong>of</strong> <strong>the</strong><br />

optimal swimming speed and bioenergetics <strong>of</strong> male and female<br />

silver eels would be <strong>of</strong> interest in future studies. The minimum cost<br />

<strong>of</strong> transport reported herein was very similar to recently reported<br />

values for silver eels (A. anguilla) <strong>of</strong> comparable sizes i.e. Palstra and<br />

co workers [40] reported a value <strong>of</strong> 40 mgO 2 kg 21 km 21 in eels<br />

not infected with <strong>the</strong> swim-bladder nematodes at 18uC. The<br />

approximately 26% increase in swimming cost (at <strong>the</strong> optimal<br />

swimming speed) that was associated with <strong>the</strong> tag found in <strong>the</strong><br />

present study translates into an increased use <strong>of</strong> body fats. The<br />

average fat content <strong>of</strong> an eel is about 20%, ranging from 10–28%<br />

[48], so an average 1 kg eel would <strong>the</strong>n have 200 grams <strong>of</strong> fat<br />

available. Swimming without and with a tag to <strong>the</strong> Sargasso Sea<br />

would cost 3.43 MJ and 4.29 MJ respectively if swimming at U opt .<br />

Assuming this is solely powered by body fat (37 kJ gram 21 ) an eel<br />

would use 93 or 115 grams <strong>of</strong> fat to complete <strong>the</strong> journey, which is<br />

57.5% <strong>of</strong> its fat reserves. Completing <strong>the</strong> journey with a tag would<br />

<strong>the</strong>n require a minimum body fat content <strong>of</strong> 12%, leaving no<br />

reserves left for gonad development and egg production. This<br />

leads to <strong>the</strong> assumption that <strong>the</strong> fat content <strong>of</strong> eels may be a critical<br />

aspect involved in <strong>the</strong>ir capacity to complete <strong>the</strong>ir migrating<br />

journey. The increased added cost <strong>of</strong> transport due to <strong>the</strong> tag can<br />

only be minimized by migrating at a lower speed (Fig. 2C). It is<br />

assumed that <strong>the</strong> duration <strong>of</strong> <strong>the</strong> <strong>European</strong> eels spawning<br />

migration is approximately 4–6 months, so if swimming speed is<br />

reduced to avoid <strong>the</strong> increased cost <strong>of</strong> a tag <strong>the</strong> result will be a<br />

much shorter distance travelled in <strong>the</strong> mean time. Aarestrup and<br />

co workers [21] reported that <strong>European</strong> eel with a PSAT attached,<br />

had an average horizontal migration speed <strong>of</strong> 13.8 km day 21 for<br />

(,0.16 Bl s 21 ), which is much lower than <strong>the</strong> speed required to<br />

complete <strong>the</strong> journey within <strong>the</strong> assumed time (about 0.5 Bl s 21 )<br />

[49]. In addition, <strong>the</strong> fur<strong>the</strong>st distance travelled was 1.300 km,<br />

compared to <strong>the</strong> necessary 6000 km to reach <strong>the</strong> hypo<strong>the</strong>tical<br />

spawning grounds. When taking <strong>the</strong> observed daily vertical<br />

movement <strong>of</strong> 800 meters additional to <strong>the</strong> horizontal migration<br />

into account [21], however, <strong>the</strong> actual swimming speed would be<br />

somewhat higher, but still far from <strong>the</strong> required cruising speed.<br />

Never<strong>the</strong>less, <strong>the</strong> drag <strong>of</strong> this type <strong>of</strong> PSAT is possibly too high<br />

even for large specimens, which was also partly concluded by<br />

Jellyman and Tsukamoto [20], who tagged <strong>the</strong> much larger New<br />

Zealand longfin eel (Anguilla dieffenbachii) with PSATs. The dummy<br />

used in <strong>the</strong> present study had a transmitter:fish mass ratio <strong>of</strong><br />

,1.7% in air, which is just under <strong>the</strong> 2% rule <strong>of</strong> thumb generally<br />

applied in tagging studies [38,50,51], and this suggests that drag is<br />

<strong>the</strong> more important factor to consider when using this type <strong>of</strong> tag<br />

on this size <strong>of</strong> fish. Although <strong>the</strong> drag <strong>of</strong> <strong>the</strong> dummy tag was only<br />

approximately 6 mN at <strong>the</strong> optimal swimming speed, it still<br />

significantly affected both <strong>the</strong> swimming performance, and <strong>the</strong><br />

energy expenditure. It shows that external tags even <strong>of</strong> relatively<br />

low drag and buoyancy can have adverse effects on <strong>the</strong> animals<br />

carrying <strong>the</strong>m. How <strong>the</strong> drag <strong>of</strong> <strong>the</strong> dummy tag relates to <strong>the</strong> drag<br />

<strong>of</strong> <strong>the</strong> eel is not known as it was not attempted to measure <strong>the</strong> drag<br />

<strong>of</strong> <strong>the</strong> eel. Unfortunately it is not straight forward to accurately<br />

measure <strong>the</strong> drag <strong>of</strong> a swimming eel due to <strong>the</strong> anguilliform<br />

swimming mode.<br />

Tail beat frequency and body wave speed were both positively<br />

correlated with swimming speed, and both increased when eels<br />

were swimming with <strong>the</strong> tag. This can explain <strong>the</strong> increased MO 2<br />

and COT that was associated with <strong>the</strong> tag, because <strong>the</strong> muscles<br />

had to contract at a higher frequency to keep <strong>the</strong> same swimming<br />

speed. Tail beat frequency as a function <strong>of</strong> U predicted tail beats <strong>of</strong><br />

0.1–0.3 Hz at zero swimming speed, and hence was not <strong>the</strong> best<br />

predictor <strong>of</strong> swimming speed in <strong>the</strong> present study. Body wave<br />

speed, on <strong>the</strong> o<strong>the</strong>r hand, was a better predictor <strong>of</strong> U since <strong>the</strong><br />

intercept was very close to zero, due to its close relationship to <strong>the</strong><br />

product <strong>of</strong> tail beat frequency and amplitude. Similar observations<br />

were reported by Tytell [52], in <strong>the</strong> closely related American eel<br />

(A. rostrata).<br />

The Strouhal number is used as a measure <strong>of</strong> <strong>the</strong> biomechanical<br />

swimming efficiency, with an optimal value <strong>of</strong> 0.3 signifying high<br />

efficiency <strong>of</strong> propulsion [31,32]. According to this, swimming<br />

without <strong>the</strong> tag was very efficient and similar to what was observed<br />

in A. rostrata [52]. Moreover, amplitude did not change with<br />

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PSATs Impair Swimming in <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

swimming speed, which was also <strong>the</strong> case with A. rostrata [52]. The<br />

Strouhal number was higher in tagged trials, indicating that <strong>the</strong><br />

tag made swimming less efficient, also reflected in <strong>the</strong> increased<br />

oxygen uptake and cost <strong>of</strong> transport. With both energetic and<br />

kinematic variables, <strong>the</strong> effect <strong>of</strong> <strong>the</strong> tag was minimal (not<br />

significant) at <strong>the</strong> lowest swimming speeds (0.3–0.4 Bl s 21 ),<br />

suggesting that <strong>the</strong> drag <strong>of</strong> <strong>the</strong> tag at those speeds (,2–3.5 mN)<br />

was negligible.<br />

It has been shown in <strong>the</strong> past that externally attached tags with<br />

even relatively low drag and buoyancy impair <strong>the</strong> swimming<br />

capacity <strong>of</strong> many diving species. Examples are Adélie penguins<br />

[53,54] and cownose ray [55]. Size and form <strong>of</strong> <strong>the</strong> PSATs<br />

currently available are very much dependent on a) <strong>the</strong> size <strong>of</strong> <strong>the</strong><br />

battery used and b) <strong>the</strong> size and form <strong>of</strong> <strong>the</strong> swimming device<br />

ensuring enough positive buoyancy to rise to surface and send <strong>the</strong><br />

data to <strong>the</strong> satellite. The actual electronic devises responsible for<br />

measurement and storage <strong>of</strong> <strong>the</strong> parameters detected only account<br />

for a fraction <strong>of</strong> <strong>the</strong> weight and <strong>the</strong> drag responsible for <strong>the</strong><br />

impairment <strong>of</strong> swimming. Additionally, an internal position <strong>of</strong> <strong>the</strong><br />

tag would only impair <strong>the</strong> free rising <strong>of</strong> <strong>the</strong> devise to <strong>the</strong> surface for<br />

transmitting <strong>the</strong> data to <strong>the</strong> satellite, as seawater blocks radio<br />

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Nature 278: 782–783.<br />

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<strong>the</strong>re a role <strong>of</strong> ocean environment in American and <strong>European</strong> eel decline?<br />

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Linden R, et al. (2004) Endurance swimming <strong>of</strong> <strong>European</strong> eel. Journal <strong>of</strong> Fish<br />

Biology 65: 312–318.<br />

6. van den Thillart GEEJ, Palstra A, van Ginneken VJT (2007) Simulated<br />

migration <strong>of</strong> <strong>European</strong> silver eel; Swim capacity and cost <strong>of</strong> transport. Journal <strong>of</strong><br />

Marine Science and Technology-Taiwan 15: 1–16.<br />

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enough to reach <strong>the</strong> Sargasso. Nature 403: 156–157.<br />

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Anguilla) in <strong>the</strong> Sargasso Sea and <strong>the</strong> Problem <strong>of</strong> <strong>the</strong> <strong>Eel</strong>s Spawning Site.<br />

Naturwissenschaften 82: 32–36.<br />

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<strong>European</strong> eels (Anguilla anguilla L.) tracked in <strong>the</strong> western North Sea. Ices<br />

Journal <strong>of</strong> Marine Science 56: 510–536.<br />

11. Tesch F (1989) Changes in swimming depth and direction <strong>of</strong> silver eels (Anguilla<br />

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2: 9–20.<br />

12. Tesch FW (1978) Telemetric Observations on <strong>the</strong> Spawning Migration <strong>of</strong> <strong>the</strong><br />

<strong>Eel</strong> Anguilla-Anguilla West <strong>of</strong> <strong>the</strong> <strong>European</strong> Continental Shelf. Environmental<br />

Biology <strong>of</strong> Fishes 3: 203–210.<br />

13. Tesch FW (1995) Vertical movements <strong>of</strong> migrating silver eels (Anguilla anguilla) in<br />

<strong>the</strong> sea. Bulletin <strong>of</strong> <strong>the</strong> Sea Fisheries<strong>Institut</strong>e 2: 23–30.<br />

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Electronic tagging and population structure <strong>of</strong> Atlantic bluefin tuna. Nature 434:<br />

1121–1127.<br />

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waves. A perfect devise for following migrating eels would have a<br />

minimal drag and size and be neutrally buoyant as not to impair<br />

<strong>the</strong> daily vertical movement <strong>of</strong> migrating eels, observed by<br />

Aarestrup et al. [21].<br />

In summary, <strong>the</strong> present study shows that <strong>the</strong> currently<br />

available PSATs are not suitable to be fitted to migrating eels,<br />

due to <strong>the</strong>ir increase in drag and <strong>the</strong> associated swimming costs by<br />

significantly affecting energy expenditure, swimming performance<br />

and efficiency in spite <strong>of</strong> <strong>the</strong> assumedly small transmitter:fish ratio<br />

and <strong>the</strong> low drag. We suggest that fur<strong>the</strong>r studies should be made<br />

with a range <strong>of</strong> different sized tags to determine <strong>the</strong> optimal tag:eel<br />

ratio that makes it possible to track eels even fur<strong>the</strong>r and longer.<br />

Author Contributions<br />

Conceived and designed <strong>the</strong> experiments: CM. Performed <strong>the</strong> experiments:<br />

CM. Analyzed <strong>the</strong> data: CM CT. Contributed reagents/materials/<br />

analysis tools: JFS. Wrote <strong>the</strong> paper: CM CT. Interpretation <strong>of</strong> data: CM<br />

CT PVS JFS. Critical revision <strong>of</strong> manuscript for important intellectual<br />

content: PVS JFS. Final approval <strong>of</strong> <strong>the</strong> version to be published: CM CT<br />

PVS JFS.<br />

21. Aarestrup K, Okland F, Hansen MM, Righton D, Gargan P, et al. (2009)<br />

Oceanic Spawning Migration <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla). Science<br />

325: 1660.<br />

22. Palstra A, Van Ginneken V, Van den Thillart G (2008) Cost <strong>of</strong> transport and<br />

optimal swimming speed in farmed and wild <strong>European</strong> silver eels (Anguilla<br />

anguilla). Comparative Biochemistry and <strong>Physiology</strong> A-Molecular & Integrative<br />

<strong>Physiology</strong> 151: 37–44.<br />

23. Durif C, Dufour S, Elie P (2005) The silvering process <strong>of</strong> Anguilla anguilla: a<br />

new classification from <strong>the</strong> yellow resident to <strong>the</strong> silver migrating stage. Journal<br />

<strong>of</strong> Fish Biology 66: 1025–1043.<br />

24. Bell WH, Terhune LDB (1970) Water tunnel design for fisheries research.<br />

Fisheries Research Board <strong>of</strong> Canada Technical Reports 195: 69.<br />

25. Schurmann H, Steffensen JF (1997) Effects <strong>of</strong> temperature, hypoxia and activity<br />

on <strong>the</strong> metabolism <strong>of</strong> juvenile Atlantic cod. Journal <strong>of</strong> Fish Biology 50:<br />

1166–1180.<br />

26. Steffensen JF, Johansen K, Bushnell PG (1984) An Automated Swimming<br />

Respirometer. Comparative Biochemistry and <strong>Physiology</strong> A-<strong>Physiology</strong> 79: 437–440.<br />

27. Steffensen JF (1989) Some Errors in Respirometry <strong>of</strong> Aquatic Brea<strong>the</strong>rs-How to<br />

Avoid and Correct for Them. Fish <strong>Physiology</strong> and Biochemistry 6: 49–59.<br />

28. Kiessling A, Johansson D, Zahl IH, Samuelsen OB (2009) Pharmacokinetics,<br />

plasma cortisol and effectiveness <strong>of</strong> benzocaine, MS-222 and isoeugenol<br />

measured in individual dorsal aorta-cannulated Atlantic salmon (Salmo salar)<br />

following bath administration. Aquaculture 286: 301–308.<br />

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Young Sockeye Salmon. Journal <strong>of</strong> <strong>the</strong> Fisheries Research Board <strong>of</strong> Canada 21:<br />

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31. Read DA, Hover FS, Triantafyllou MS (2003) Forces on oscillating foils for<br />

propulsion and maneuvering. Journal <strong>of</strong> Fluids and Structures 17: 163–183.<br />

32. Triantafyllou GS, Triantafyllou MS, Grosenbaugh MA (1993) Optimal Thrust<br />

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swimming. Annual Review <strong>of</strong> Fluid Mechanics 32: 33-+.<br />

34. Degani G, Gallagher ML, Meltzer A (1989) The Influence <strong>of</strong> Body Size and<br />

Temperature on Oxygen-Consumption <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong>, Anguilla-Anguilla.<br />

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35. Walsh PJ, Foster GD, Moon TW (1983) The Effects <strong>of</strong> Temperature on<br />

Metabolism <strong>of</strong> <strong>the</strong> American <strong>Eel</strong> Anguilla-Rostrata (Lesueur)-Compensation in<br />

<strong>the</strong> Summer and Torpor in <strong>the</strong> Winter. Physiological Zoology 56: 532–540.<br />

36. Lewis AE, Muntz WRA (1984) The Effects <strong>of</strong> External Ultrasonic Tagging on<br />

<strong>the</strong> Swimming Performance <strong>of</strong> Rainbow-Trout, Salmo-Gairdneri Richardson.<br />

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Rainbow-Trout (Salmo-Gairdneri) and White Perch (Morone-Americana)-<br />

Effects <strong>of</strong> Attaching Telemetry Transmitters. Canadian Journal <strong>of</strong> Fisheries<br />

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38. Winter JD (1983) Underwater telemetry. In: Nielsen LA, Johnson DL, eds.<br />

Fisheries Tecniques. Be<strong>the</strong>sdaMD: American Fisheries Society. pp 371–395.<br />

39. Steinhausen MF, Andersen NG, Steffensen JF (2006) The effect <strong>of</strong> external<br />

dummy transmitters on oxygen consumption and performance <strong>of</strong> swimming<br />

Atlantic cod. Journal <strong>of</strong> Fish Biology 69: 951–956.<br />

PLoS ONE | www.plosone.org 7 June 2011 | Volume 6 | Issue 6 | e20797


PSATs Impair Swimming in <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />

40. Palstra AP, Heppener DFM, van Ginneken VJT, Szekely C, van den<br />

Thillart GEEJ (2007) Swimming performance <strong>of</strong> silver eels is severely impaired<br />

by <strong>the</strong> swim-bladder parasite Anguillicola crassus. Journal <strong>of</strong> Experimental<br />

Marine Biology and Ecology 352: 244–256.<br />

41. Quintella BR, Mateus CS, Costa JL, Domingos I, Almeida PR (2010) Critical<br />

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42. Brett JR (1971) Energetic Responses <strong>of</strong> Salmon to Temperature-Study <strong>of</strong> Some<br />

Thermal Relations in <strong>Physiology</strong> and Freshwater Ecology <strong>of</strong> Sockeye Salmon<br />

(Oncorhynchus-Nerka). American Zoologist 11: 99-&.<br />

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45. Peake SJ, Farrell AP (2004) Locomotory behaviour and post-exercise physiology<br />

in relation to swimming speed, gait transition and metabolism in free-swimming<br />

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critical swimming speeds by increasing burst-glide swimming duration in carp<br />

Cyprinus carpio, L. Journal <strong>of</strong> Fish Biology 71: 1630–1638.<br />

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Indications <strong>of</strong> insufficient energetic stores for migration and gonadal development.<br />

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Linden R, et al. (2004) Endurance swimming <strong>of</strong> <strong>European</strong> eel. Journal <strong>of</strong> Fish<br />

Biology 65: 312–318.<br />

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juvenile coho salmon. American Fisheries Society Symposium 353-356.<br />

51. Thorstad EB, Okland F, Finstad B (2000) Effects <strong>of</strong> telemetry transmitters on<br />

swimming performance <strong>of</strong> adult Atlantic salmon. Journal <strong>of</strong> Fish Biology 57:<br />

531–535.<br />

52. Tytell ED (2004) The hydrodynamics <strong>of</strong> eel swimming II. Effect <strong>of</strong> swimming<br />

speed. Journal <strong>of</strong> Experimental Biology 207: 3265–3279.<br />

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Instrumented Adelie Penguins (Pygoscelis-Adeliae). Journal <strong>of</strong> Experimental<br />

Biology 158: 355–368.<br />

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PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e20797


T H E F A C U L T Y O F S C I E N C E ,<br />

RES E A R C H A N D I N N O V A T I O N<br />

U N I V E R S I T Y O F C O P E N H A G E N<br />

PhD School <strong>of</strong> Science<br />

Co-authorship statement<br />

All papers/manuscripts with multiple authors enclosed as annexes to a PhD <strong>the</strong>sis synopsis<br />

should contain a co-author statement, stating <strong>the</strong> PhD student’s contribution to <strong>the</strong> paper.<br />

1.General information<br />

PhD student<br />

Name<br />

Caroline Methling<br />

Civ.reg.no. (If not applicable, <strong>the</strong>n birth date)<br />

16.12.1977<br />

E-mail<br />

cmethling@bio.ku.dk<br />

Department<br />

Department <strong>of</strong> Biology<br />

Principal supervisor<br />

Name<br />

John Fleng Steffensen<br />

Position<br />

Pr<strong>of</strong>essor<br />

E-mail<br />

jfsteffensen@bio.ku.dk<br />

2.Title <strong>of</strong> PhD <strong>the</strong>sis<br />

<strong>Cardio</strong>-<strong>respiratory</strong> <strong>Physiology</strong> <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla) in Extreme Environments<br />

3.This co-authorship declaration applies to <strong>the</strong> following paper<br />

Methling, C., Tudorache, C., Skov, P.V., and Steffensen, J.F., 2011. Pop Up Satellite Tags Impair Swimming<br />

Performance and Energetics <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla). Plos One 6.<br />

4.The student's contribution to <strong>the</strong> paper:<br />

CM designed and performed <strong>the</strong> experiments, analysed <strong>the</strong> data and wrote <strong>the</strong> manuscript<br />

5. Material in <strong>the</strong> paper from ano<strong>the</strong>r degree / <strong>the</strong>sis :<br />

Articles/work published in connection with ano<strong>the</strong>r degree/<strong>the</strong>sis must not form part <strong>of</strong> <strong>the</strong> PhD <strong>the</strong>sis.<br />

1/2<br />

Revised on 10 September 2012


T H E F A C U L T Y O F S C I E N C E ,<br />

RES E A R C H A N D I N N O V A T I O N<br />

U N I V E R S I T Y O F C O P E N H A G E N<br />

PhD School <strong>of</strong> Science<br />

Co-authorship statement<br />

All papers/manuscripts with multiple authors enclosed as annexes to a PhD <strong>the</strong>sis synopsis<br />

should contain a co-author statement, stating <strong>the</strong> PhD student’s contribution to <strong>the</strong> paper.<br />

1.General information<br />

PhD student<br />

Name<br />

Caroline Methling<br />

Civ.reg.no. (If not applicable, <strong>the</strong>n birth date)<br />

16.12.1977<br />

E-mail<br />

cmethling@bio.ku.dk<br />

Department<br />

Department <strong>of</strong> Biology<br />

Principal supervisor<br />

Name<br />

John Fleng Steffensen<br />

Position<br />

Pr<strong>of</strong>essor<br />

E-mail<br />

jfsteffensen@bio.ku.dk<br />

2.Title <strong>of</strong> PhD <strong>the</strong>sis<br />

<strong>Cardio</strong>-<strong>respiratory</strong> <strong>Physiology</strong> <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla) in Extreme Environments<br />

3.This co-authorship declaration applies to <strong>the</strong> following paper<br />

Methling, C., Pedersen, P.B., Steffensen, J.F., and Skov, P.V., 2012. Tolerance towards hypercapnia does<br />

not preclude a negative impact on metabolism and postprandial proceses in <strong>the</strong> <strong>European</strong> eel (Anguilla<br />

anguilla). (Draft manuscript)<br />

4.The student's contribution to <strong>the</strong> paper:<br />

CM designed and performed <strong>the</strong> experiments, analysed <strong>the</strong> data and wrote <strong>the</strong> manuscript<br />

1/2<br />

Revised on 10 September 2012


T H E F A C U L T Y O F S C I E N C E ,<br />

RES E A R C H A N D I N N O V A T I O N<br />

U N I V E R S I T Y O F C O P E N H A G E N<br />

PhD School <strong>of</strong> Science<br />

Co-authorship statement<br />

All papers/manuscripts with multiple authors enclosed as annexes to a PhD <strong>the</strong>sis synopsis<br />

should contain a co-author statement, stating <strong>the</strong> PhD student’s contribution to <strong>the</strong> paper.<br />

1.General information<br />

PhD student<br />

Name<br />

Caroline Methling<br />

Civ.reg.no. (If not applicable, <strong>the</strong>n birth date)<br />

16.12.1977<br />

E-mail<br />

cmethling@bio.ku.dk<br />

Department<br />

Department <strong>of</strong> Biology<br />

Principal supervisor<br />

Name<br />

John Fleng Steffensen<br />

Position<br />

Pr<strong>of</strong>essor<br />

E-mail<br />

jfsteffensen@bio.ku.dk<br />

2.Title <strong>of</strong> PhD <strong>the</strong>sis<br />

<strong>Cardio</strong>-<strong>respiratory</strong> <strong>Physiology</strong> <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla) in Extreme Environments<br />

3.This co-authorship declaration applies to <strong>the</strong> following paper<br />

Methling, C., Skov, P.V. and Steffensen, J.F., 2012. The influence <strong>of</strong> temperature and hypoxia on oxygen<br />

consumption in <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla) over a wide range <strong>of</strong> temperatures. (Draft<br />

manuscript)<br />

4.The student's contribution to <strong>the</strong> paper:<br />

CM designed and performed <strong>the</strong> experiments, analysed <strong>the</strong> data and wrote <strong>the</strong> manuscript<br />

1/2<br />

Revised on 10 September 2012


T H E F A C U L T Y O F S C I E N C E ,<br />

RES E A R C H A N D I N N O V A T I O N<br />

U N I V E R S I T Y O F C O P E N H A G E N<br />

PhD School <strong>of</strong> Science<br />

Co-authorship statement<br />

All papers/manuscripts with multiple authors enclosed as annexes to a PhD <strong>the</strong>sis synopsis<br />

should contain a co-author statement, stating <strong>the</strong> PhD student’s contribution to <strong>the</strong> paper.<br />

1.General information<br />

PhD student<br />

Name<br />

Caroline Methling<br />

Civ.reg.no. (If not applicable, <strong>the</strong>n birth date)<br />

16.12.1977<br />

E-mail<br />

cmethling@bio.ku.dk<br />

Department<br />

Department <strong>of</strong> Biology<br />

Principal supervisor<br />

Name<br />

John Fleng Steffensen<br />

Position<br />

Pr<strong>of</strong>essor<br />

E-mail<br />

jfsteffensen@bio.ku.dk<br />

2.Title <strong>of</strong> PhD <strong>the</strong>sis<br />

<strong>Cardio</strong>-<strong>respiratory</strong> <strong>Physiology</strong> <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla) in Extreme Environments<br />

3.This co-authorship declaration applies to <strong>the</strong> following paper<br />

Methling, C., Steffensen, J.F., and Skov, P.V., 2012. The temperature challenges on cardiac performance in<br />

winter-quiescent and migration-stage eels Anguilla anguilla. Comparative Biochemistry and <strong>Physiology</strong> A-<br />

Molecular & Integrative <strong>Physiology</strong> 163, 66-73.<br />

4.The student's contribution to <strong>the</strong> paper:<br />

CM designed and performed <strong>the</strong> experiments, analysed <strong>the</strong> data and wrote <strong>the</strong> manuscript<br />

1/2<br />

Revised on 10 September 2012

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