Cardio-respiratory Physiology of the European Eel ... - Biologisk Institut
Cardio-respiratory Physiology of the European Eel ... - Biologisk Institut
Cardio-respiratory Physiology of the European Eel ... - Biologisk Institut
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
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
REFERENCES<br />
Aarestrup,K., Okland,F., Hansen,M.M., Righton,D., Gargan,P., Castonguay,M., Bernatchez,L.,<br />
Howey,P., Sparholt,H., Pedersen,M.I., McKinley,R.S., 2009. Oceanic Spawning Migration <strong>of</strong><br />
<strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla anguilla). Science 325(5948), 1660.<br />
Agnisola,C., McKenzie,D.J., Pellegrino,D., Bronzi,P., Tota,B., Taylor,E.W., 1999. <strong>Cardio</strong>vascular<br />
responses to hypoxia in <strong>the</strong> Adriatic sturgeon (Acipenser naccarii). Journal <strong>of</strong> Applied<br />
Ichthyology-Zeitschrift fur Angewandte Ichthyologie 15(4-5), 67-72.<br />
Aho,E., Vornanen,M., 1999. Contractile properties <strong>of</strong> atrial and ventricular myocardium <strong>of</strong> <strong>the</strong> heart <strong>of</strong><br />
rainbow trout Oncorhynchus mykiss: Effects <strong>of</strong> <strong>the</strong>rmal acclimation. Journal <strong>of</strong> Experimental<br />
Biology 202(19), 2663-2677.<br />
Aho,E., Vornanen,M., 2001. Cold acclimation increases basal heart rate but decreases its <strong>the</strong>rmal<br />
tolerance in rainbow trout (Oncorhynchus mykiss). Journal <strong>of</strong> Comparative <strong>Physiology</strong><br />
B-Biochemical Systemic and Environmental <strong>Physiology</strong> 171(2), 173-179.<br />
Albert,V., Jonsson,B., Bernatchez,L., 2006. Natural hybrids in Atlantic eels (Anguilla anguilla,<br />
A-rostrata): evidence for successful reproduction and fluctuating abundance in space and time<br />
1. Molecular Ecology 15(7), 1903-1916.<br />
Alsop,D.H., Kieffer,J.D., Wood,C.M., 1999. The effects <strong>of</strong> temperature and swimming speed on<br />
instantaneous fuel use and nitrogenous waste excretion <strong>of</strong> <strong>the</strong> Nile tilapia. Physiological and<br />
Biochemical Zoology 72(4), 474-483.<br />
Avise,J.C., Nelson,W.S., Arnold,J., Koehn,R.K., Williams,G.C., Thorsteinsson,V., 1990. The<br />
Evolutionary Genetic Status <strong>of</strong> Icelandic <strong>Eel</strong>s. Evolution 44(5), 1254-1262.<br />
Axelsson,M., Fritsche,R., 1991. Effects <strong>of</strong> Exercise, Hypoxia and Feeding on <strong>the</strong> Gastrointestinal<br />
Blood-Flow in <strong>the</strong> Atlantic Cod Gadus-Morhua. Journal <strong>of</strong> Experimental Biology 158, 181-198.<br />
Axelsson,M., Davison,W., Forster,M.E., Farrell,A.P., 1992. <strong>Cardio</strong>vascular-Responses <strong>of</strong> <strong>the</strong><br />
Red-Blooded Antarctic Fishes Pago<strong>the</strong>nia-Bernacchii and P-Borchgrevinki. Journal <strong>of</strong><br />
Experimental Biology 167, 179-201.<br />
Axelsson,M., Altimiras,J., Claireaux,G., 2002. Post-prandial blood flow to <strong>the</strong> gastrointestinal tract is<br />
not compromised during hypoxia in <strong>the</strong> sea bass Dicentrarchus labrax. Journal <strong>of</strong> Experimental<br />
Biology 205(18), 2891-2896.<br />
Axelsson,M., 2005. The circulatory system and its control. In: Farrell,A.P., Steffensen,J.F. (Eds.), The<br />
<strong>Physiology</strong> <strong>of</strong> Polar Fishes. Elsevier, San Diego, pp. 239-280.<br />
Baden,S.P., Loo,L.O., Pihl,L., Rosenberg,R., 1990. Effects <strong>of</strong> Eutrophication on Benthic Communities<br />
Including Fish - Swedish West-Coast. Ambio 19(3), 113-122.<br />
Baker,D.W., Brauner,C.J., 2012. Metabolic changes associated with acid-base regulation during<br />
hypercarbia in <strong>the</strong> CO2-tolerant chondrostean, white sturgeon (Acipenser transmontanus)<br />
1. Comparative Biochemistry and <strong>Physiology</strong> A-Molecular & Integrative <strong>Physiology</strong> 161(1),<br />
61-68.<br />
Barnes,R., King,H., Carter,C.G., 2011. Hypoxia tolerance and oxygen regulation in Atlantic salmon,<br />
Salmo salar from a Tasmanian population. Aquaculture 318(3-4), 397-401.<br />
Beamish,F.W.H., 1978. Swimming Capacity. In: Hoar,W.S., Randall,D.J. (Eds.), Fish <strong>Physiology</strong> Vol.<br />
VII. Academic Press, New York, pp. 101-187.<br />
Beamish,F.W.H., Trippel,E.A., 1990. Heat Increment - A Static Or Dynamic Dimension in<br />
Bioenergetic Models. Transactions <strong>of</strong> <strong>the</strong> American Fisheries Society 119(4), 649-661.<br />
44
Behrens,J.W., Steffensen,J.F., 2007. The effect <strong>of</strong> hypoxia on behavioural and physiological aspects <strong>of</strong><br />
lesser sandeel, Ammodytes tobianus (Linnaeus, 1785). Marine Biology 150(6), 1365-1377.<br />
Berg,T., Steen,J.B., 1965. Physiological Mechanisms for Aerial Respiration in <strong>Eel</strong>. Comparative<br />
Biochemistry and <strong>Physiology</strong> 15(4), 469-&.<br />
Bers,D.M., 2002. Cardiac excitation-contraction coupling. Nature 415(6868), 198-205.<br />
Boëtius,J., 1980. Atlantic Anguilla. A presentation <strong>of</strong> new and old data <strong>of</strong> total numbers <strong>of</strong> vertebrae<br />
with special reference to <strong>the</strong> occurence <strong>of</strong> Anguilla rostrata in Europe. DANA 1, 93-112.<br />
Boijink,C.D., Florindo,L.H., Leite,C.A.C., Kalinin,A.L., Milsom,W.K., Rantin,F.T., 2010. Hypercarbic<br />
cardio<strong>respiratory</strong> reflexes in <strong>the</strong> facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus):<br />
<strong>the</strong> role <strong>of</strong> branchial CO2 chemoreceptors. Journal <strong>of</strong> Experimental Biology 213(16),<br />
2797-2807.<br />
Bonhommeau,S., Castonguay,M., Rivot,E., Sabatie,R., Le Pape,O., 2010. The duration <strong>of</strong> migration <strong>of</strong><br />
Atlantic Anguilla larvae. Fish and Fisheries 11(3), 289-306.<br />
Booth,J.H., 1979. Effects <strong>of</strong> Oxygen-Supply, Epinephrine, and Acetylcholine on <strong>the</strong> Distribution <strong>of</strong><br />
Blood-Flow in Trout Gills. Journal <strong>of</strong> Experimental Biology 83(DEC), 31-39.<br />
Boyce,S.J., Clarke,A., 1997. Effect <strong>of</strong> body size and ration on specific dynamic action in <strong>the</strong> Antarctic<br />
plunderfish, Harpagifer antarcticus Nybelin 1947. Physiological Zoology 70(6), 679-690.<br />
Brett,J.R., 1964. The Respiratory Metabolism and Swimming Performance <strong>of</strong> Young Sockeye Salmon.<br />
Journal <strong>of</strong> <strong>the</strong> Fisheries Research Board <strong>of</strong> Canada 21(5), 1183-1226.<br />
Brett,J.R., 1971. Energetic Responses <strong>of</strong> Salmon to Temperature - Study <strong>of</strong> Some Thermal Relations in<br />
<strong>Physiology</strong> and Freshwater Ecology <strong>of</strong> Sockeye Salmon (Oncorhynchus-Nerka). American<br />
Zoologist 11(1), 99-&.<br />
Brett,J.R., 1979. Environmental factors and growth. In: Hoar,W.S., Randall,D.J., Brett,J.R. (Eds.),<br />
Bioenergetics and Growth. Academic Press, New York, pp. 599-675.<br />
Brett,J.R., Groves,T.D.D., 1979. Physiological energetics. In: Hoar,W.S., Randall,D.J., Brett,J.R.<br />
(Eds.), Bioenergetics and Growth. Academic Press, New York, pp. 279-352.<br />
Brown,C.R., Cameron,J.N., 1991. The Relationship Between Specific Dynamic Action (Sda) and<br />
Protein-Syn<strong>the</strong>sis Rates in <strong>the</strong> Channel Catfish. Physiological Zoology 64(1), 298-309.<br />
Bruun,A.F., 1963. The breeding <strong>of</strong> <strong>the</strong> North Atlantic freshwater eels. Advances in Marine Biology 1,<br />
137-169.<br />
Burleson,M.L., Smatresk,N.J., Milsom,W.K., 1992. Afferent inputs associated with cardioventilatory<br />
control in fish. In: Hoar,W.S., Randall,D.J., Farrell,A.P. (Eds.), The <strong>Cardio</strong>vascular System.<br />
Academic Press, San Diego, pp. 389-426.<br />
Cecchini,S., Saroglia,M., Caricato,G., Terova,G., Sileo,L., 2001. Effects <strong>of</strong> graded environmental<br />
hypercapnia on sea bass (Dicentrarchus labrax L.) feed intake and acid-base balance.<br />
Aquaculture Research 32(6), 499-502.<br />
Cerezo,J., Garcia,B.G., 2004. The effects <strong>of</strong> oxygen levels on oxygen consumption, survival and<br />
ventilatory frequency <strong>of</strong> sharpsnout sea bream (Diplodus puntazzo Gmelin, 1789) at different<br />
conditions <strong>of</strong> temperature and fish weight. Journal <strong>of</strong> Applied Ichthyology 20(6), 488-492.<br />
Chan,D.K.O., 1986. <strong>Cardio</strong>vascular, Respiratory, and Blood Adjustments to Hypoxia in <strong>the</strong> Japanese<br />
<strong>Eel</strong>, Anguilla-Japonica. Fish <strong>Physiology</strong> and Biochemistry 2(1-4), 179-193.<br />
Claiborne,J.B., Heisler,N., 1986. Acid-Base Regulation and Ion Transfers in <strong>the</strong> Carp<br />
(Cyprinus-Carpio) - Ph Compensation During Graded Long-Term and Short-Term<br />
Environmental Hypercapnia, and <strong>the</strong> Effect <strong>of</strong> Bicarbonate Infusion. Journal <strong>of</strong> Experimental<br />
Biology 126, 41-61.<br />
45
Claireaux,G., Lagardere,J.P., 1999. Influence <strong>of</strong> temperature, oxygen and salinity on <strong>the</strong> metabolism <strong>of</strong><br />
<strong>the</strong> <strong>European</strong> sea bass. Journal <strong>of</strong> Sea Research 42(2), 157-168.<br />
Claireaux,G., Webber,D.M., Lagardere,J.P., Kerr,S.R., 2000. Influence <strong>of</strong> water temperature and<br />
oxygenation on <strong>the</strong> aerobic metabolic scope <strong>of</strong> Atlantic cod (Gadus morhua). Journal <strong>of</strong> Sea<br />
Research 44(3-4), 257-265.<br />
Claireaux,G., Lefrancois,C., 2007. Linking environmental variability and fish performance: integration<br />
through <strong>the</strong> concept <strong>of</strong> scope for activity. Philosophical Transactions <strong>of</strong> <strong>the</strong> Royal Society<br />
B-Biological Sciences 362(1487), 2031-2041.<br />
Clarke,A., Johnston,N.M., 1999. Scaling <strong>of</strong> metabolic rate with body mass and temperature in teleost<br />
fish. Journal <strong>of</strong> Animal Ecology 68(5), 893-905.<br />
Corkum,C.P., Gamperl,A.K., 2009. Does <strong>the</strong> Ability to Metabolically Downregulate Alter <strong>the</strong> Hypoxia<br />
Tolerance <strong>of</strong> Fishes?: A Comparative Study Using Cunner (T. adspersus) and Greenland Cod<br />
(G. ogac). Journal <strong>of</strong> Experimental Zoology Part A-Ecological Genetics and <strong>Physiology</strong><br />
311A(4), 231-239.<br />
Crocker,C.E., Cech,J.J., 1996. The effects <strong>of</strong> hypercapnia on <strong>the</strong> growth <strong>of</strong> juvenile white sturgeon,<br />
Acipenser transmontanus. Aquaculture 147(3-4), 293-299.<br />
Crocker,C.E., Farrell,A.P., Gamperi,A.K., Cech,J.J., 2000. <strong>Cardio</strong><strong>respiratory</strong> responses <strong>of</strong> white<br />
sturgeon to environmental hypercapnia. American Journal <strong>of</strong> <strong>Physiology</strong>-Regulatory Integrative<br />
and Comparative <strong>Physiology</strong> 279(2), R617-R628.<br />
Crossin,G.T., Hinch,S.G., Farrell,A.P., Higgs,D.A., Healey,M.C., 2004. Somatic energy <strong>of</strong> sockeye<br />
salmon Oncorhynchus nerka at <strong>the</strong> onset <strong>of</strong> upriver migration: a comparison among ocean<br />
climate regimes. Fisheries Oceanography 13(5), 345-349.<br />
CruzNeto,A.P., Steffensen,J.F., 1997. The effects <strong>of</strong> acute hypoxia and hypercapnia on oxygen<br />
consumption <strong>of</strong> <strong>the</strong> freshwater <strong>European</strong> eel. Journal <strong>of</strong> Fish Biology 50(4), 759-769.<br />
Dalla Via,J., van den Thillart,G., Cattani,O., Cortesi,P., 1998. Behavioural responses and biochemical<br />
correlates in Solea solea to gradual hypoxic exposure. Canadian Journal <strong>of</strong> Zoology-Revue<br />
Canadienne de Zoologie 76(11), 2108-2113.<br />
Deigweiher,K., Koschnick,N., Portner,H.O., Lucassen,M., 2008. Acclimation <strong>of</strong> ion regulatory<br />
capacities in gills <strong>of</strong> marine fish under environmental hypercapnia. American Journal <strong>of</strong><br />
<strong>Physiology</strong>-Regulatory Integrative and Comparative <strong>Physiology</strong> 295(5), R1660-R1670.<br />
Dekker,W., 2000. The fractal geometry <strong>of</strong> <strong>the</strong> <strong>European</strong> eel stock. Ices Journal <strong>of</strong> Marine Science<br />
57(1), 109-121.<br />
Diaz,R.J., Breitburg,D., 2009. The Hypoxic Environment. In: Richards,G.R., Farrell,A.P., Brauner,C.J.<br />
(Eds.), Hypoxia. Academic Press, Amsterdam, pp. 2-23.<br />
Domenici,P., Steffensen,J.F., Batty,R.S., 2000. The effect <strong>of</strong> progressive hypoxia on swimming activity<br />
and schooling in Atlantic herring. Journal <strong>of</strong> Fish Biology 57(6), 1526-1538.<br />
Driedzic,W.R., Gesser,H., 1994. Energy-Metabolism and Contractility in Ecto<strong>the</strong>rmic Vertebrate<br />
Hearts - Hypoxia, Acidosis, and Low-Temperature. Physiological Reviews 74(1), 221-258.<br />
Driedzic,W.R., Bailey,J.R., Sephton,D.H., 1996. Cardiac adaptations to low temperature non-polar<br />
teleost fish. Journal <strong>of</strong> Experimental Zoology 275(2-3), 186-195.<br />
Durif,C.M.F., Dufour,S., Elie,P., 2006. Impact <strong>of</strong> silvering stage, age, body size and condition on<br />
reproductive potential <strong>of</strong> <strong>the</strong> <strong>European</strong> eel. Marine Ecology-Progress Series 327, 171-181.<br />
Durif,C.M.F., van Ginneken,V.J.T., Dufour,S., Müller,T., Elie,P., 2009. Seasonal evolution and<br />
individual differences in silveering eels from different locations. In: van den Thillart,G.E.E.J.,<br />
Dufour,S., Rankin,J.C. (Eds.), Spawning Migration <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong>. Springer, pp. 13-38.<br />
46
Egginton,S., Johnston,I.A., 1984. Effects <strong>of</strong> Acclimation Temperature on Routine Metabolism Muscle<br />
Mitochondrial Volume Density and Capillary Supply in <strong>the</strong> Elver (Anguilla-Anguilla L).<br />
Journal <strong>of</strong> Thermal Biology 9(3), 165-170.<br />
Elliott,J.M., 1976. Energetics <strong>of</strong> Feeding, Metabolism and Growth <strong>of</strong> Brown Trout(Salmo-Trutta-L) in<br />
Relation to Body-Weight, Water Temperature and Ration Size. Journal <strong>of</strong> Animal Ecology<br />
45(3), 923-948.<br />
Farrell,A.P., Steffensen,J.F., 1987. An Analysis <strong>of</strong> <strong>the</strong> Energetic Cost <strong>of</strong> <strong>the</strong> Branchial and Cardiac<br />
Pumps During Sustained Swimming in Trout. Fish <strong>Physiology</strong> and Biochemistry 4(2), 73-79.<br />
Farrell,A.P., 1991. From Hagfish to Tuna - A Perspective on Cardiac-Function in Fish. Physiological<br />
Zoology 64(5), 1137-1164.<br />
Farrell,A.P., Jones,D.R., 1992. The Heart. In: Hoar,W.S., Randall,D.J., Farrell,A.P. (Eds.), The<br />
<strong>Cardio</strong>vascular System. Academic Press, San Diego, pp. 1-88.<br />
Farrell,A.P., 2007. Tribute to P. L. Lutz: a message from <strong>the</strong> heart - why hypoxic bradycardia in fishes?<br />
Journal <strong>of</strong> Experimental Biology 210(10), 1715-1725.<br />
Farrell,A.P., Richards,J.G., 2009. Defining Hypoxia: An Integrative Syn<strong>the</strong>sis <strong>of</strong> <strong>the</strong> Responses <strong>of</strong> Fish<br />
to Hypoxia. In: Richards,J.G., Farrell,A.P., Brauner,C.J. (Eds.), Hypoxia. Academic Press,<br />
Amsterdam, pp. 487-503.<br />
Fernandes,M.N., Rantin,F.T., 1989. Respiratory Responses <strong>of</strong> Oreochromis-Niloticus (Pisces,<br />
Cichlidae) to Environmental Hypoxia Under Different Thermal Conditions. Journal <strong>of</strong> Fish<br />
Biology 35(4), 509-519.<br />
Fine,J.M., Drilhon,A., Ridgway,G.J., Amouch,P., B<strong>of</strong>fa,G., 1967. Les Groupes de Transferrines Dans<br />
Le Genre Anguilla . Differences Dans les Frequences Phenotypiques de Transferrines Chez<br />
Anguilla Anguilla et Anguilla Rostrata. Comptes Rendus Hebdomadaires des Seances de l<br />
Academie des Sciences Serie D 265(1), 58-&.<br />
Fivelstad,S., Waagbo,R., Stefansson,S., Olsen,A.B., 2007. Impacts <strong>of</strong> elevated water carbon dioxide<br />
partial pressure at two temperatures on Atlantic salmon (Salmo salar L.) parr growth and<br />
haematology172. Aquaculture 269(1-4), 241-249.<br />
Fonselius,S.H., 1969. Hydrography <strong>of</strong> <strong>the</strong> Baltic deep basins III. Fishery Board <strong>of</strong> Sweden,<br />
Go<strong>the</strong>nberg, pp. 1-97.<br />
Foss,A., Rosnes,B.A., Oiestad,V., 2003. Graded environmental hypercapnia in juvenile spotted<br />
wolffish (Anarhichas minor Olafsen): effects on growth, food conversion efficiency and<br />
nephrocalcinosis. Aquaculture 220(1-4), 607-617.<br />
Fricke,H., Kaese,R., 1995. Tracking <strong>of</strong> Artificially Matured <strong>Eel</strong>s (Anguilla-Anguilla) in <strong>the</strong> Sargasso<br />
Sea and <strong>the</strong> Problem <strong>of</strong> <strong>the</strong> <strong>Eel</strong>s Spawning Site. Naturwissenschaften 82(1), 32-36.<br />
Fritsche,R., Nilsson,S., 1993. <strong>Cardio</strong>vascular and ventilatory control during hypoxia. In: Rankin,J.C.,<br />
Jensen,F.B. (Eds.), Fish Ecophysiology. Chapman and Hall, London, pp. 180-206.<br />
Fry,F.E.J., 1971. The effect <strong>of</strong> environmental factors on <strong>the</strong> physiology <strong>of</strong> fish. In: Hoar,W.S.,<br />
Randall,D.J. (Eds.), Environmental Relations and Behavior. Academic Press, New York, pp.<br />
1-98.<br />
Fry,F.E.J., 1947. Effect <strong>of</strong> <strong>the</strong> environment on animal actitvity. pp. 1-62.<br />
Fu,S.J., Xie,X.J., Cao,Z.D., 2005. Effect <strong>of</strong> meal size on postprandial metabolic response in sou<strong>the</strong>rn<br />
catfish (Silurus meridionalis). Comparative Biochemistry and <strong>Physiology</strong> A-Molecular &<br />
Integrative <strong>Physiology</strong> 140(4), 445-451.<br />
Fu,S.J., Xie,X.J., Cao,Z.D., 2005. Effect <strong>of</strong> dietary composition on specific dynamic action in sou<strong>the</strong>rn<br />
catfish Silurus meridionalis Chen. Aquaculture Research 36(14), 1384-1390.<br />
47
Gallaugher,P., Farrell,A.P., 1998. Hematocrit and Blood Oxygen-Carrying Capacity. In: Perry,S.F.,<br />
Tufts,B.L. (Eds.), Fish Respiration. Academic Press, San Diego, pp. 185-217.<br />
Gamperl,A.K., Driedzic,W.R., 2009. <strong>Cardio</strong>vascular Function and Cardiac Metabolism<br />
390. In: Richards,J.G., Farrell,A.P., Brauner,C.J. (Eds.), Hypoxia. Academic Press, Amsterdam,<br />
pp. 301-360.<br />
Gesser,H., 1996. Cardiac force-interval relationship, adrenaline and sarcoplasmic reticulum in rainbow<br />
trout. Journal <strong>of</strong> Comparative <strong>Physiology</strong> B-Biochemical Systemic and Environmental<br />
<strong>Physiology</strong> 166(4), 278-285.<br />
Gillis,G.B., 1998. Neuromuscular control <strong>of</strong> anguilliform locomotion: Patterns <strong>of</strong> red and white muscle<br />
activity during swimming in <strong>the</strong> American eel Anguilla rostrata. Journal <strong>of</strong> Experimental<br />
Biology 201(23), 3245-3256.<br />
Gilmour,K.A., Milsom,W.K., Rantin,F.T., Reid,S.G., Perry,S.F., 2005. <strong>Cardio</strong><strong>respiratory</strong> responses to<br />
hypercarbia in tambaqui Colossoma macropomum: chemoreceptor orientation and specificity<br />
2. Journal <strong>of</strong> Experimental Biology 208(6), 1095-1107.<br />
Gnaiger,E., Bitterlich,G., 1984. Proximate Biochemical-Composition and Caloric Content Calculated<br />
from Elemental Chn Analysis - A Stoichiometric Concept. Oecologia 62(3), 289-298.<br />
Gollock,M.J., Currie,S., Petersen,L.H., Gamperl,A.K., 2006. <strong>Cardio</strong>vascular and haematological<br />
responses <strong>of</strong> Atlantic cod (Gadus morhua) to acute temperature increase. Journal <strong>of</strong><br />
Experimental Biology 209(15), 2961-2970.<br />
Goolish,E.M., 1987. Cold-Acclimation Increases <strong>the</strong> Ventricle Size <strong>of</strong> Carp, Cyprinus-Carpio<br />
164. Journal <strong>of</strong> Thermal Biology 12(3), 203-205.<br />
Graham,J.B., 1997. Air Breathing Fishes: Evolution, Diversity, and Adaptation. Academic Press, San<br />
Diego.<br />
Graham,J.B., 2006. Aquatic and aerial respiration. In: Evans,D.H., Claiborne,J.B. (Eds.), The<br />
<strong>Physiology</strong> <strong>of</strong> Fishes. CRC Press, Boca Raton, FL, pp. 85-118.<br />
Graham,M., Farrell,A., 1985. The Seasonal Intrinsic Cardiac-Performance <strong>of</strong> A Marine Teleost.<br />
Journal <strong>of</strong> Experimental Biology 118, 173-183.<br />
Graham,M.S., Farrell,A.P., 1989. The Effect <strong>of</strong> Temperature-Acclimation and Adrenaline on <strong>the</strong><br />
Performance <strong>of</strong> A Perfused Trout Heart. Physiological Zoology 62(1), 38-61.<br />
Guderley,H., Demers,A., Couture,P., 1994. Acclimatization <strong>of</strong> Blue-Mussel, (Mytilus-Edulis Linnaeus,<br />
1758) to Intertidal Conditions - Effects on Mortality and Gaping During Air Exposure. Journal<br />
<strong>of</strong> Shellfish Research 13(2), 379-385.<br />
Hardewig,I., Peck,L.S., Pörtner,H.O., 1999. Thermal sensitivity <strong>of</strong> mitochondrial function in <strong>the</strong><br />
Antarctic Noto<strong>the</strong>nioid, Lepidonoto<strong>the</strong>n nudifrons. Journal <strong>of</strong> Comparative <strong>Physiology</strong> B 169,<br />
597-604.<br />
Haverinen,J., Vornanen,M., 2007. Temperature acclimation modifies sinoatrial pacemaker mechanism<br />
<strong>of</strong> <strong>the</strong> rainbow trout heart. American Journal <strong>of</strong> <strong>Physiology</strong>-Regulatory Integrative and<br />
Comparative <strong>Physiology</strong> 292(2), R1023-R1032.<br />
Heisler,N., 1984. Acid-base regulation in fishes. In: Hoar,W.S., Randall,D.J. (Eds.), Gills: Anatomy,<br />
Gas Transfer, and Acid-Base Regulation. Academic Press, New York, pp. 315-401.<br />
Heisler,N., 1993. Acid-base regulation. In: Evans,D.H. (Ed.), The physiology <strong>of</strong> fishes. pp. 343-378.<br />
Helly,J.J., Levin,L.A., 2004. Global distribution <strong>of</strong> naturally occurring marine hypoxia on continental<br />
margins. Deep-Sea Research Part I-Oceanographic Research Papers 51(9), 1159-1168.<br />
Hochochka,P.W., Somero,G.N., 2002. Biochemical Adaptation: Mechanism and Process in<br />
Physiological Evolution. Oxford University Press, New York.<br />
48
Holeton,G.F., 1974. Metabolic Cold Adaptation <strong>of</strong> Polar Fish - Fact Or Artifact. Physiological Zoology<br />
47(3), 137-152.<br />
Huang,J.B., van Breemen,C., Kuo,K.H., Hove-Madsen,L., Tibbits,G.F., 2006. Store-operated Ca2+<br />
entry modulates sarcoplasmic reticulum Ca2+ loading in neonatal rabbit cardiac ventricular<br />
myocytes. American Journal <strong>of</strong> <strong>Physiology</strong>-Cell <strong>Physiology</strong> 290(6), C1572-C1582.<br />
Hyde,D.A., Perry,S.F., 1987. Acid-Base and Ionic Regulation in <strong>the</strong> American <strong>Eel</strong> (Anguilla-Rostrata)<br />
During and After Prolonged Aerial Exposure - Branchial and Renal Adjustments. Journal <strong>of</strong><br />
Experimental Biology 133, 429-447.<br />
Hyde,D.A., Moon,T.W., Perry,S.F., 1987. Physiological Consequences <strong>of</strong> Prolonged Aerial Exposure<br />
in <strong>the</strong> American <strong>Eel</strong>, Anguilla-Rostrata - Blood Respiratory and Acid-Base Status. Journal <strong>of</strong><br />
Comparative <strong>Physiology</strong> B-Biochemical Systemic and Environmental <strong>Physiology</strong> 157(5),<br />
635-642.<br />
Imbrogno,S., Angelone,T., Adamo,C., Pulera,E., Tota,B., Cerra,M.C., 2006. Beta3-adrenoceptor in <strong>the</strong><br />
eel (Anguilla anguilla) heart: negative inotropy and NO-cGMP-dependent mechanism. Journal<br />
<strong>of</strong> Experimental Biology 209(24), 4966-4973.<br />
Ishimatsu,A., Hayashi,M., Lee,K.S., Kikkawa,T., Kita,J., 2005. Physiological effects on fishes in a<br />
high-CO2 world. Journal <strong>of</strong> Geophysical Research-Oceans 110(C9).<br />
Ishimatsu,A., Hayashi,M., Kikkawa,T., 2008. Fishes in high-CO2, acidified oceans. Marine<br />
Ecology-Progress Series 373, 295-302.<br />
Iversen,N.K., McKenzie,D.J., Malte,H., Wang,T., 2010. Reflex bradycardia does not influence oxygen<br />
consumption during hypoxia in <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla). Journal <strong>of</strong> Comparative<br />
<strong>Physiology</strong> B-Biochemical Systemic and Environmental <strong>Physiology</strong> 180(4), 495-502.<br />
Jobling,M., Davies,P.S., 1980. Effects <strong>of</strong> Feeding on Metabolic-Rate, and <strong>the</strong> Specific Dynamic Action<br />
in Plaice, Pleuronectes-Platessa l. Journal <strong>of</strong> Fish Biology 16(6), 629-638.<br />
Jobling,M., 1981. The Influences <strong>of</strong> Feeding on <strong>the</strong> Metabolic-Rate <strong>of</strong> Fishes - A Short Review.<br />
Journal <strong>of</strong> Fish Biology 18(4), 385-400.<br />
Jordan,A.D., Steffensen,J.F., 2007. Effects <strong>of</strong> ration size and hypoxia on specific dynamic action in <strong>the</strong><br />
cod. Physiological and Biochemical Zoology 80(2), 178-185.<br />
Jourdan-Pineau,H., Dupont-Prinet,A., Claireaux,G., McKenzie,D.J., 2010. An Investigation <strong>of</strong><br />
Metabolic Prioritization in <strong>the</strong> <strong>European</strong> Sea Bass, Dicentrarchus labrax. Physiological and<br />
Biochemical Zoology 83(1), 68-77.<br />
Karlson,K., Rosenberg,R., Bonsdorff,E., 2002. Temporal and spatial large-scale effects <strong>of</strong><br />
eutrophication and oxygen deficiency on benthic fauna in Scandinavian and Baltic waters - A<br />
review. Oceanography and Marine Biology, Vol 40 40, 427-489.<br />
Kiceniuk,J.W., Jones,D.R., 1977. The oxygen transport system in rainbow trout (Salmo gairdneri)<br />
during sustained excercise. Journal <strong>of</strong> Experimental Biology 69, 247-260.<br />
Kideys,A.E., 2002. Fall and rise <strong>of</strong> <strong>the</strong> Black Sea ecosystem. Science 297(5586), 1482-1484.<br />
Kieffer,J.D., Alsop,D., Wood,C.M., 1998. A respirometric analysis <strong>of</strong> fuel use during aerobic<br />
swimming at different temperatures in rainbow trout (Oncorhynchus mykiss). Journal <strong>of</strong><br />
Experimental Biology 201(22), 3123-3133.<br />
Klaiman,J.M., Fenna,A.J., Shiels,H.A., Macri,J., Gillis,T.E., 2011. Cardiac Remodeling in Fish:<br />
Strategies to Maintain Heart Function during Temperature Change. Plos One 6(9).<br />
Kramer,D.L., Mcclure,M., 1982. Aquatic Surface Respiration, A Widespread Adaptation to Hypoxia in<br />
Tropical Fresh-Water Fishes. Environmental Biology <strong>of</strong> Fishes 7(1), 47-55.<br />
49
Langenbuch,M., Portner,H.O., 2003. Energy budget <strong>of</strong> hepatocytes from Antarctic fish (Pachycara<br />
brachycephalum and Lepidonoto<strong>the</strong>n kempi) as a function <strong>of</strong> ambient CO2: pH-dependent<br />
limitations <strong>of</strong> cellular protein biosyn<strong>the</strong>sis? Journal <strong>of</strong> Experimental Biology 206(22),<br />
3895-3903.<br />
Lauff,R.F., Wood,C.M., 1996. Respiratory gas exchange, nitrogenous waste excretion, and fuel usage<br />
during starvation in juvenile rainbow trout, Oncorhynchus mykiss. Journal <strong>of</strong> Comparative<br />
<strong>Physiology</strong> B-Biochemical Systemic and Environmental <strong>Physiology</strong> 165(7), 542-551.<br />
Lauff,R.F., Wood,C.H., 1996. Respiratory gas exchange, nitrogenous waste excretion, and fuel usage<br />
during aerobic swimming in juvenile rainbow trout. Journal <strong>of</strong> Comparative <strong>Physiology</strong><br />
B-Biochemical Systemic and Environmental <strong>Physiology</strong> 166(8), 501-509.<br />
Laursen,J.S., Andersen,N.A., Lykkeboe,G., 1985. Temperature-Acclimation and Oxygen<br />
Binding-Properties <strong>of</strong> Blood <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong>, Anguilla-Anguilla. Comparative<br />
Biochemistry and <strong>Physiology</strong> A-<strong>Physiology</strong> 81(1), 79-86.<br />
Lee,C.G., Farrell,A.P., Lotto,A., MacNutt,M.J., Hinch,S.G., Healey,M.C., 2003. The effect <strong>of</strong><br />
temperature on swimming performance and oxygen consumption in adult sockeye<br />
(Oncorhynchus nerka) and coho (O-kisutch) salmon stocks. Journal <strong>of</strong> Experimental Biology<br />
206(18), 3239-+.<br />
Lee,K.S., Kita,J., Ishimatsu,A., 2003. Effects <strong>of</strong> lethal levels <strong>of</strong> environmental hypercapnia on<br />
cardiovascular and blood-gas status in yellowtail, Seriola quinqueradiata. Zoological Science<br />
20(4), 417-422.<br />
Lemoigne,J., Soulier,P., Peyraudwaitzenegger,M., Peyraud,C., 1986. Cutaneous and Gill O-2 Uptake in<br />
<strong>the</strong> <strong>European</strong> <strong>Eel</strong> (Anguilla-Anguilla L) in Relation to Ambient P-O2, 10-400 Torr. Respiration<br />
<strong>Physiology</strong> 66(3), 341-354.<br />
Liew,H.J., Sinha,A.K., Mauro,N., Diricx,M., Blust,R., De Boeck,G., 2012. Fasting goldfish, Carassius<br />
auratus, and common carp, Cyprinus carpio, use different metabolic strategies when swimming.<br />
Comparative Biochemistry and <strong>Physiology</strong> A-Molecular & Integrative <strong>Physiology</strong> 163(3-4),<br />
327-335.<br />
Lin,Y.S., Poh,Y.P., Tzeng,C.S., 2001. A phylogeny <strong>of</strong> freshwater eels inferred from mitochondrial<br />
genes. Molecular Phylogenetics and Evolution 20(2), 252-261.<br />
Luo,Y.P., Xie,X.J., 2008. Effects <strong>of</strong> temperature on <strong>the</strong> specific dynamic action <strong>of</strong> <strong>the</strong> sou<strong>the</strong>rn catfish,<br />
Silurus meridionalis. Comparative Biochemistry and <strong>Physiology</strong> A-Molecular & Integrative<br />
<strong>Physiology</strong> 149(2), 150-156.<br />
Maes,G.E., Pujolar,J.M., Hellemans,B., Volckaert,F.A.M., 2006. Evidence for isolation by time in <strong>the</strong><br />
<strong>European</strong> eel (Anguilla anguilla L.). Molecular Ecology 15(8), 2095-2107.<br />
Maes,G.E., van Vo,B., Crivelli,A.J., Volckaert,F.A.M., 2009. Morphological and genetic seasonal<br />
dynamics <strong>of</strong> <strong>European</strong> eel Anguilla anguilla recruitment in sou<strong>the</strong>rn France (vol 74, pg 2047,<br />
2009). Journal <strong>of</strong> Fish Biology 75(4), 944.<br />
Matikainen,N., Vornanen,M., 1992. Effect <strong>of</strong> Season and Temperature-Acclimation on <strong>the</strong> Function <strong>of</strong><br />
Crucian Carp (Carassius-Carassius) Heart. Journal <strong>of</strong> Experimental Biology 167, 203-220.<br />
McCleave,J.D., Arnold,G.P., 1999. Movements <strong>of</strong> yellow- and silver-phase <strong>European</strong> eels (Anguilla<br />
anguilla L.) tracked in <strong>the</strong> western North Sea. Ices Journal <strong>of</strong> Marine Science 56(4), 510-536.<br />
McCue,M.D., 2006. Specific dynamic action: a century <strong>of</strong> investigation. Comparative Biochemistry<br />
and <strong>Physiology</strong> A-Molecular & Integrative <strong>Physiology</strong> 144(4), 381-394.<br />
50
McKendry,J.E., Milsom,W.K., Perry,S.F., 2001. Branchial CO2 receptors and cardio<strong>respiratory</strong><br />
adjustments during hypercarbia in Pacific spiny dogfish Squalus acanthias. Journal <strong>of</strong><br />
Experimental Biology 204(8), 1519-1527.<br />
McKenzie,D.J., Piccolella,M., la Valle,A.Z., Taylor,E.W., Bolis,C.L., Steffensen,J.F., 2003. Tolerance<br />
<strong>of</strong> chronic hypercapnia by <strong>the</strong> <strong>European</strong> eel Anguilla anguilla. Journal <strong>of</strong> Experimental Biology<br />
206(10), 1717-1726.<br />
McKenzie,D.J., Skov,P.V., Taylor,E.W.T., Wang,T., Steffensen,J.F., 2009. Abolition <strong>of</strong> reflex<br />
bradycardia by cardiac vagotomy has no effect on <strong>the</strong> regulation <strong>of</strong> oxygen uptake by Atlantic<br />
cod in progressive hypoxia. Comparative Biochemistry and <strong>Physiology</strong> A-Molecular &<br />
Integrative <strong>Physiology</strong> 153(3), 332-338.<br />
Melzner,F., Gutowska,M.A., Langenbuch,M., Dupont,S., Lucassen,M., Thorndyke,M.C., Bleich,M.,<br />
Portner,H.O., 2009. Physiological basis for high CO2 tolerance in marine ecto<strong>the</strong>rmic animals:<br />
pre-adaptation through lifestyle and ontogeny?. Biogeosciences 6(10), 2313-2331.<br />
Minegishi,Y., Aoyama,J., Inoue,J.G., Miya,M., Nishida,M., Tsukamoto,K., 2005. Molecular phylogeny<br />
and evolution <strong>of</strong> <strong>the</strong> freshwater eels genus Anguilla based on <strong>the</strong> whole mitochondrial genome<br />
sequences. Molecular Phylogenetics and Evolution 34(1), 134-146.<br />
Moran,D., Stottrup,J.G., 2011. The effect <strong>of</strong> carbon dioxide on growth <strong>of</strong> juvenile Atlantic cod Gadus<br />
morhua L. Aquatic Toxicology 102(1-2), 24-30.<br />
Nauen,J.C., Lauder,G.V., 2002. Hydrodynamics <strong>of</strong> caudal fin locomotion by chub mackerel, Scomber<br />
japonicus (Scombridae). Journal <strong>of</strong> Experimental Biology 205(12), 1709-1724.<br />
Nielsen,K.E., Gesser,H., 1984. <strong>Eel</strong> and Rainbow-Trout Myocardium Under Anoxia And/Or<br />
Hypercapnic Acidosis, with Changes in (Ca-2+)0 and (Na+)0. Molecular <strong>Physiology</strong> 5(3-4),<br />
189-198.<br />
Nilsson,G.E., Rosen,P., Johansson,D., 1993. Anoxic Depression <strong>of</strong> Spontaneous Locomotor-Activity in<br />
Crucian Carp Quantified by A Computerized Imaging Technique. Journal <strong>of</strong> Experimental<br />
Biology 180, 153-162.<br />
Nilsson,G.E., Crawley,N., Lunde,I.G., Munday,P.L., 2009. Elevated temperature reduces <strong>the</strong><br />
<strong>respiratory</strong> scope <strong>of</strong> coral reef fishes. Global Change Biology 15(6), 1405-1412.<br />
Nilsson,G.E., Ostlund-Nilsson,S., Munday,P.L., 2010. Effects <strong>of</strong> elevated temperature on coral reef<br />
fishes: Loss <strong>of</strong> hypoxia tolerance and inability to acclimate. Comparative Biochemistry and<br />
<strong>Physiology</strong> A-Molecular & Integrative <strong>Physiology</strong> 156(4), 389-393.<br />
Nilsson,S., 1983. Autonomic nerve function in <strong>the</strong> vertebrates. Springer, Berlin, Heidelberg New York.<br />
Northcote,T.G., 1978. Migratory strategies and production in freswater fishes. In: Gerking,S.D. (Ed.),<br />
Ecology <strong>of</strong> Freshwater Production. Blackwell, Oxford, pp. 326-359.<br />
Nyman,L., 1972. Some Effects <strong>of</strong> Temperature on <strong>Eel</strong> Anguilla Behavior. <strong>Institut</strong>e <strong>of</strong> Freshwater<br />
Research Drottningholm Report(52), 90-102.<br />
Olson,K.R., Farrell,A.P., 2006. The <strong>Cardio</strong>vascular System. In: Evans,D.H., Claiborne,J.B. (Eds.), The<br />
<strong>Physiology</strong> <strong>of</strong> Fishes. Taylor & Francis Group, Boca Raton, FL, pp. 119-152.<br />
Ott,M.E., Heisler,N., Ultsch,G.R., 1980. A Re-Evaluation <strong>of</strong> <strong>the</strong> Relationship Between Temperature<br />
and <strong>the</strong> Critical Oxygen-Tension in Fresh-Water Fishes. Comparative Biochemistry and<br />
<strong>Physiology</strong> A-<strong>Physiology</strong> 67(3), 337-340.<br />
Pennec,J.P., Peyraud,C., 1983. Effects <strong>of</strong> Adrenaline on Isolated Heart <strong>of</strong> <strong>the</strong> <strong>Eel</strong> (Anguilla-Anguilla L)<br />
During Winter. Comparative Biochemistry and <strong>Physiology</strong> C-Pharmacology Toxicology &<br />
Endocrinology 74(2), 477-480.<br />
51
Peres,H., Oliva-Teles,A., 2001. Effect <strong>of</strong> dietary protein and lipid level on metabolic utilization <strong>of</strong> diets<br />
by european sea bass (Dicentrarchus labrax) juveniles. Fish <strong>Physiology</strong> and Biochemistry 25(4),<br />
269-275.<br />
Perez-Casanova,J.C., Lall,S.P., Gamperl,A.K., 2010. Effects <strong>of</strong> dietary protein and lipid level, and<br />
water temperature, on <strong>the</strong> post-feeding oxygen consumption <strong>of</strong> Atlantic cod and haddock.<br />
Aquaculture Research 41(2), 198-209.<br />
Perry,S.E., Fritsche,R., Hoagland,T.M., Duff,D.W., Olson,K.R., 1999. The control <strong>of</strong> blood pressure<br />
during external hypercapnia in <strong>the</strong> rainbow trout (Oncorhynchus mykiss). Journal <strong>of</strong><br />
Experimental Biology 202(16), 2177-2190.<br />
Perry,S.F., McKendry,J.E., 2001. The relative roles <strong>of</strong> external and internal CO2 versus H+ in eliciting<br />
<strong>the</strong> cardio<strong>respiratory</strong> responses <strong>of</strong> Salmo salar and Squalus acanthias to hypercarbia. Journal <strong>of</strong><br />
Experimental Biology 204(22), 3963-3971.<br />
Perry,S.F., Jonz,M.G., Gilmour,K.M., 2009. Oxygen sensing and hypoxic ventilatory response. In:<br />
Richards,G.R., Farrell,A.P., Brauner,C.J. (Eds.), Hypoxia. Academic Press, Amsterdam, pp.<br />
193-254.<br />
Peyraud-Waitzenegger,M., Bar<strong>the</strong>lemy,L., Peyraud,C., 1980. <strong>Cardio</strong>vascular and Ventilatory Effects <strong>of</strong><br />
Catecholamines in Unrestrained <strong>Eel</strong>s (Anguilla-Anguilla L) - A Study <strong>of</strong> Seasonal-Changes in<br />
Reactivity. Journal <strong>of</strong> Comparative <strong>Physiology</strong> 138(4), 367-375.<br />
Peyraud-Waitzenegger,M., Soulier,P., 1989. Ventilatory and Circulatory Adjustments in <strong>the</strong> <strong>European</strong><br />
<strong>Eel</strong> (Anguilla-Anguilla L) Exposed to Short-Term Hypoxia. Experimental Biology 48(2),<br />
107-122.<br />
Pörtner,H.O., 2001. Climate change and temperature-dependent biogeography: oxygen limitation <strong>of</strong><br />
<strong>the</strong>rmal tolerance in animals. Naturwissenschaften 88(4), 137-146.<br />
Pörtner,H.O., 2002. Climate variations and <strong>the</strong> physiological basis <strong>of</strong> temperature dependent<br />
biogeography: systemic to molecular hierarchy <strong>of</strong> <strong>the</strong>rmal tolerance in animals<br />
2. Comparative Biochemistry and <strong>Physiology</strong> A-Molecular and Integrative <strong>Physiology</strong> 132(4),<br />
739-761.<br />
Pörtner,H.O., Langenbuch,M., Reipschlager,A., 2004. Biological impact <strong>of</strong> elevated ocean CO2<br />
concentrations: Lessons from animal physiology and earth history<br />
11. Journal <strong>of</strong> Oceanography 60(4), 705-718.<br />
Pörtner,H.O., Knust,R., 2007. Climate change affects marine fishes through <strong>the</strong> oxygen limitation <strong>of</strong><br />
<strong>the</strong>rmal tolerance. Science 315(5808), 95-97.<br />
Pörtner,H.O., Farrell,A.P., 2008. ECOLOGY <strong>Physiology</strong> and Climate Change. Science 322(5902),<br />
690-692.<br />
Pörtner,H.O., 2008. Ecosystem effects <strong>of</strong> ocean acidification in times <strong>of</strong> ocean warming: a<br />
physiologist's view. Marine Ecology-Progress Series 373, 203-217.<br />
Pujolar,J.M., Maes,G.E., Volckaert,F.A.M., 2006. Genetic patchiness among recruits in <strong>the</strong> <strong>European</strong><br />
eel Anguilla anguilla. Marine Ecology-Progress Series 307, 209-217.<br />
Pujolar,J.M., De Leo,G.A., Ciccotti,E., Zane,L., 2009. Genetic composition <strong>of</strong> Atlantic and<br />
Mediterranean recruits <strong>of</strong> <strong>European</strong> eel Anguilla anguilla based on EST-linked microsatellite<br />
loci. Journal <strong>of</strong> Fish Biology 74(9), 2034-2046.<br />
Randall,D., 1982. The Control <strong>of</strong> Respiration and Circulation in Fish During Exercise and Hypoxia<br />
1. Journal <strong>of</strong> Experimental Biology 100(OCT), 275-&.<br />
Riemann,L., Alfredsson,H., Hansen,M.M., Als,T.D., Nielsen,T.G., Munk,P., Aarestrup,K., Maes,G.E.,<br />
Sparholt,H., Petersen,M.I., Bachler,M., Castonguay,M., 2010. Qualitative assessment <strong>of</strong> <strong>the</strong> diet<br />
52
<strong>of</strong> <strong>European</strong> eel larvae in <strong>the</strong> Sargasso Sea resolved by DNA barcoding. Biology Letters 6(6),<br />
819-822.<br />
Root,R.W., 1931. The <strong>respiratory</strong> function <strong>of</strong> <strong>the</strong> blood <strong>of</strong> marine fishes. Biological Bulletin 61(3),<br />
427-456.<br />
Ross,L.G., Mckinney,R.W., Cardwell,S.K., Fullarton,J.G., Roberts,S.E.J., Ross,B., 1992. The Effects<br />
<strong>of</strong> Dietary-Protein Content, Lipid-Content and Ration Level on Oxygen-Consumption and<br />
Specific Dynamic Action in Oreochromis-Niloticus l. Comparative Biochemistry and<br />
<strong>Physiology</strong> A-<strong>Physiology</strong> 103(3), 573-578.<br />
Rousseau,K., Aroua,S., Schmitz,M., Elie,P., Dufour,S., 2009. Silvering: Metamorphosis or Puberty. In:<br />
van den Thillart,G.E.E.J., Dufour,S., Rankin,J.C. (Eds.), Spawning Migration <strong>of</strong> <strong>the</strong> <strong>European</strong><br />
<strong>Eel</strong>. Springer, pp. 39-64.<br />
Sandblom,E., Axelsson,M., 2005. Effects <strong>of</strong> hypoxia on <strong>the</strong> venous circulation in rainbow trout<br />
(Oncorhynchus mykiss). Comparative Biochemistry and <strong>Physiology</strong> A-Molecular & Integrative<br />
<strong>Physiology</strong> 140(2), 233-239.<br />
Sandblom,E., Axelsson,M., 2007. Venous hemodynamic responses to acute temperature increase in <strong>the</strong><br />
rainbow trout (Oncorhynchus mykiss). American Journal <strong>of</strong> <strong>Physiology</strong>-Regulatory Integrative<br />
and Comparative <strong>Physiology</strong> 292(6), R2292-R2298.<br />
Santer,R.M., 1985. Morphology and Innervation <strong>of</strong> <strong>the</strong> Fish Heart. Advances in Anatomy Embryology<br />
and Cell Biology 89, 1-99.<br />
Satoh,S., 2002. <strong>Eel</strong>, Anguilla spp. In: Webster,C.D., Lim,C. (Eds.), Nutrient reguirements and and<br />
feeding for finfish in aquaculture. CABI Publishing, New York, pp. 319-326.<br />
Schmidt,J., 1923. The breeding places <strong>of</strong> <strong>the</strong> eel. Philosophical Transactions <strong>of</strong> <strong>the</strong> Royal Society B<br />
211, 179-208.<br />
Schurmann,H., Steffensen,J.F., 1997. Effects <strong>of</strong> temperature, hypoxia and activity on <strong>the</strong> metabolism <strong>of</strong><br />
juvenile Atlantic cod. Journal <strong>of</strong> Fish Biology 50(6), 1166-1180.<br />
Secor,S.M., 2009. Specific dynamic action: a review <strong>of</strong> <strong>the</strong> postprandial metabolic response. Journal <strong>of</strong><br />
Comparative <strong>Physiology</strong> B-Biochemical Systemic and Environmental <strong>Physiology</strong> 179(1), 1-56.<br />
Seibert,H., 1979. Thermal Adaptation <strong>of</strong> Heart-Rate and Its Parasympa<strong>the</strong>tic Control in <strong>the</strong> <strong>European</strong><br />
<strong>Eel</strong> Anguilla-Anguilla (L). Comparative Biochemistry and <strong>Physiology</strong> C-Pharmacology<br />
Toxicology & Endocrinology 64(2), 275-278.<br />
Sephton,D.H., Driedzic,W.R., 1991. Effect <strong>of</strong> Acute and Chronic Temperature Transition on Enzymes<br />
<strong>of</strong> Cardiac Metabolism in White Perch (Morone-Americana), Yellow Perch (Perca-Flavescens),<br />
and Smallmouth Bass (Micropterus-Dolomieui). Canadian Journal <strong>of</strong> Zoology-Revue<br />
Canadienne de Zoologie 69(1), 258-262.<br />
Shiels,H.A., Vornanen,M., Farrell,A.P., 2002. The force-frequency relationship in fish hearts - a<br />
review. Comparative Biochemistry and <strong>Physiology</strong> A-Molecular and Integrative <strong>Physiology</strong><br />
132(4), II.<br />
Sims,D.W., Davies,S.J., 1994. Does Specific Dynamic Action (Sda) Regulate Return <strong>of</strong> Appetite in <strong>the</strong><br />
Lesser Spotted Dogfish, Scyliorhinus-Canicula. Journal <strong>of</strong> Fish Biology 45(2), 341-348.<br />
Skov,P.V., Bushnell,P., Tirsgaard,B., Steffensen,J.F., 2009. The role <strong>of</strong> adrenaline as a modulator <strong>of</strong><br />
cardiac performance in two Antarctic fishes. Polar Biology 32(2), 215-223.<br />
Sorokin,V.P., Konstantinov,K.G., 1960. The eel Anguilla anguilla in <strong>the</strong> Kola Bay. Zool. Zh. 39,<br />
621-622.<br />
53
St-Pierre,J., Charest,P.M., Guderley,H., 1998. Relative contribution <strong>of</strong> quantitative and qualitative<br />
changes in mitochondria to metabolic compensation during seasonal acclimatisation <strong>of</strong> rainbow<br />
trout Oncorhynchus mykiss. Journal <strong>of</strong> Experimental Biology 201(21), 2961-2970.<br />
Steffensen,J.F., 1989. Some Errors in Respirometry <strong>of</strong> Aquatic Brea<strong>the</strong>rs - How to Avoid and Correct<br />
for Them. Fish <strong>Physiology</strong> and Biochemistry 6(1), 49-59.<br />
Steffensen,J.F., Lomholt,J.P., 1990. Accumulation <strong>of</strong> carbon dioxide in fish farms with recirculating<br />
water. In: Ryans,R.C. (Ed.), Fish physiolohy, fish toxicology and fisheries management.<br />
Environmental Research Laboratories -U.S. Environmental Protection Agency,<br />
EPA/600/9-90/011, A<strong>the</strong>ns, pp. 157-161.<br />
Steinhausen,M.F., Sandblom,E., Eliason,E.J., Verhille,C., Farrell,A.P., 2008. The effect <strong>of</strong> acute<br />
temperature increases on <strong>the</strong> cardio<strong>respiratory</strong> performance <strong>of</strong> resting and swimming sockeye<br />
salmon (Oncorhynchus nerka). Journal <strong>of</strong> Experimental Biology 211(24), 3915-3926.<br />
Svedang,H., Wickstrom,H., 1997. Low fat contents in female silver eels: Indications <strong>of</strong> insufficient<br />
energetic stores for migration and gonadal development. Journal <strong>of</strong> Fish Biology 50(3),<br />
475-486.<br />
Takeuchi,T., Satoh,S., Kiron,V., 2002. Common carp, Cyprinus carbio. In: Webster,C.D., Lim,C.<br />
(Eds.), Nutrient reguirements and and feeding for finfish in aquaculture. CABI Publishing, New<br />
York, pp. 245-261.<br />
Taylor,E.W., 1985. Control and co-ordination <strong>of</strong> gill ventillation and perfusion. pp. 123-161.<br />
Taylor,E.W., 1992. Nerveous control <strong>of</strong> <strong>the</strong> heart and cardio<strong>respiratory</strong> interactions. In: Hoar,W.S.,<br />
Randall,D.J., Farrell,A.P. (Eds.), The <strong>Cardio</strong>vascular System. Academic Press, San Diego, pp.<br />
343-387.<br />
Taylor,E.W., Jordan,D., Coote,J.H., 1999. Central control <strong>of</strong> <strong>the</strong> cardiovascular and <strong>respiratory</strong> systems<br />
and <strong>the</strong>ir interactions in vertebrates. Physiological Reviews 79(3), 855-916.<br />
Teichmann,H., 1959. Uber Die Leistung des Geruchssinnes Beim Aal [Anguilla-Anguilla (L)].<br />
Zeitschrift fur Vergleichende Physiologie 42(3), 206-254.<br />
Tesch,F., 1989. Changes in swimming depth and direction <strong>of</strong> silver eels (Anguilla anguilla L.) from <strong>the</strong><br />
continental shelf to <strong>the</strong> deep sea. Aquatic Living Resources 2(1), 9-20.<br />
Tesch,F.W., 1978. Telemetric Observations on <strong>the</strong> Spawning Migration <strong>of</strong> <strong>the</strong> <strong>Eel</strong> Anguilla-Anguilla<br />
West <strong>of</strong> <strong>the</strong> <strong>European</strong> Continental Shelf. Environmental Biology <strong>of</strong> Fishes 3(2), 203-210.<br />
Tesch,F.W., 1995. Vertical movements <strong>of</strong> migrating silver eels (Anguilla anguilla) in <strong>the</strong> sea.<br />
284. Bulletin <strong>of</strong> <strong>the</strong> Sea Fisheries<strong>Institut</strong>e 2, 23-30.<br />
Tesch,F.W., 2003. The <strong>Eel</strong>, 5 ed. Blackwell Publishing, Oxford.<br />
Tiitu,V., Vornanen,M., 2001. Cold adaptation suppresses <strong>the</strong> contractility <strong>of</strong> both atrial and ventricular<br />
muscle <strong>of</strong> <strong>the</strong> crucian carp heart. Journal <strong>of</strong> Fish Biology 59(1), 141-156.<br />
Tiitu,V., Vornanen,M., 2003. Ryanodine and dihydropyridine receptor binding in ventricular cardiac<br />
muscle <strong>of</strong> fish with different temperature preferences. Journal <strong>of</strong> Comparative <strong>Physiology</strong><br />
B-Biochemical Systemic and Environmental <strong>Physiology</strong> 173(4), 285-291.<br />
Tsadik,G.G., Kutty,M.N., 1987. Influence <strong>of</strong> ambient oxygen on feeding and growth <strong>of</strong> Tilapia<br />
(Oreochromis niloticus) (Linneaus).<br />
Tsukuda,H., Liu,B., Fujii,K., 1985. Pulsation Rate and Oxygen-Consumption <strong>of</strong> Isolated Hearts <strong>of</strong> <strong>the</strong><br />
Goldfish, Carassius-Auratus, Acclimated to Different Temperatures. Comparative Biochemistry<br />
and <strong>Physiology</strong> A-<strong>Physiology</strong> 82(2), 281-283.<br />
Tytell,E.D., Lauder,G.V., 2004. The hydrodynamics <strong>of</strong> eel swimming - I. Wake structure. Journal <strong>of</strong><br />
Experimental Biology 207(11), 1825-1841.<br />
54
Ultsch,G.R., 1996. Gas exchange, hypercarbia and acid-base balance, paleoecology, and <strong>the</strong><br />
evolutionary transition from water-breathing to air-breathing among vertebrates.<br />
Palaeogeography Palaeoclimatology Palaeoecology 123(1-4), 1-27.<br />
Valverde,J.C., Lopez,F.J.M., Garcia,B.G., 2006. Oxygen consumption and ventilatory frequency<br />
responses to gradual hypoxia in common dentex (Dentex dentex): Basis for suitable oxygen<br />
level estimations. Aquaculture 256(1-4), 542-551.<br />
van den Thillart,G.E.E.J., van Raaij,M., 1995. Endogenous fuels; non-invasive versus invasive<br />
approaches. In: Hochochka,P.W., Mommsen,T.P. (Eds.), Metabolic biochemistry. Elsevier,<br />
Amsterdam, pp. 33-63.<br />
Van Ginneken,V., Antonissen,E., Muller,U.K., Booms,R., Eding,E., Verreth,J., van den Thillart,G.,<br />
2005. <strong>Eel</strong> migration to <strong>the</strong> Sargasso: remarkably high swimming efficiency and low energy<br />
costs. Journal <strong>of</strong> Experimental Biology 208(7), 1329-1335.<br />
van Ginneken,V.J.T., Onderwater,M., Olivar,O.L., van den Thillart,G.E.E.J., 2001. Metabolic<br />
depression and investigation <strong>of</strong> glucose/ethanol conversion in <strong>the</strong> <strong>European</strong> eel (Anguilla<br />
anguilla Linnaeus 1758) during anaerobiosis. Thermochimica Acta 373(1), 23-30.<br />
van Ginneken,V.J.T., Maes,G.E., 2005. The european eel (Anguilla anguilla, Linnaeus), its lifecycle,<br />
evolution and reproduction: a literature review. Reviews in Fish Biology and Fisheries 15(4),<br />
367-398.<br />
van Waarde,A., van den Thillart,G.E.E.J., Kesbeke,F., 1983. Anaerobic Energy-Metabolism <strong>of</strong> <strong>the</strong><br />
<strong>European</strong> <strong>Eel</strong>, Anguilla-Anguilla l. Journal <strong>of</strong> Comparative <strong>Physiology</strong> 149(4), 469-475.<br />
Vanella,F.A., Boy,C.C., Lattuca,M.E., Calvo,J., 2010. Temperature influence on post-prandial<br />
metabolic rate <strong>of</strong> sub-Antarctic teleost fish. Comparative Biochemistry and <strong>Physiology</strong><br />
A-Molecular & Integrative <strong>Physiology</strong> 156(2), 247-254.<br />
Videler,J.J., 1993. Fish Swimming. Chapman & Hall, London.<br />
Von Herbing,I.H., White,L., 2002. The effects <strong>of</strong> body mass and feeding on metabolic rate in small<br />
juvenile Atlantic cod. Journal <strong>of</strong> Fish Biology 61(4), 945-958.<br />
Vornanen,M., 1998. L-type Ca2+ current in fish cardiac myocytes: Effects <strong>of</strong> <strong>the</strong>rmal acclimation and<br />
beta-adrenergic stimulation. Journal <strong>of</strong> Experimental Biology 201(4), 533-547.<br />
Vornanen,M., 1999. Na+/Ca2(+) exchange current in ventricular myocytes <strong>of</strong> fish heart: Contribution<br />
to sarcolemmal Ca2+ influx. Journal <strong>of</strong> Experimental Biology 202(13), 1763-1775.<br />
Vornanen,M., Shiels,H.A., Farrell,A.P., 2002. Plasticity <strong>of</strong> excitation-contraction coupling in fish<br />
cardiac myocytes. Comparative Biochemistry and <strong>Physiology</strong> A-Molecular and Integrative<br />
<strong>Physiology</strong> 132(4), II.<br />
Walsh,P.J., Foster,G.D., Moon,T.W., 1983. The Effects <strong>of</strong> Temperature on Metabolism <strong>of</strong> <strong>the</strong><br />
American <strong>Eel</strong> Anguilla-Rostrata (Lesueur) - Compensation in <strong>the</strong> Summer and Torpor in <strong>the</strong><br />
Winter<br />
1. Physiological Zoology 56(4), 532-540.<br />
Wang,Q.Q., Wang,W., Huang,Q.D., Zhang,Y.R., Luo,Y.P., 2012. Effect <strong>of</strong> meal size on <strong>the</strong> specific<br />
dynamic action <strong>of</strong> <strong>the</strong> juvenile snakehead (Channa argus). Comparative Biochemistry and<br />
<strong>Physiology</strong> A-Molecular & Integrative <strong>Physiology</strong> 161(4), 401-405.<br />
Wea<strong>the</strong>rley,A.H., 1976. Factors Affecting Maximization <strong>of</strong> Fish Growth. Journal <strong>of</strong> <strong>the</strong> Fisheries<br />
Research Board <strong>of</strong> Canada 33(4), 1046-1058.<br />
Wells,R.M.G., Weber,R.E., 1990. The Spleen in Hypoxic and Exercised Rainbow-Trout<br />
1. Journal <strong>of</strong> Experimental Biology 150, 461-466.<br />
55
Winberg,G.G., 1956. Rate <strong>of</strong> Metabolism and Food Requirements <strong>of</strong> Fishes. Belorussian State<br />
University, Minsk. (Transl. by Fish. Res. Borad. Can. Transl. Ser. No. 194, 1960).<br />
Wood,C.M., 2001. Influence on feeding, exercise, and temperature on nitrogen metabolism and<br />
excretion. In: Wright,P., Anderson,P. (Eds.), Nitrogen Excretion. Academic Press, San Diego,<br />
pp. 201-238.<br />
Zhou,B.S., Wu,R.S.S., Randall,D.J., Lam,P.K.S., 2001. Bioenergetics and RNA/DNA ratios in <strong>the</strong><br />
common carp (Cyprinus carpio) under hypoxia. Journal <strong>of</strong> Comparative <strong>Physiology</strong><br />
B-Biochemical Systemic and Environmental <strong>Physiology</strong> 171(1), 49-57.<br />
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 />
References<br />
Andersen,N.A., Laursen,J.S., Lykkeboe,G., 1985. Seasonal-Variations in Hematocrit, Red-Cell Hemoglobin<br />
and Nucleoside Triphosphate Concentrations, in <strong>the</strong> <strong>European</strong> <strong>Eel</strong> Anguilla-Anguilla. Comparative<br />
Biochemistry and <strong>Physiology</strong> A-<strong>Physiology</strong> 81(1), 87-92.<br />
Barnes,R., King,H., Carter,C.G., 2011. Hypoxia tolerance and oxygen regulation in Atlantic salmon, Salmo<br />
salar from a Tasmanian population. Aquaculture 318(3-4), 397-401.<br />
Bertin,L., 1956. <strong>Eel</strong>s: A Biological Study. Cleaver-Hume Press, London.<br />
Brauner,C.J., Weber,R.E., 1998. Hydrogen ion titrations <strong>of</strong> <strong>the</strong> anodic and cathodic haemoglobin<br />
components <strong>of</strong> <strong>the</strong> <strong>European</strong> eel Anguilla anguilla. Journal <strong>of</strong> Experimental Biology 201(17), 2507-<br />
2514.<br />
Bruun,A.F., 1963. The breeding <strong>of</strong> <strong>the</strong> north Atlantic freshwater eel. Adv. Mar. Biol. 1, 137-169.<br />
Busk,M., Boutilier,R.G., 2005. Metabolic arrest and its regulation in anoxic eel hepatocytes. Physiological<br />
and Biochemical Zoology 78(6), 926-936.<br />
Cerezo,J., Garcia,B.G., 2004. The effects <strong>of</strong> oxygen levels on oxygen consumption, survival and ventilatory<br />
frequency <strong>of</strong> sharpsnout sea bream (Diplodus puntazzo Gmelin, 1789) at different conditions <strong>of</strong><br />
temperature and fish weight. Journal <strong>of</strong> Applied Ichthyology 20(6), 488-492.<br />
Chabot,D., Claireaux,G., 2008. Environmental hypoxia as a metabolic constraint on fish: The case <strong>of</strong><br />
Atlantic cod, Gadus morhua. Marine Pollution Bulletin 57(6-12), 287-294.<br />
12
Chan,D.K.O., 1986. <strong>Cardio</strong>vascular, Respiratory, and Blood Adjustments to Hypoxia in <strong>the</strong> Japanese <strong>Eel</strong>,<br />
Anguilla-Japonica. Fish <strong>Physiology</strong> and Biochemistry 2(1-4), 179-193.<br />
Chapman,L.J., Chapman,C.A., Nordlie,F.G., Rosenberger,A.E., 2002. Physiological refugia: swamps,<br />
hypoxia tolerance and maintenance <strong>of</strong> fish diversity in <strong>the</strong> Lake Victoria region. Comparative<br />
Biochemistry and <strong>Physiology</strong> A-Molecular and Integrative <strong>Physiology</strong> 133(3), 421-437.<br />
Claireaux,G., Lefrancois,C., 2007. Linking environmental variability and fish performance: integration<br />
through <strong>the</strong> concept <strong>of</strong> scope for activity. Philosophical Transactions <strong>of</strong> <strong>the</strong> Royal Society B-<br />
Biological Sciences 362(1487), 2031-2041.<br />
Corkum,C.P., Gamperl,A.K., 2009. Does <strong>the</strong> Ability to Metabolically Downregulate Alter <strong>the</strong> Hypoxia<br />
Tolerance <strong>of</strong> Fishes?: A Comparative Study Using Cunner (T. adspersus) and Greenland Cod (G.<br />
ogac). Journal <strong>of</strong> Experimental Zoology Part A-Ecological Genetics and <strong>Physiology</strong> 311A(4), 231-<br />
239.<br />
CruzNeto,A.P., Steffensen,J.F., 1997. The effects <strong>of</strong> acute hypoxia and hypercapnia on oxygen consumption<br />
<strong>of</strong> <strong>the</strong> freshwater <strong>European</strong> eel. Journal <strong>of</strong> Fish Biology 50(4), 759-769.<br />
Degani,G., Gallagher,M.L., Meltzer,A., 1989. The in fluence <strong>of</strong> body size and temperature on oxygen<br />
consumption <strong>of</strong> <strong>the</strong> <strong>European</strong> eel, Anguilla anguilla. Journal <strong>of</strong> Fish Biology 34(1), 19-24.<br />
Diaz,R.J., Breitburg,D., 2009. The Hypoxic Environment. In: Richards,G.R., Farrell,A.P., Brauner,C.J.<br />
(Eds.), Hypoxia. Academic Press, Amsterdam, pp. 2-23.<br />
Fago,A., Carratore,V., Diprisco,G., Feuerlein,R.J., Sottrupjensen,L., Weber,R.E., 1995. The Cathodic<br />
Hemoglobin <strong>of</strong> Anguilla-Anguilla - Amino-Acid-Sequence and Oxygen Equilibria <strong>of</strong> A Reverse<br />
Bohr Effect Hemoglobin with High Oxygen-Affinity and High Phosphate Sensitivity. Journal <strong>of</strong><br />
Biological Chemistry 270(32), 18897-18902.<br />
Farrell,A.P., Hinch,S.G., Cooke,S.J., Patterson,D.A., Crossin,G.T., Lapointe,M., Ma<strong>the</strong>s,M.T., 2008. Pacific<br />
Salmon in Hot Water: Applying Aerobic Scope Models and Biotelemetry to Predict <strong>the</strong> Success <strong>of</strong><br />
Spawning Migrations. Physiological and Biochemical Zoology 81(6), 697-708.<br />
Farrell,A.P., Steffensen,J.F., 1987. An Analysis <strong>of</strong> <strong>the</strong> Energetic Cost <strong>of</strong> <strong>the</strong> Branchial and Cardiac Pumps<br />
During Sustained Swimming in Trout. Fish <strong>Physiology</strong> and Biochemistry 4(2), 73-79.<br />
Fernandes,M.N., Rantin,F.T., 1989. Respiratory Responses <strong>of</strong> Oreochromis-Niloticus (Pisces, Cichlidae) to<br />
Environmental Hypoxia Under Different Thermal Conditions. Journal <strong>of</strong> Fish Biology 35(4), 509-<br />
519.<br />
Freyh<strong>of</strong>,J., Kottelat,M., 2010. Anguilla anguilla. IUCN 2012.IUCN Red List <strong>of</strong> Threatened Species.Version<br />
2012.1. www.iuncredlist.org.<br />
Fry,F.E.J., 1971. The effect <strong>of</strong> environmental factors on <strong>the</strong> physiology <strong>of</strong> fish. In: Hoar,W.S., Randall,D.J.<br />
(Eds.), Environmental Relations and Behavior. Academic Press, New York, pp. 1-98.<br />
Gleeson,T.T., 1996. Post-exercise lactate metabolism: A comparative review <strong>of</strong> sites, pathways, and<br />
regulation. Annual Review <strong>of</strong> <strong>Physiology</strong> 58, 565-581.<br />
Goolish,E.M., 1991. Aerobic and Anaerobic Scaling in Fish. Biological Reviews <strong>of</strong> <strong>the</strong> Cambridge<br />
Philosophical Society 66(1), 33-56.<br />
Hochachka,P.W., Buck,L.T., Doll,C.J., Land,S.C., 1996. Unifying <strong>the</strong>ory <strong>of</strong> hypoxia tolerance: Molecular<br />
metabolic defense and rescue mechanisms for surviving oxygen lack. Proceedings <strong>of</strong> <strong>the</strong> National<br />
Academy <strong>of</strong> Sciences <strong>of</strong> <strong>the</strong> United States <strong>of</strong> America 93(18), 9493-9498.<br />
Hyde,D.A., Moon,T.W., Perry,S.F., 1987a. Physiological Consequences <strong>of</strong> Prolonged Aerial Exposure in <strong>the</strong><br />
American <strong>Eel</strong>, Anguilla-Rostrata - Blood Respiratory and Acid-Base Status. Journal <strong>of</strong> Comparative<br />
<strong>Physiology</strong> B-Biochemical Systemic and Environmental <strong>Physiology</strong> 157(5), 635-642.<br />
Hyde,D.A., Perry,S.F., 1987b. Acid-Base and Ionic Regulation in <strong>the</strong> American <strong>Eel</strong> (Anguilla-Rostrata)<br />
During and After Prolonged Aerial Exposure - Branchial and Renal Adjustments. Journal <strong>of</strong><br />
Experimental Biology 133, 429-447.<br />
Iversen,N.K., McKenzie,D.J., Malte,H., Wang,T., 2010. Reflex bradycardia does not influence oxygen<br />
consumption during hypoxia in <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla). Journal <strong>of</strong> Comparative<br />
<strong>Physiology</strong> B-Biochemical Systemic and Environmental <strong>Physiology</strong> 180(4), 495-502.<br />
Lemoigne,J., Soulier,P., PeyraudWaitzenegger,M., Peyraud,C., 1986. Cutaneous and Gill O-2 Uptake in <strong>the</strong><br />
<strong>European</strong> <strong>Eel</strong> (Anguilla-Anguilla L) in Relation to Ambient P-O2, 10-400 Torr. Respiration<br />
<strong>Physiology</strong> 66(3), 341-354.<br />
13
Mandic,M., Lau,G.Y., Nijjar,M.M.S., Richards,J.G., 2008. Metabolic recovery in goldfish: A comparison <strong>of</strong><br />
recovery from severe hypoxia exposure and exhaustive exercise. Comparative Biochemistry and<br />
<strong>Physiology</strong> C-Toxicology & Pharmacology 148(4), 332-338.<br />
Maxime,V., Pichavant,K., Boeuf,G., Nonnotte,G., 2000. Effects <strong>of</strong> hypoxia on <strong>respiratory</strong> physiology <strong>of</strong><br />
turbot, Scophthalmus maximus. Fish <strong>Physiology</strong> and Biochemistry 22(1), 51-59.<br />
Mccue,M.D., 2006. Specific dynamic action: A century <strong>of</strong> investigation. Comparative Biochemistry and<br />
<strong>Physiology</strong> A-Molecular & Integrative <strong>Physiology</strong> 144(4), 381-394.<br />
McKenzie,D.J., Piccolella,M., la Valle,A.Z., Taylor,E.W., Bolis,C.L., Steffensen,J.F., 2003. Tolerance <strong>of</strong><br />
chronic hypercapnia by <strong>the</strong> <strong>European</strong> eel Anguilla anguilla. Journal <strong>of</strong> Experimental Biology<br />
206(10), 1717-1726.<br />
McKenzie,D.J., Piraccini,G., Piccolella,M., Steffensen,J.F., Bolis,C.L., Taylor,E.W., 2000. Effects <strong>of</strong> dietary<br />
fatty acid composition on metabolic rate and responses to hypoxia in <strong>the</strong> <strong>European</strong> eel (Anguilla<br />
anguilla). Fish <strong>Physiology</strong> and Biochemistry 22(4), 281-296.<br />
Methling,C., Pedersen,P.B., Steffensen,J.F., Skov,P.V., 2012a. Tolerance towards hypercapnia does not<br />
preclude a negative impact on metabolism and postprandial proceses in <strong>the</strong> <strong>European</strong> eel (Anguilla<br />
anguilla). (draft manuscript).<br />
Methling,C., Steffensen,J.F., Skov,P.V., 2012b. The temperature challenges on cardiac performance in<br />
winter-quiescent and migration-stage eels Anguilla anguilla. Comparative Biochemistry and<br />
<strong>Physiology</strong> A-Molecular & Integrative <strong>Physiology</strong> 163(1), 66-73.<br />
Nilsson,G.E., Crawley,N., Lunde,I.G., Munday,P.L., 2009. Elevated temperature reduces <strong>the</strong> <strong>respiratory</strong><br />
scope <strong>of</strong> coral reef fishes. Global Change Biology 15(6), 1405-1412.<br />
Nonnotte,G., Maxime,V., Truchot,J.P., Williot,P., Peyraud,C., 1993. Respiratory Responses to Progressive<br />
Ambient Hypoxia in <strong>the</strong> Sturgeon, Acipenser-Baeri. Respiration <strong>Physiology</strong> 91(1), 71-82.<br />
Nyman,L., 1972. Some Effects <strong>of</strong> Temperature on <strong>Eel</strong> Anguilla Behavior. <strong>Institut</strong>e <strong>of</strong> Freshwater Research<br />
Drottningholm Report (52), 90-102.<br />
Ott,M.E., Heisler,N., Ultsch,G.R., 1980. A Re-Evaluation <strong>of</strong> <strong>the</strong> Relationship Between Temperature and <strong>the</strong><br />
Critical Oxygen-Tension in Fresh-Water Fishes. Comparative Biochemistry and <strong>Physiology</strong> A-<br />
<strong>Physiology</strong> 67(3), 337-340.<br />
Peyraud-Waitzenegger,M., Soulier,P., 1989. Ventilatory and Circulatory Adjustments in <strong>the</strong> <strong>European</strong> <strong>Eel</strong><br />
(Anguilla-Anguilla L) Exposed to Short-Term Hypoxia. Experimental Biology 48(2), 107-122.<br />
Portner,H.O., 2002. Climate variations and <strong>the</strong> physiological basis <strong>of</strong> temperature dependent biogeography:<br />
systemic to molecular hierarchy <strong>of</strong> <strong>the</strong>rmal tolerance in animals. Comparative Biochemistry and<br />
<strong>Physiology</strong> A-Molecular and Integrative <strong>Physiology</strong> 132(4), 739-761.<br />
Portner,H.O., Farrell,A.P., 2008. ECOLOGY <strong>Physiology</strong> and Climate Change. Science 322(5902), 690-692.<br />
Portner,H.O., Knust,R., 2007. Climate change affects marine fishes through <strong>the</strong> oxygen limitation <strong>of</strong> <strong>the</strong>rmal<br />
tolerance. Science 315(5808), 95-97.<br />
Priede,I.G., 1985. Metabolic scope in fishes. In: Tytler,E.P., Calow,P. (Eds.), Fish energetics: New<br />
perspectives. Croom Helm, London, pp. 33-64.<br />
Richards,J.G., 2009. Metabolic and Molecular Responses <strong>of</strong> Fish to Hypoxia. In: Richards,J.G., Farrell,A.P.,<br />
Brauner,C.J. (Eds.), Hypoxia. Academic Press, Amsterdam, pp. 443-485.<br />
Sadler,K., 1979a. Effects <strong>of</strong> Temperature on <strong>the</strong> Growth and Survival <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong>, Anguilla-<br />
Anguilla l. Journal <strong>of</strong> Fish Biology 15(4), 499-507.<br />
Sadler,K., 1979b. Effects <strong>of</strong> Temperature on <strong>the</strong> Growth and Survival <strong>of</strong> <strong>the</strong> <strong>European</strong> <strong>Eel</strong>, Anguilla-<br />
Anguilla l. Journal <strong>of</strong> Fish Biology 15(4), 499-507.<br />
Scarabello,M., Heigenhauser,G.J.F., Wood,C.M., 1991. The Oxygen Debt Hypo<strong>the</strong>sis in Juvenile Rainbow-<br />
Trout After Exhaustive Exercise. Respiration <strong>Physiology</strong> 84(2), 245-259.<br />
Scarabello,M., Heigenhauser,G.J.F., Wood,C.M., 1992. Gas-Exchange, Metabolite Status and Excess<br />
Postexercise Oxygen-Consumption After Repetitive Bouts <strong>of</strong> Exhaustive Exercise in Juvenile<br />
Rainbow-Trout. Journal <strong>of</strong> Experimental Biology 167, 155-169.<br />
Schurmann,H., Steffensen,J.F., 1997. Effects <strong>of</strong> temperature, hypoxia and activity on <strong>the</strong> metabolism <strong>of</strong><br />
juvenile Atlantic cod. Journal <strong>of</strong> Fish Biology 50(6), 1166-1180.<br />
Steffensen,J.F., 1989. Some Errors in Respirometry <strong>of</strong> Aquatic Brea<strong>the</strong>rs - How to Avoid and Correct for<br />
Them. Fish <strong>Physiology</strong> and Biochemistry 6(1), 49-59.<br />
14
Svendsen,J.C., Steffensen,J.F., Aarestrup,K., Frisk,M., Etzerodt,A., Jyde,M., 2012. Excess posthypoxic<br />
oxygen consumption in rainbow trout (Oncorhynchus mykiss): recovery in normoxia and hypoxia.<br />
Canadian Journal <strong>of</strong> Zoology-Revue Canadienne de Zoologie 90(1), 1-11.<br />
Tuurala,H., Egginton,S., Soivio,A., 1998. Cold exposure increases branchial water-blood barrier thickness in<br />
<strong>the</strong> eel. Journal <strong>of</strong> Fish Biology 53(2), 451-455.<br />
Valverde,J.C., Lopez,F.J.M., Garcia,B.G., 2006. Oxygen consumption and ventilatory frequency responses<br />
to gradual hypoxia in common dentex (Dentex dentex): Basis for suitable oxygen level estimations<br />
1. Aquaculture 256(1-4), 542-551.<br />
van den Thillart,G., Verbeek,R., 1991. Anoxia-Induced Oxygen Debt <strong>of</strong> Goldfish (Carassius-Auratus L)<br />
1. Physiological Zoology 64(2), 525-540.<br />
van Ginneken,V.J.T., Onderwater,M., Olivar,O.L., van den Thillart,G.E.E.J., 2001. Metabolic depression and<br />
investigation <strong>of</strong> glucose/ethanol conversion in <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla Linnaeus 1758)<br />
during anaerobiosis. Thermochimica Acta 373(1), 23-30.<br />
van Waarde,A., van den Thillart,G.E.E.J., Kesbeke,F., 1983. Anaerobic Energy-Metabolism <strong>of</strong> <strong>the</strong> <strong>European</strong><br />
<strong>Eel</strong>, Anguilla-Anguilla l. Journal <strong>of</strong> Comparative <strong>Physiology</strong> 149(4), 469-475.<br />
Virani,N.A., Rees,B.B., 2000. Oxygen consumption, blood lactate and inter-individual variation in <strong>the</strong> gulf<br />
killifish, Fundulus grandis, during hypoxia and recovery. Comparative Biochemistry and <strong>Physiology</strong><br />
A-Molecular and Integrative <strong>Physiology</strong> 126(3), 397-405.<br />
Walsh,P.J., Foster,G.D., Moon,T.W., 1983. The Effects <strong>of</strong> Temperature on Metabolism <strong>of</strong> <strong>the</strong> American <strong>Eel</strong><br />
Anguilla-Rostrata (Lesueur) - Compensation in <strong>the</strong> Summer and Torpor in <strong>the</strong> Winter. Physiological<br />
Zoology 56(4), 532-540.<br />
Walsh,P.J., Moon,T.W., 1982. The Influence <strong>of</strong> Temperature on Extracellular and Intracellular Ph in <strong>the</strong><br />
American <strong>Eel</strong>, Anguilla-Rostrata (Le-Sueur). Respiration <strong>Physiology</strong> 50(2), 129-140.<br />
Weber,R.E., Fago,A., 2004. Functional adaptation and its molecular basis in vertebrate hemoglobins,<br />
neuroglobins and cytoglobins. Respiratory <strong>Physiology</strong> & Neurobiology 144(2-3), 141-159.<br />
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 />
References<br />
Aarestrup, K., Okland, F., Hansen, M.M., Righton, D., Gargan, P., Castonguay, M.,<br />
Bernatchez, L., Howey, P., Sparholt, H., Pedersen, M.I., McKinley, R.S., 2009. Oceanic<br />
spawning migration <strong>of</strong> <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla). Science 325, 1660.<br />
Aho, E., Vornanen, M., 1999. Contractile properties <strong>of</strong> atrial and ventricular myocardium<br />
<strong>of</strong> <strong>the</strong> heart <strong>of</strong> rainbow trout Oncorhynchus mykiss: effects <strong>of</strong> <strong>the</strong>rmal acclimation.<br />
J. Exp. Biol. 202, 2663–2677.<br />
Aho, E., Vornanen, M., 2001. Cold acclimation increases basal heart rate but decreases<br />
its <strong>the</strong>rmal tolerance in rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. B<br />
171, 173–179.<br />
Bailey, J., Sephton, D., Driedzic, W.R., 1991. Impact <strong>of</strong> an acute temperature-change on<br />
performance and metabolism <strong>of</strong> pickerel (Esox niger) and eel (Anguilla rostrata)<br />
hearts. Physiol. Zool. 64, 697–716.<br />
Bailey,J.R.,Barter,T.,Driedzic,W.R.,2000.Maintenance<strong>of</strong>restingtensionin<strong>the</strong><br />
American eel (Anguilla rostrata L.) heart is dependent upon exogenous fuel and<br />
<strong>the</strong> sarcoplasmic reticulum. J. Exp. Zool. 286, 707–717.<br />
Bers, D.M., 2002. Cardiac excitation–contraction coupling. Nature 415, 198–205.<br />
Bertin, L., 1956. <strong>Eel</strong>s: A Biological Study. Cleaver-Hume Press, London.<br />
Driedzic, W.R., Gesser, H., 1988. Differences in force frequency relationships and<br />
calcium dependency between elasmobranch and teleost hearts. J. Exp. Biol. 140,<br />
227–241.<br />
Driedzic, W.R., Bailey, J.R., Sephton, D.H., 1996. Cardiac adaptations to low temperature<br />
non-polar teleost fish. J. Exp. Zool. 275, 186–195.<br />
Farrell, A.P., Jones, D.R., 1992. The heart. Fish Physiol. 12, 1–88.<br />
Farrell, A.P., Gamperl, A.K., Hicks, J.M.T., Shiels, H.A., Jain, K.E., 1996. Maximum cardiac<br />
performance <strong>of</strong> rainbow trout (Oncorhynchus mykiss) at temperatures approaching<br />
<strong>the</strong>ir upper lethal limit. J. Exp. Biol. 199, 663–672.<br />
Gar<strong>of</strong>alo, F., Parisella, M.L., Amelio, D., Tota, B., Imbrogno, S., 2009. Phospholamban<br />
S-nitrosylation modulates starling response in fish heart. Proc. R. Soc. B Biol. Sci.<br />
276, 4043–4052.<br />
Gesser, H., Andresen, P., Brams, P., Sundlaursen, J., 1982. Inotropic effects <strong>of</strong> adrenaline<br />
on <strong>the</strong> anoxic or hypercapnic myocardium <strong>of</strong> rainbow trout and eel. J. Comp. Physiol.<br />
147, 123–128.<br />
Goolish, E.M., 1987. Cold acclimation increases <strong>the</strong> ventricle size <strong>of</strong> carp, Cyprinus<br />
carpio. J. Therm. Biol. 12, 203–205.<br />
Graham, M., Farrell, A., 1985. The seasonal intrinsic cardiac-performance <strong>of</strong> a marine<br />
teleost. J. Exp. Biol. 118, 173–183.<br />
Graham, M.S., Farrell, A.P., 1989. The effect <strong>of</strong> temperature-acclimation and adrenaline<br />
on <strong>the</strong> performance <strong>of</strong> a perfused trout heart. Physiol. Zool. 62, 38–61.<br />
Hovemadsen, L., 1992. The influence <strong>of</strong> temperature on ryanodine sensitivity and <strong>the</strong><br />
force frequency relationship in <strong>the</strong> myocardium <strong>of</strong> rainbow trout. J. Exp. Biol. 167,<br />
47–60.<br />
Hovemadsen, L., Gesser, H., 1989. Force frequency relation in <strong>the</strong> myocardium <strong>of</strong> rainbow<br />
trout — effects <strong>of</strong> K + and adrenaline. J. Comp. Physiol. B 159, 61–69.<br />
Huang, J.B., van Breemen, C., Kuo, K.H., Hove-Madsen, L., Tibbits, G.F., 2006. Store-operated<br />
Ca 2+ entry modulates sarcoplasmic reticulum Ca 2+ loading in neonatal rabbit cardiac<br />
ventricular myocytes. Am. J. Physiol. 290, C1572–C1582.<br />
Imbrogno, S., De Iuri, L., Mazza, R., Tota, B., 2001. Nitric oxide modulates cardiac performance<br />
in <strong>the</strong> heart <strong>of</strong> Anguilla anguilla. J. Exp. Biol. 204, 1719–1727.<br />
Imbrogno, S., Angelone, T., Adamo, C., Pulera, E., Tota, B., Cerra, M.C., 2006. Beta3-<br />
adrenoceptor in <strong>the</strong> eel (Anguilla anguilla) heart: negative inotropy and NO-cGMPdependent<br />
mechanism. J. Exp. Biol. 209, 4966–4973.<br />
Keen, J.E., Vianzon, D.M., Farrell, A.P., Tibbits, G.F., 1993. Thermal acclimation alters<br />
both adrenergic sensitivity and adrenoceptor density in cardiac tissue <strong>of</strong> rainbow<br />
trout. J. Exp. Biol. 181, 27–47.<br />
Kurebayashi, N., Ogawa, Y., 2001. Depletion <strong>of</strong> Ca 2+ in <strong>the</strong> sarcoplasmic reticulum stimulates<br />
Ca 2+ entry into mouse skeletal muscle fibres. J. Physiol. 533, 185–199 London.<br />
Landeira-Fernandez, A.M., Morrissette, J.M., Blank, J.M., Block, B.A., 2004. Temperature<br />
dependence <strong>of</strong> <strong>the</strong> Ca 2+ ‐ATPase (SERCA2) in <strong>the</strong> ventricles <strong>of</strong> tuna and mackerel.<br />
Am. J. Physiol. 286, R398–R404.<br />
Matikainen, N., Vornanen, M., 1992. Effect <strong>of</strong> season and temperature-acclimation on<br />
<strong>the</strong> function <strong>of</strong> crucian carp (Carassius carassius) heart. J. Exp. Biol. 167, 203–220.<br />
McCleave, J.D., 2003. Spawning areas <strong>of</strong> <strong>the</strong> Atlantic eels. In: Aida, K., Tsukamoto, K.,<br />
Yamauchi, K. (Eds.), <strong>Eel</strong> Biology. Springer-Verlag, Tokyo, pp. 141–155.<br />
Nilsson, S., 1983. Autonomic Nerve Function in <strong>the</strong> Vertebrates. Springer-Verlag, Berlin.<br />
Nyman, L., 1972. Some effects <strong>of</strong> temperature on eel Anguilla behavior. <strong>Institut</strong>e <strong>of</strong><br />
Freshwater Research Drottningholm Report, pp. 90–102.<br />
Pennec, J.P., Peyraud, C., 1983. Effects <strong>of</strong> adrenaline on isolated heart <strong>of</strong> <strong>the</strong> eel (Anguilla<br />
anguilla l) during winter. Comp. Biochem. Physiol. C 74, 477–480.<br />
Peyraud-Waitzenegger, M., Bar<strong>the</strong>lemy, L., Peyraud, C., 1980. <strong>Cardio</strong>vascular and<br />
ventilatory effects <strong>of</strong> catecholamines in unrestrained eels (Anguilla anguilla L) —<br />
a study <strong>of</strong> seasonal changes in reactivity. J. Comp. Physiol. 138, 367–375.<br />
Putney, J.W., 1986. A model for receptor-regulated calcium entry. Cell Calcium 7, 1–12.<br />
Rodnick, K.J., Sidell, B.D., 1997. Structural and biochemical analyses <strong>of</strong> cardiac ventricular<br />
enlargement in cold-acclimated striped bass. Am. J. Physiol. 273, R252–R258.<br />
Seibert, H., 1979. Thermal adaptation <strong>of</strong> heart rate and its parasympa<strong>the</strong>tic control in<br />
<strong>the</strong> <strong>European</strong> eel Anguilla anguilla (L). Comp. Biochem. Physiol. C 64, 275–278.<br />
Sephton, D.H., Driedzic, W.R., 1991. Effect <strong>of</strong> acute and chronic temperature transition<br />
on enzymes <strong>of</strong> cardiac metabolism in white perch (Morone americana), yellow perch<br />
(Perca flavescens), and smallmouth bass (Micropterus dolomieui). Can. J. Zool./ Rev.<br />
Can. Zool. 69, 258–262.<br />
Shiels, H.A., Farrell, A.P., 1997. The effect <strong>of</strong> temperature and adrenaline on <strong>the</strong> relative<br />
importance <strong>of</strong> <strong>the</strong> sarcoplasmic reticulum in contributing Ca 2+ to force development<br />
in isolated ventricular trabeculae from rainbow trout. J. Exp. Biol. 200, 1607–1621.<br />
Shiels, H.A., Farrell, A.P., 2000. The effect <strong>of</strong> ryanodine on isometric tension development<br />
in isolated ventricular trabeculae from Pacific mackerel (Scomber japonicus).<br />
Comp. Biochem. Physiol. A 125, 331–341.<br />
Shiels, H.A., Vornanen, M., Farrell, A.P., 2002. The force–frequency relationship in fish<br />
hearts — a review. Comp. Biochem. Physiol. A 132, 811–826.<br />
Shiels, H.A., Vornanen, M., Farrell, A.P., 2003. Acute temperature change modulates<br />
<strong>the</strong> response <strong>of</strong> I-Ca to adrenergic stimulation in fish cardiomyocytes. Physiol.<br />
Biochem. Zool. 76, 816–824.<br />
Skov, P.V., Bushnell, P., Tirsgaard, B., Steffensen, J.F., 2009. The role <strong>of</strong> adrenaline as a<br />
modulator <strong>of</strong> cardiac performance in two Antarctic fishes. Polar Biol. 32, 215–223.<br />
Tiitu, V., Vornanen, M., 2001. Cold adaptation suppresses <strong>the</strong> contractility <strong>of</strong> both atrial<br />
and ventricular muscle <strong>of</strong> <strong>the</strong> crucian carp heart. J. Fish Biol. 59, 141–156.<br />
Tsukuda, H., Liu, B., Fujii, K., 1985. Pulsation rate and oxygen-consumption <strong>of</strong> isolated<br />
hearts <strong>of</strong> <strong>the</strong> goldfish, Carassius auratus, acclimated to different temperatures. Comp.<br />
Biochem. Physiol. A 82, 281–283.
C. Methling et al. / Comparative Biochemistry and <strong>Physiology</strong>, Part A 163 (2012) 66–73<br />
73<br />
Vornanen, M., 1989. Regulation <strong>of</strong> contractility <strong>of</strong> <strong>the</strong> fish (Carassius carassius L) heart<br />
ventricle. Comp. Biochem. Physiol. C 94, 477–483.<br />
Vornanen, M., 1994. Seasonal and temperature-induced changes in myosin heavychain<br />
composition <strong>of</strong> crucian carp hearts. Am. J. Physiol. 267, R1567–R1573.<br />
Vornanen, M., 1998. L-type Ca 2+ current in fish cardiac myocytes: effects <strong>of</strong> <strong>the</strong>rmal<br />
acclimation and beta-adrenergic stimulation. J. Exp. Biol. 201, 533–547.<br />
Vornanen, M., 1999. Na + /Ca 2(+) exchange current in ventricular myocytes <strong>of</strong> fish<br />
heart: contribution to sarcolemmal Ca 2+ influx. J. Exp. Biol. 202, 1763–1775.<br />
Vornanen, M., Shiels, H.A., Farrell, A.P., 2002. Plasticity <strong>of</strong> excitation–contraction coupling<br />
in fish cardiac myocytes. Comp. Biochem. Physiol. A 132, 827–846.<br />
Walsh, P.J., Foster, G.D., Moon, T.W., 1983. The effects <strong>of</strong> temperature on metabolism <strong>of</strong><br />
<strong>the</strong> American eel Anguilla rostrata (LeSueur) — compensation in <strong>the</strong> summer and<br />
torpor in <strong>the</strong> winter. Physiol. Zool. 56, 532–540.<br />
Woo, S.H., Morad, R., 2001. Bimodal regulation <strong>of</strong> Na + –Ca 2+ exchanger by betaadrenergic<br />
signaling pathway in shark ventricular myocytes. Proc. Natl. Acad. Sci.<br />
U.S.A. 98, 2023–2028.
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 />
References<br />
Axelsson, M. and Fritsche, R. (1991). Effects <strong>of</strong> Exercise, Hypoxia and Feeding on <strong>the</strong> Gastrointestinal<br />
Blood-Flow in <strong>the</strong> Atlantic Cod Gadus-Morhua. Journal <strong>of</strong> Experimental Biology, 158, 181-198.<br />
Baker, D. W., Matey, V., Huynh, K. T., Wilson, J. M., Morgan, J. D., and Brauner, C. J. (2009). Complete<br />
intracellular pH protection during extracellular pH depression is associated with hypercarbia<br />
tolerance in white sturgeon, Acipenser transmontanus. American Journal <strong>of</strong> <strong>Physiology</strong>-Regulatory<br />
Integrative and Comparative <strong>Physiology</strong>, 296, R1868-R1880.<br />
Beamish, F. W. H. and Trippel, E. A. (1990). Heat Increment - A Static Or Dynamic Dimension in<br />
Bioenergetic Models. Transactions <strong>of</strong> <strong>the</strong> American Fisheries Society, 119, 649-661.<br />
Bower, C. E. and Holm-Hansen, T. (1980). A Salicylate-Hypochlorite Method for Determining Ammonia in<br />
Seawater. Canadian Journal <strong>of</strong> Fisheries and Aquatic Sciences, 37, 794-798.<br />
Brauner, C. J., Wang, T., Wang, Y., Richards, J. G., Gonzalez, R. J., Bernier, N. J. et al. (2004). Limited<br />
extracellular but complete intracellular acid-base regulation during short-term environmental<br />
hypercapnia in <strong>the</strong> armoured catfish, Liposarcus pardalis. Journal <strong>of</strong> Experimental Biology, 207,<br />
3381-3390.<br />
Bridges, C. R., Hlastala, M. P., Riepl, G., and Scheid, P. (1983). Root Effect Induced by Co2 and by Fixed<br />
Acid in <strong>the</strong> Blood <strong>of</strong> <strong>the</strong> <strong>Eel</strong>, Anguilla-Anguilla. Respiration <strong>Physiology</strong>, 51, 275-286.<br />
Brown, C. R. and Cameron, J. N. (1991). The Relationship Between Specific Dynamic Action (Sda) and<br />
Protein-Syn<strong>the</strong>sis Rates in <strong>the</strong> Channel Catfish. Physiological Zoology, 64, 298-309.<br />
Buchmann, K. and Bjerregaard, J. (1990). Comparative Efficacies <strong>of</strong> Commercially Available<br />
Benzimidazoles Against Pseudodactylogyrus Infestations in <strong>Eel</strong>s. Diseases <strong>of</strong> Aquatic Organisms, 9,<br />
117-120.<br />
Cecchini, S., Saroglia, M., Caricato, G., Terova, G., and Sileo, L. (2001). Effects <strong>of</strong> graded environmental<br />
hypercapnia on sea bass (Dicentrarchus labrax L.) feed intake and acid-base balance. Aquaculture<br />
Research, 32, 499-502.<br />
Chabot, D. and Claireaux, G. (2008). Quantification <strong>of</strong> SMR and SDA in aquatic animals using quantiles and<br />
non-linear quantile regression. Comparative Biochemistry and <strong>Physiology</strong> A-Molecular and<br />
Integrative <strong>Physiology</strong>, 150, S99.<br />
Claiborne, J. B. and Heisler, N. (1986). Acid-Base Regulation and Ion Transfers in <strong>the</strong> Carp (Cyprinus-<br />
Carpio) - Ph Compensation During Graded Long-Term and Short-Term Environmental Hypercapnia,<br />
and <strong>the</strong> Effect <strong>of</strong> Bicarbonate Infusion. Journal <strong>of</strong> Experimental Biology, 126, 41-61.<br />
Crocker, C. E. and Cech, J. J. (1996). The effects <strong>of</strong> hypercapnia on <strong>the</strong> growth <strong>of</strong> juvenile white sturgeon,<br />
Acipenser transmontanus. Aquaculture, 147, 293-299.<br />
Crocker, C. E., Farrell, A. P., Gamperi, A. K., and Cech, J. J. (2000). <strong>Cardio</strong><strong>respiratory</strong> responses <strong>of</strong> white<br />
sturgeon to environmental hypercapnia. American Journal <strong>of</strong> <strong>Physiology</strong>-Regulatory Integrative and<br />
Comparative <strong>Physiology</strong>, 279, R617-R628.<br />
CruzNeto, A. P. and Steffensen, J. F. (1997). The effects <strong>of</strong> acute hypoxia and hypercapnia on oxygen<br />
consumption <strong>of</strong> <strong>the</strong> freshwater <strong>European</strong> eel. Journal <strong>of</strong> Fish Biology, 50, 759-769.<br />
Danley, M. L., Kenney, P. B., Mazik, P. M., Kiser, R., and Hankins, J. A. (2005). Effects <strong>of</strong> carbon dioxide<br />
exposure on intensively cultured rainbow trout Oneorhynchus mykiss: Physiological responses and<br />
fillet attributes. Journal <strong>of</strong> <strong>the</strong> World Aquaculture Society, 36, 249-261.<br />
16
Deigweiher, K., Koschnick, N., Portner, H. O., and Lucassen, M. (2008). Acclimation <strong>of</strong> ion regulatory<br />
capacities in gills <strong>of</strong> marine fish under environmental hypercapnia. American Journal <strong>of</strong> <strong>Physiology</strong>-<br />
Regulatory Integrative and Comparative <strong>Physiology</strong>, 295, R1660-R1670.<br />
Engin, K. and Carter, C. G. (2001). Ammonia and urea excretion rates <strong>of</strong> juvenile Australian short-finned eel<br />
(Anguilla australis australis) as influenced by dietary protein level. Aquaculture, 194, 123-136.<br />
Farrell, A. P. and Lutz, P. L. (1975). Apparent Anion Imbalance in Fresh-Water Adapted <strong>Eel</strong><br />
1. Journal <strong>of</strong> Comparative <strong>Physiology</strong>, 102, 159-166.<br />
Fivelstad, S., Olsen, A. B., Asgard, T., Baeverfjord, G., Rasmussen, T., et al. (2003). Long-term sublethal<br />
effects <strong>of</strong> carbon dioxide on Atlantic salmon smolts (Salmo salar L.): ion regulation, haematology,<br />
element composition, nephrocalcinosis and growth parameters. Aquaculture, 215, 301-319.<br />
Fivelstad, S., Waagbo, R., Stefansson, S., and Olsen, A. B. (2007). Impacts <strong>of</strong> elevated water carbon dioxide<br />
partial pressure at two temperatures on Atlantic salmon (Salmo salar L.) parr growth and<br />
haematology. Aquaculture, 269, 241-249.<br />
Foss, A., Rosnes, B. A., and Oiestad, V. (2003). Graded environmental hypercapnia in juvenile spotted<br />
wolffish (Anarhichas minor Olafsen): effects on growth, food conversion efficiency and<br />
nephrocalcinosis. Aquaculture, 220, 607-617.<br />
Gesser, H., Andresen, P., Brams, P., and Sundlaursen, J. (1982). Inotropic Effects <strong>of</strong> Adrenaline on <strong>the</strong><br />
Anoxic Or Hypercapnic Myocardium <strong>of</strong> Rainbow-Trout and <strong>Eel</strong>. Journal <strong>of</strong> Comparative<br />
<strong>Physiology</strong>, 147, 123-128.<br />
Gnaiger, E. (1983). Calculations <strong>of</strong> energetic and biochemical equivalents <strong>of</strong> respirometry oxygen<br />
consumption. In E.Gnaiger and H. Forstener (Eds.), Polarographic Oxygen Sensors (pp. 337-345).<br />
Berlin: Springer.<br />
Gnaiger, E. and Bitterlich, G. (1984). Proximate Biochemical-Composition and Caloric Content Calculated<br />
from Elemental Chn Analysis - A Stoichiometric Concept. Oecologia, 62, 289-298.<br />
Heinsbroek, L. T. N., Van Ho<strong>of</strong>f, P. L. A., Swinkels, W., Tanck, M. W. T., Schrama, J. W., and Verreth, J.<br />
A. J. (2007). Effects <strong>of</strong> feed composition on life history developments in feed intake, metabolism,<br />
growth and body composition <strong>of</strong> <strong>European</strong> eel, Anguilla anguilla. Aquaculture, 267, 175-187.<br />
Heinsbroek, L. T. N., van Thoor, R. M. H., and Elizondo, L. J. (1991). The effect <strong>of</strong> feeding level on <strong>the</strong><br />
apparant digestibilities <strong>of</strong> nutrients and energy <strong>of</strong> a reference diet for <strong>the</strong> <strong>European</strong> eel, Anguilla<br />
anguilla L., and <strong>the</strong> African catfish ( Clarias gariepinus Burchell). In (pp. 175-188).<br />
Heisler, N. (1982). Intracellular and Extracellular Acid-Base Regulation in <strong>the</strong> Tropical Fresh-Water Teleost<br />
Fish Synbranchus-Marmoratus in Response to <strong>the</strong> Transition from Water Breathing to Air Breathing<br />
1. Journal <strong>of</strong> Experimental Biology, 99, 9-28.<br />
Heisler, N. (1984). Acid-base regulation in fishes. In W.S.Hoar and D. J. Randall (Eds.), Gills: Anatomy,<br />
Gas Transfer, and Acid-Base Regulation (pp. 315-401). New York: Academic Press.<br />
Heisler, N. (1993). Acid-base regulation. In D.H.Evans (Ed.), The physiology <strong>of</strong> fishes (pp. 343-378).<br />
Hyde, D. A. and Perry, S. F. (1989). Differential Approaches to Blood Acid-Base Regulation During<br />
Exposure to Prolonged Hypercapnia in 2 Fresh-Water Teleosts - <strong>the</strong> Rainbow-Trout (Salmo-<br />
Gairdneri) and <strong>the</strong> American <strong>Eel</strong> (Anguilla-Rostrata). Physiological Zoology, 62, 1164-1186.<br />
Ishimatsu, A., Hayashi, M., and Kikkawa, T. (2008). Fishes in high-CO2, acidified oceans<br />
3. Marine Ecology-Progress Series, 373, 295-302.<br />
Ishimatsu, A., Hayashi, M., Lee, K. S., Kikkawa, T., and Kita, J. (2005). Physiological effects on fishes in a<br />
high-CO2 world. Journal <strong>of</strong> Geophysical Research-Oceans, 110.<br />
Jobling, M. (1981). The Influences <strong>of</strong> Feeding on <strong>the</strong> Metabolic-Rate <strong>of</strong> Fishes - A Short Review<br />
1. Journal <strong>of</strong> Fish Biology, 18, 385-400.<br />
Jordan, A. D. and Steffensen, J. F. (2007). Effects <strong>of</strong> ration size and hypoxia on specific dynamic action in<br />
<strong>the</strong> cod. Physiological and Biochemical Zoology, 80, 178-185.<br />
Jourdan-Pineau, H., Dupont-Prinet, A., Claireaux, G., and McKenzie, D. J. (2010). An Investigation <strong>of</strong><br />
Metabolic Prioritization in <strong>the</strong> <strong>European</strong> Sea Bass, Dicentrarchus labrax. Physiological and<br />
Biochemical Zoology, 83, 68-77.<br />
Langenbuch, M. and Portner, H. O. (2002). Changes in metabolic rate and N excretion in <strong>the</strong> marine<br />
invertebrate Sipunculus nudus under conditions <strong>of</strong> environmental hypercapnia: identifying effective<br />
acid-base variables. Journal <strong>of</strong> Experimental Biology, 205, 1153-1160.<br />
17
Langenbuch, M. and Portner, H. O. (2003). Energy budget <strong>of</strong> hepatocytes from Antarctic fish (Pachycara<br />
brachycephalum and Lepidonoto<strong>the</strong>n kempi) as a function <strong>of</strong> ambient CO2: pH-dependent<br />
limitations <strong>of</strong> cellular protein biosyn<strong>the</strong>sis?. Journal <strong>of</strong> Experimental Biology, 206, 3895-3903.<br />
Leung, K. M. Y., Chu, J. C. W., and Wu, R. S. S. (1999). Effects <strong>of</strong> body weight, water temperature and<br />
ration size on ammonia excretion by <strong>the</strong> areolated grouper (Epinephelus areolatus) and mangrove<br />
snapper (Lutjanus argentimaculatus). Aquaculture, 170, 215-227.<br />
McCue, M. D. (2006). Specific dynamic action: A century <strong>of</strong> investigation<br />
2. Comparative Biochemistry and <strong>Physiology</strong> A-Molecular and Integrative <strong>Physiology</strong>, 144, 381-<br />
394.<br />
McKenzie, D. J., Piccolella, M., la Valle, A. Z., Taylor, E. W., Bolis, C. L., and Steffensen, J. F. (2003).<br />
Tolerance <strong>of</strong> chronic hypercapnia by <strong>the</strong> <strong>European</strong> eel Anguilla anguilla. Journal <strong>of</strong> Experimental<br />
Biology, 206, 1717-1726.<br />
McKenzie, D. J., Taylor, E. W., la Valle, A. Z., and Steffensen, J. F. (2002). Tolerance <strong>of</strong> acute hypercapnic<br />
acidosis by <strong>the</strong> <strong>European</strong> eel (Anguilla anguilla). Journal <strong>of</strong> Comparative <strong>Physiology</strong> B-Biochemical<br />
Systemic and Environmental <strong>Physiology</strong>, 172, 339-346.<br />
Methling, C., Steffensen, J. F., and Skov, P. V. (2012). The temperature challenges on cardiac performance<br />
in winter-quiescent and migration-stage eels Anguilla anguilla. Comparative Biochemistry and<br />
<strong>Physiology</strong> A-Molecular and Integrative <strong>Physiology</strong>, 163, 66-73.<br />
Moran, D. and Stottrup, J. G. (2011). The effect <strong>of</strong> carbon dioxide on growth <strong>of</strong> juvenile Atlantic cod Gadus<br />
morhua L. Aquatic Toxicology, 102, 24-30.<br />
Owen, S. F. (2001). Meeting energy budgets by modulation <strong>of</strong> behaviour and physiology in <strong>the</strong> eel (Anguilla<br />
anguilla L.). Comparative Biochemistry and <strong>Physiology</strong> A-Molecular and Integrative <strong>Physiology</strong>,<br />
128, 631-644.<br />
Owen, S. F., Houlihan, D. F., Rennie, M. J., and van Weerd, J. H. (1998). Bioenergetics and nitrogen balance<br />
<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 />
Fisheries and Aquatic Sciences, 55, 2365-2375.<br />
Petochi, T., Di Marco, P., Priori, A., Finoia, M. G., Mercatali, I., and Marino, G. (2011). Coping strategy and<br />
stress response <strong>of</strong> <strong>European</strong> sea bass Dicentrarchus labrax to acute and chronic environmental<br />
hypercapnia under hyperoxic conditions. Aquaculture, 315, 312-320.<br />
Portner, H. O. (2008). Ecosystem effects <strong>of</strong> ocean acidification in times <strong>of</strong> ocean warming: a physiologist's<br />
view. Marine Ecology-Progress Series, 373, 203-217.<br />
Portner, H. O., Langenbuch, M., and Reipschlager, A. (2004). Biological impact <strong>of</strong> elevated ocean CO2<br />
concentrations: Lessons from animal physiology and earth history. Journal <strong>of</strong> Oceanography, 60,<br />
705-718.<br />
Secor, S. M. (2009). Specific dynamic action: a review <strong>of</strong> <strong>the</strong> postprandial metabolic response<br />
2. Journal <strong>of</strong> Comparative <strong>Physiology</strong> B-Biochemical Systemic and Environmental <strong>Physiology</strong>, 179,<br />
1-56.<br />
Steffensen, J. F. (1989). Some Errors in Respirometry <strong>of</strong> Aquatic Brea<strong>the</strong>rs - How to Avoid and Correct for<br />
Them. Fish <strong>Physiology</strong> and Biochemistry, 6, 49-59.<br />
Steffensen, J. F. and Lomholt, J. P. (1990). Accumulation <strong>of</strong> carbon dioxide in fish farms with recirculating<br />
water. In R.C.Ryans (Ed.), Fish physiolohy, fish toxicology and fisheries management (pp. 157-161).<br />
A<strong>the</strong>ns: Environmental Research Laboratories -U.S. Environmental Protection Agency, EPA/600/9-<br />
90/011.<br />
Wood, C. M. (2001). Influence <strong>of</strong> feeding, exercise, and temperature on nitrogen metabolism and excretion.<br />
In P.A.Wrigth and P. M. Anderson (Eds.), Nitrogen excretion (pp. 201-238). New York: Academic<br />
press.<br />
Zhang, W., Cao, Z. D., Peng, J. L., Chen, B. J., and Fu, S. J. (2010). The effects <strong>of</strong> dissolved oxygen level on<br />
<strong>the</strong> metabolic interaction between digestion and locomotion in juvenile sou<strong>the</strong>rn catfish (Silurus<br />
meridionalis Chen). Comparative Biochemistry and <strong>Physiology</strong> A-Molecular and Integrative<br />
<strong>Physiology</strong>, 157, 212-219.<br />
Zhou, B. S., Wu, R. S. S., Randall, D. J., and Lam, P. K. S. (2001). Bioenergetics and RNA/DNA ratios in<br />
<strong>the</strong> common carp (Cyprinus carpio) under hypoxia. Journal <strong>of</strong> Comparative <strong>Physiology</strong> B-<br />
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 />
PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20797
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 />
PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e20797
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 />
PLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20797
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 />
PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e20797
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 />
References<br />
1. McCleave JD, Harden Jones FR (1979) <strong>Eel</strong>s-New Interest in An Old Problem.<br />
Nature 278: 782–783.<br />
2. Schmidt J (1923) Breeding places and migrations <strong>of</strong> <strong>the</strong> eel. Nature 111: 51–54.<br />
3. Schmidt J (1925) The breeding places <strong>of</strong> <strong>the</strong> eel. Smithsonian Report for 1924.<br />
pp 279–316.<br />
4. Castonguay M, Hodson PV, Moriarty C, Drinkwater KF, Jessop BM (1994) Is<br />
<strong>the</strong>re a role <strong>of</strong> ocean environment in American and <strong>European</strong> eel decline?<br />
Fisheries Oceanography 3: 197–203.<br />
5. van den Thillart GEEJ, van Ginneken VJT, Korner F, Heijmans R, van der<br />
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 />
7. van Ginneken VJT, van den Thillart GEEJ (2000) <strong>Physiology</strong>-<strong>Eel</strong> fat stores are<br />
enough to reach <strong>the</strong> Sargasso. Nature 403: 156–157.<br />
8. van Ginneken VJT, Antonissen E, Muller UK, Booms R, Eding E, et al. (2005)<br />
<strong>Eel</strong> migration to <strong>the</strong> Sargasso: remarkably high swimming efficiency and low<br />
energy costs. Journal <strong>of</strong> Experimental Biology 208: 1329–1335.<br />
9. Fricke H, Kaese R (1995) Tracking <strong>of</strong> Artificially Matured <strong>Eel</strong>s (Anguilla-<br />
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 />
10. McCleave JD, Arnold GP (1999) Movements <strong>of</strong> yellow- and silver-phase<br />
<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 />
anguilla L.) from <strong>the</strong> continental shelf to <strong>the</strong> deep sea. Aquatic Living Resources<br />
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 />
14. Block BA, Teo SLH, Walli A, Boustany A, Stokesbury MJW, et al. (2005)<br />
Electronic tagging and population structure <strong>of</strong> Atlantic bluefin tuna. Nature 434:<br />
1121–1127.<br />
15. Bonfil R, Meyer M, Scholl MC, Johnson R, O’Brien S, et al. (2005)<br />
Transoceanic migration, spatial dynamics, and population linkages <strong>of</strong> white<br />
sharks. Science 310: 100–103.<br />
16. Marcinek DJ, Blackwell SB, Dewar H, Freund EV, Farwell C, et al. (2001)<br />
Depth and muscle temperature <strong>of</strong> Pacific bluefin tuna examined with acoustic<br />
and pop-up satellite archival tags. Marine Biology 138: 869–885.<br />
17. Seitz AC, Weng KC, Boustany AM, Block BA (2002) Behaviour <strong>of</strong> a sharptail<br />
mola in <strong>the</strong> Gulf <strong>of</strong> Mexico. Journal <strong>of</strong> Fish Biology 60: 1597–1602.<br />
18. Weng KC, Block BA (2004) Diel vertical migration <strong>of</strong> <strong>the</strong> bigeye thresher shark<br />
(Alopias superciliosus), a species possessing orbital retia mirabilia. Fishery<br />
Bulletin 102: 221–229.<br />
19. Wilson SG, Lutcavage ME, Brill RW, Genovese MP, Cooper AB, et al. (2005)<br />
Movements <strong>of</strong> bluefin tuna (Thunnus thynnus) in <strong>the</strong> northwestern Atlantic<br />
Ocean recorded by pop-up satellite archival tags. Marine Biology 146: 409–423.<br />
20. Jellyman D, Tsukamoto K (2002) First use <strong>of</strong> archival transmitters to track<br />
migrating freshwater eels Anguilla dieffenbachii at sea. Marine Ecology-Progress<br />
Series 233: 207–215.<br />
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 />
29. Beamish FWH (1978) Swimming capacity. In: Randall DJ, Randall DJ, eds. Fish<br />
<strong>Physiology</strong> vol 7. New York: Academic Press. pp 101–187.<br />
30. Brett JR (1964) The Respiratory Metabolism and Swimming Performance <strong>of</strong><br />
Young Sockeye Salmon. Journal <strong>of</strong> <strong>the</strong> Fisheries Research Board <strong>of</strong> Canada 21:<br />
1183–1226.<br />
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 />
Development in Oscillating Foils with Application to Fish Propulsion. Journal <strong>of</strong><br />
Fluids and Structures 7: 205–224.<br />
33. Triantafyllou MS, Triantafyllou GS, Yue DKP (2000) Hydrodynamics <strong>of</strong> fishlike<br />
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 />
Journal <strong>of</strong> Fish Biology 34: 19–24.<br />
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 />
Journal <strong>of</strong> Fish Biology 25: 577–585.<br />
37. Mellas EJ, Haynes JM (1985) Swimming Performance and Behavior <strong>of</strong><br />
Rainbow-Trout (Salmo-Gairdneri) and White Perch (Morone-Americana)-<br />
Effects <strong>of</strong> Attaching Telemetry Transmitters. Canadian Journal <strong>of</strong> Fisheries<br />
and Aquatic Sciences 42: 488–493.<br />
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 />
swimming speed <strong>of</strong> yellow- and silver-phase <strong>European</strong> eel (Anguilla anguilla, L.).<br />
Journal <strong>of</strong> Applied Ichthyology 26: 432–435.<br />
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 />
43. Randall D, Brauner C (1991) Effects <strong>of</strong> Environmental-Factors on Exercise in<br />
Fish. Journal <strong>of</strong> Experimental Biology 160: 113–126.<br />
44. Haro AJ (1991) Thermal Preferenda and Behavior <strong>of</strong> Atlantic <strong>Eel</strong>s (Genus<br />
Anguilla) in Relation to Their Spawning Migration. Environmental Biology <strong>of</strong><br />
Fishes 31: 171–184.<br />
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 />
smallmouth bass (Micropterus dolomieu). Journal <strong>of</strong> Experimental Biology 207:<br />
1563–1575.<br />
46. Tudorache C, Viaenen P, Blust R, De Boeck G (2007) Longer flumes increase<br />
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 />
47. Tudorache C, O’K Eefe RA, Benfey TJ (2010) Flume length and post-exercise<br />
impingement affect anaerobic metabolism in brook charr Salvelinus fontinalis.<br />
Journal <strong>of</strong> Fish Biology 76: 729–733.<br />
48. Svedang H, Wickstrom H (1997) Low fat contents in female silver eels:<br />
Indications <strong>of</strong> insufficient energetic stores for migration and gonadal development.<br />
Journal <strong>of</strong> Fish Biology 50: 475–486.<br />
49. Van den Thillart G, Van Ginneken V, Korner F, Heijmans R, Van der<br />
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 />
50. Moser M, Olson A, Quinn T (1990) Effects <strong>of</strong> dummy ultrasonic transmitters on<br />
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 />
53. Culik B, Wilson RP (1991) Swimming Energetics and Performance <strong>of</strong><br />
Instrumented Adelie Penguins (Pygoscelis-Adeliae). Journal <strong>of</strong> Experimental<br />
Biology 158: 355–368.<br />
54. Ropert-Coudert Y, Wilson RP, Yoda K, Kato A (2007) Assessing performance<br />
constraints in penguins with externally-attached devices. Marine Ecology-<br />
Progress Series 333: 281–289.<br />
55. Grusha DS (2005) Investigation <strong>of</strong> <strong>the</strong> life history <strong>of</strong> <strong>the</strong> cownose ray Rhinoptera<br />
bongsus (Mitchill 1815) [dissertation]. The College <strong>of</strong> William and Mary in<br />
Virginia.<br />
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