Aquaculture 236 (2004) 659 – 675 Www.elsevier.com/Locate/Aqua-Online

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Tolerance, growth and haloplasticity of the

Atlantic wolffish (Anarhichas lupus)

exposed to various salinities

N.R. Le Franc

oisa,b,

*, S.G. Lamarrea,b

, P.U. Bliera

aLaboratoire de biologie evolutive, Departement de biologie, Universite du Quebec a Rimouski (UQAR),

300 Allee des Ursulines, Rimouski, Quebec, Canada G5L 3A1bMinistere de lAgriculture, des Pecheries et de lAlimentation du Quebec (MAPAQ),

Centre Aquacole Marin de Grande-Riviere, 6, rue du Parc, Grande-Riviere, Quebec, Canada G0C 1V0

Received 28 August 2003; received in revised form 14 February 2004; accepted 24 February 2004

Abstract

Tolerance, growth, ionic and osmotic response and gill enzymatic activity adjustments

(Na+K+ATPase, COX, CS, PK and LDH) of Atlantic wolffish ( Anarhichas lupus) exposed to

different salinities (0x, 7x, 14x, 21x, 28xand 35x) were examined. Direct transfer

from seawater (SW) to freshwater (FW) of 0+ juveniles situated the L50 for salinity tolerance

between 5xand 6x. The data from prolonged exposure to various salinities indicate efficient

homeostatic control in Atlantic wolffish A. lupus and improved growth performance at 14x

compared to 28xafter 70 days (calculated growth trajectories in length and in weight). The

latter is linked to indications of reduced metabolic costs of ion regulation, i.e. significantly

reduced Na+K+ATPase and LDH activity at 14xcompared to 28x(Na+K+ATPase: 5.67F 1.41

compared to 7.27F 1.23 and 5.44F 0.86 compared to 6.86F 1.34 U/mg protein at weeks 14

and 19, respectively, p = 0.05 and 0.026, respectively; LDH: 10.4F

2.14 compared to12.39F 2.02 U/mg protein at week 14 and 12.46F 1.34 compared to 17.17F 2.67 U/mg

protein for LDH, p = 0.026 and 0.008, respectively). A strong positive relationship between

Na+K+ATPase, LDH and PK activity was also observed [log y = 1.107log LDH 0.914,

p = 0.0000, r2 =0.46 (n = 33) at weeks 14 and 19 and with PK (n = 18) at week 19, log y = 1.143

log PK 0.595, p = 0.001, r2 = 0.497]. This indicates that from week 14 onward, the activity of

branchial Na+K+ATPase is coupled to adjustments in the level of activity of the glycolytic

enzymes. Our results indicate that Atlantic wolffish is a strong osmoregulator that demonstrates

0044-8486/$ - see front matterD 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2004.02.021

* Corresponding author. UQAR/MAPAQ, Centre Aquacole Marin, 6, rue du Parc, Grande-Riviere, Quebec,

Canada G0C 1V0. Tel.: +1-418-385-2251(222); fax: +1-418-385-3343.

E-mail address: [email protected] (N.R. Le Francois).

www.elsevier.com/locate/aqua-online

Aquaculture 236 (2004) 659675


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haloplasticity which enables cultivation of this species at reduced salinities with expected benefits

in productivity.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Atlantic wolffish; Metabolic enzyme; Na+K+ATPase; Haloplasticity and growth

1. Introduction

The wolffishes are part of a small family of marine fishes inhabiting moderately

deep waters of the North Atlantic and North Pacific oceans (amphiboreal distribution).

They are carnivorous, consuming a variety of bottom-living crustaceans and inverte-

brates. Some species are of commercial importance especially those from the Atlantic

Ocean and Barents Sea (Scott and Scott, 1988). Two of the three members of the

Anarhichadidae found in the Atlantic area, the Atlantic wolffish ( Anarhichas lupus)

and the spotted wolffish ( Anarhichas minor), have been recently listed as species at

risk in Canadian waters (COSEWIC, 2002; Kulka, 2002). Biological and technical

efforts aimed at the emergence of a sustainable mariculture industry based on those

species are actually being applied in the northern hemisphere (Le Francois et al., 2002;

Falk-Petersen et al., 1999). More specifically, the cultivation potential of both species

( A. lupus and A. minor) is being evaluated in the eastern coastal regions of Quebec,

Canada, through a series of concerted actions aimed at the diversification of itsmariculture industry. The maritime regions of Quebec are, however, characterised by

local and large-scale seasonal variations in salinity and temperature along the axis of

the St-Lawrence estuary where most of the potential sites for mariculture are located,

implying careful selection of candidate species which are tolerant of cold-water and

salinity variations (Le Francois et al., 2002).

In the wild, adults are known to frequent the coastal environment for malefemale

pairing in the spring, in the fall for spawning (Templeman, 1986) and for the duration

of the egg mass incubation period (c 900j/days) and early juvenile residency (Keats

et al., 1985). Efficient osmoregulatory strategies will therefore be of importance in the

regulation of salts during these prolonged exposures to varying salinities. In general,marine fish do not tolerate prolonged exposure to FW. However, several species of

marine fish (Sparus sarba: Wu and Woo, 1983; Woo and Kelly, 1995; Gadus morhua:

Dutil et al., 1992, 1997; Mylio macrocephalus: Kelly et al., 1999a; Scophtalmus

maximus: Imsland et al., 2001, 2002; Dicentrarchus labrax: Saillant et al., 2003;

Jensen et al., 1998; A. minor: Foss et al., 2001) have displayed good tolerance to low

salinities.

Despite inferior growth performance under cultivation conditions (Moksness, 1994)

compared to the spotted wolffish, the Atlantic wolffish remains a species of interest for

mariculture in a North American context for several reasons: (1) it is a coastal species

that is a priori more tolerant than the spotted wolffish to the low and fluctuatingsalinity and temperature that characterise the estuarine coastal areas; (2) known ability

to synthesize antifreeze proteins indicates that this species could be a good candidate

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for sea cage farming operations in subzero environments (northwest Atlantic coastal

regions) and biomass valorization (Le Francois and Blier, 2002); and (3) offers good

potential for knowledge transfer towards spotted wolffish (physiology of reproduction,

nutrition and rearing technology) (Oiestad, 1999).The key enzyme of osmoregulation, Na+K+ATPase is located at the level of the chloride

cells (CC) of the gills. Those cells, rich in mitochondria in order to fulfil the high energetic

demand of the gill tissue, are the site for effective excretion of monovalent ions (Na+, Cl) of

fish adapted to SW. Na+K+ATPase is active in the absorption of ions in FW and excretion in

SW (McCormick, 1995), and its activity is generally correlated to the number of CC (Perry

and Walsh, 1989). In salmonids, Na+K+ATPase activity is directly related to salinity

elevation (Atlantic salmon, Salmo salar: Langdon and Thorpe, 1985; rainbow trout,

Oncorhynchus mykiss: Madsen and Naamansen, 1989; brook charr, Salvelinus fontinalis:

Le Francois and Blier, 2000), whereas in marine fish, Na+K+ATPase activity level seems

dictated by the numerical gradient between internal and external osmolality (370480 and

8001200 mOsm kg 1, respectively; Gaumet et al., 1995; Jensen et al., 1998). A minimal

Na+K+ATPase activity value should occur around an internal osmolality value between

10xand 15x(isoosmotic salinity) (Brett, 1979).

It has been proposed that the energetic cost for homeostasis (i.e. ionic and osmotic

regulation) is reduced in isoosmotic environments and that these energy savings are

translated in growth enhancement (Lambert et al., 1994; Gaumet et al., 1995; Woo and

Kelly, 1995; Dutil et al., 1997; Boeuf and Payan, 2001; Imsland et al., 2001). We

propose that the growth rate of juvenile Atlantic wolffish may be limited by the energy

cost associated with growth and maintenance processes (Blier et al., 1997; Lemieux etal., 2003) and energy expenditure linked to osmoregulatory processes which for gill

tissue can account for around 4% of the animals total energy budget (Kirschner, 1993;

Morgan and Iwama, 1999). Few studies have linked growth enhancement of a marine

fish species at lower salinities to adjustments in the level of activity of Na+K+ATPase

and key-metabolic enzyme activities.

In hypoosmotic environments, fish are in hyperosmoregulatory mode, trying to

maintain their internal osmolality at values higher than the ambient environment. At

the opposite, in hypersaline waters, fish try to maintain their internal osmolality at levels

inferior to the external environment (hypoosmoregulatory mode). These regulatory

mechanisms at the gill level require substantial amounts of energy (Kirschner, 1993).Many biochemical studies suggest that much of gill tissue metabolism is attributable to

the oxidative demands of the chloride cells, relying mainly on glucose and lactate as

metabolic fuels (Mommsen, 1984; Perry and Walsh, 1989; Soengas et al., 1995; Morgan

and Iwama, 1999). In salmonids, several enzymes of the branchial intermediary

metabolism display a pronounced elevation of their level of activity in relation to

increases in salinity or during smoltification (Langdon and Thorpe, 1985; McCormick et

al., 1989; Soengas et al., 1995). A recent study on 1+ brook charr (Le Francois and

Blier, 2003) revealed a clear elevation of Na+K+ATPase activity following prolonged

exposure to SW that was not correlated with a proportional elevation in activity levels of

key enzymes of energy metabolism (LDH, PK, CS and COX). Kelly et al. (1999b),experimenting on sea bream, reported the lowest isocitrate dehydrogenase (ICDH) and

Na+K+ATPase activities at isoosmotic salinity and highest at extreme salinities, while

N.R. Le Francois et al. / Aquaculture 236 (2004) 659675 661


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other metabolic enzymes (lactate dehydrogenase and glucose-6-phosphate dehydroge-

nase) failed to display similar trends.

To our knowledge, no study has been conducted on the physiological tolerance of the

Atlantic wolffish to low salinities, their time-course effects on branchial Na+K+ATPaseactivity and associated energy metabolism and potential impacts on survival and growth.

Most available studies on fish osmoregulatory capacities in relation to salinity are aimed at

salmonids following transfer from FW to SW. By comparison, few have dealt with

acclimation strategies and mechanisms exploited by marine fish species. In the present

study, we propose to: (1) evaluate the tolerance, growth and physiological state of the

Atlantic wolffish at different stages of development following exposure to low and

intermediate salinities; (2) evaluate more closely the short-term adaptation responses; and

(3) characterise the enzymatic adjustments at the branchial tissue level and the ionoosmotic

adjustments at regular intervals.

In addition to Na+K+ATPase, the activities of key metabolic enzymes cytochrome c

oxidase (COX), citrate synthase (CS), pyruvate kinase (PK) and lactate dehydrogenase

(LDH) will be assessed. Mitochondrial enzymes (COX and CS) are indicative of tissue

aerobic capacity, and gill CS activity is indicative of chloride cell (CC) number and

Na+K+ATPase activity (Perry and Walsh, 1989). PK and LDH measurements are being

used as indicators of the glycolytic capacity of the gill tissue and of lactate oxidation

capacity.

2. Materials and methods

Wild fertilized egg masses of Atlantic wolffish were collected in Conception Bay

(Newfoundland, Canada) and transported to the Centre Aquacole Marin facilities (Grande-

Riviere, Quebec, Canada). The wild egg masses were fragmented in small pieces and

incubated in modified Heath-tray incubators a 45 jC until hatching. The fish hatched in

March 2000 and were transferred to experimental low-level raceways for first-feeding and

on-growing phase (Strand et al., 1995).

2.1. Experiment I. Challenge tests 0+ common wolffish juveniles

Duplicates of 20 newly hatched Atlantic wolffish (mean mass of 0.10 g), fed

exclusively with enriched Artemia for a period of 10 days, were directly transferred to

closed systems at 10 jC at salinity 0, 7, 14, 21, 28 and 35. Oxygen, temperature and

salinity were closely monitored and 75% of the water was renewed every day. Mortality

was recorded every 2 3 h during a first 72-h challenge test. The fish that survived

(83.3%) were then used in a 20-day growth trial at 7x, 14x, 21x, 28xand 35x

in the same experimental units. Fourteen fish were used per replicate per salinity. Fish

were fed with enriched Artemia (Super Selcok INVE) three times a day at concen-

trations of 100 Artemia/l. Oxygen, temperature and salinity were monitored twice daily.

A second 72-h challenge test was done using 0.40-g fish at 0x, 1x, 2x, 3x, 4x,5x, 7xand 28xin order to locate the L50. Initial, intermediate and final length and

weight were measured and final water and protein content were also measured. Table 1



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presents the ionic composition of the five experimental salinities (7x, 14x, 21x,

28xand 35x).

2.2. Experiment II. Long-term survival, growth and haloplasticity (1+ wolffish juveniles)

A second group of fish of mean mass of 28.01 F 5.93 g were randomly assigned to four

experimental salinities in flow-through raceway units (two replicates of 7x, 14x, 21x

and 28x, n = 100 per replicate) for a prolonged exposure (19 weeks) in order to

characterise tolerance, growth and haloplasticity of branchial enzymes activity. An initial

2-week acclimation to the rearing units was provided prior to direct exposure. Experi-

mental salinities were obtained by mixing ambient SW (28x) with dechlorinated FW of

equal temperature.The current velocity was fixed at 0.5 cm s 1, and the water level was kept at 810

cm to facilitate food ingestion. Temperature, salinity, oxygen and mortality were

recorded daily. Natural photoperiod was used (sunrise sunset) The fish were fed on

a commercial feed three times a day to satiation. Every 2 weeks for the first 8 weeks,

five fish were randomly collected from each replicate (n = 10/salinity), anaesthetized

(benzocaine at 50 mg/l) in water of equivalent temperature and salinity, weighed (g)

and measured (total length mm). Maximal blood volume was collected using heparin-

ized syringes and microtubes and immediately centrifuged at 7000g for 10 min at

4 jC. The plasmatic fraction was collected and immediately frozen at 80 jC. The

second gill arch was then sampled and rinsed with ice-cold SEI buffer (300 mMsucrose, 30 mM EDTA, 100 mM Imidazole, pH 7.1), carefully blotted dry and

immersed in SEI buffer in 2-ml microtubes. A portion of white muscle from the dorsal

region was also sampled, and all samples were rapidly frozen at 80 jC until analysed.

Two additional samplings were conducted at weeks 14 and 19. Mean experimental salinities

during the trial were 7.51F 0.44x, 14.88F 0.88x, 21.86F 0.73xand 29.31F 0.54x,

respectively.

White muscle water, lipid (Barnes and Blackstock, 1973) and protein (Smith et al.,

1985) content were determined. Growth performances in length and weight at the group

level were estimated using 25 fish per replicates from November 2000 to March 2001 at

four occasions (after 0, 70, 97 and 128 days, respectively).Branchial Na+K+ATPase (EC 3.6.1.37) activity (McCormick and Bern, 1989),

cytochrome c oxidase (COX: EC 1.9.3.1), citrate synthase (CS: EC 4.1.3.7), pyruvate

Table 1

Osmolality and ionic composition of the four experimental salinities (7x, 14x, 21xand 28x)

Salinity (x) Osmolalitya

(mOsm kg 1

)

Na+

(mmol L 1

)a

Cl

(mmol L 1

)a

K+

(mmol L 1

)7 210 88 113 2.01

14 420 178 228 4.00

21 616 267 345 6.03

28 840 356 456 8.00

aSalinity expressed in osmolality and Na+ and Cl ionic concentration: y = 29.8x; y = 12.8x and y = 16.29x,

respectively.



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kinase (PK: EC 2.7.1.40) and lactate dehydrogenase (LDH: EC 1.1.1.27) activities

were measured at 10 jC according to Le Francois and Blier (2003) using a Lambda 40

(Perkin Elmer) equipped with a thermostated cell holder connected to a circulating

water bath and expressed in units per milligram of protein per hour. Plasma osmolality(mOsm kg 1) and ionic concentration were measured using a single-chamber micro-

osmometer (model 3300 from Advanced instrument) and an ion analyser (Corning

Model 644, Bayer), respectively. Osmolality and ionic composition of the experimental

salinities are shown in Table 1.

In parallel, intestinal transit time (rate of food movement through the intestine) at

the different salinities was evaluated using 10 fish (in isolation) per salinity previously

starved for a week and then force-fed with three food items. Occurrence of faeces

production was carefully monitored every 2 h for a period of 70 h. Trypsin activity

(EC 3.4.21.4) was also assessed at time 0 and after 8 and 19 weeks (Bergmeyer,

1983).

2.3. Experiment III. Direct transfer to hypoosmotic conditions, short-term osmotic

response

A challenge test of 120 h on 45 Atlantic wolffish juveniles of 70.02F 9.56 g (45 2

replicates 4 salinities) at 10 jC (9.73F 0.15 jC) was conducted in order to follow

gradual acclimation (survival, osmolality, ionic composition) in the hours following

direct transfer to water of 0, 7, 14, 21 and 28 of salinity. After 120 h, due to lack of

mortality at salinities equal or higher than 7, it was decided to extend the trial to a totalof 41 days (984 h) to allow a clearer evaluation of osmotic adjustments. The

experimental units consisted of plastic containers of 40 l aligned inside a raceway unit

that acted as a water bath. Rearing units were individually aerated. Salinity was obtained

by mixing SW at 28 of salinity with dechlorinated FW. Eighty percent of the water was

renewed every second day at intervals with feeding. The percentage of oxygen saturation

was maintained at 93.14F 0.27%. Blood was collected initially (time zero) on 10 fish

followed by samplings after 6, 12, 24, 48, 120, 500 and 984 h (n = 4 X 2 per salinity).

Plasma osmolality and plasmatic sodium (Na+), chloride (Cl) and potassium (K+) were

measured immediately.

2.4. Statistical analysis

Statistical analyses were done using Systat 10.2 software (SPSS, 1998). The mean

values of the studied variables have been compared using nested ANOVA with salinity

nested within tanks. In some cases, three factors nested ANOVA were performed with the

term week as an additional factor. The hypothesis analyses were corrected following Kirby

(1993). A multiple comparison test of Tukey was used when significant differences were

observed. For some statistical tests, statistical conformity required that the data be log-

transformed. Growth trajectory slopes were compared using covariance analysis

(ANCOVA). Experiments II and III were conducted using randomised complete blockdesign. Differences present at the 5% level were considered significant. All statistical tests

are described in Zar (1984).



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3. Results

3.1. Survival and osmotic adjustments in hypoosmotic conditions 0+

Atlantic wolffish

(Experiment I)

The 72-h challenge tests on 0+ Atlantic wolffish and subsequent 20-day growth trial at

different salinities indicated no significant detrimental effects of hypoosmotic stress on

survival at salinities equal to or higher than 7x. Lethal limit of salinity for 0+ Atlantic

wolffish was under 7xat 10 jC (100% mortality after 6 h in FW) (results not shown). No

mortality occurred at the other salinities. The second challenge test from 0xto 7x(0x,

1x, 2x, 3x, 4x, 5x, 6x, 7x) and 28xindicated that the L50 for salinity tolerance

was located between 5xand 6xwith a clear negative relation between decreasing salinity

levels and cumulative mortality. Hundred percent mortality occurred after 10, 14, 20, 24 and

54 h at 0x, 1x, 2x, 3x, 4xand 5x, respectively (Fig. 1) No mortality was recorded

at 7xand 28x.

3.2. 1+ Atlantic wolffish juveniles (long-term adaptation, Experiment II)

During the total duration of the prolonged growth trial, no mortality was recorded.

Initial mean plasma osmolality value was 341.9 mOsm kg 1. After 4 weeks of exposure,

the group of fish held at 7xexhibited significantly lower plasma osmolality level, 245.56

Fig. 1. Cumulative mortality (%) of Atlantic wolffish juveniles after a 72-h challenge test (direct transfer) at

various salinities (0x, 1x, 2x, 3x, 4x, 5xand 6x).



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mOsm kg 1 (a reduction of 35%), compared with the groups at 21xand 28xthat

remained unaltered. After 19 weeks, this group reached values equivalent to 75% of the

group held at 28x. At this time, plasma osmolality level was 254.42F 7.36 mOsm kg 1

compared to 318.61F15.19, 332.65F 19.72 and 339.38F 20.05 mOsm kg 1 at 14x,21xand 28x, respectively (Fig. 2).

3.3. 1+ Atlantic wolffish juveniles (short-term adaptation, Experiment III)

Besides the mass mortality observed after 12 h in FW, the most prominent feature of the

physiological response (osmoionic adjustments) to abrupt transfer to hypoosmotic

salinities was the pronounced decrease of plasma Na+ and Cl concentrations at 7x

after 6 h and of osmolality values after 12 h (Fig. 3). These low values persisted for the

duration of the trial (40 days) and, on an overall basis, differed significantly from the fish

held at 21xand 28x. A relative steady state followed the initial osmotic stress of the

fish held at 14xand 21xas they all reached osmolality and plasmatic Na+ and Cl

values similar to the fish held at 28x(Fig. 3). No mortality occurred at any of the

salinities. Plasmatic K+ and water content did not display any adjustments following

exposure to the various salinities (results not shown).

3.4. Growth performances in hypoosmotic conditions

3.4.1. 0+ Atlantic wolffish juveniles

After the 20-day growth trial, final mass was significantly higher at 7x

compared to28xand 35x, but tissue water content was also significantly higher in the group held at

7x. Dry weight did not differ significantly among the experimental groups (general mean

mass of 0.0509 g). However, tissue protein content and total length were significantly

higher at 7x(see Table 2).

Fig. 2. Plasma osmolality (mOsm kg 1) in Atlantic wolffish reared at four salinities (7x, 14x, 21xand 28x)

for 19 weeks (n = 10).



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3.4.2. 1+

Atlantic wolffish juvenilesDuring Experiment III, growth performance in length revealed, from day 70, a

significant difference between the experimental salinities 14xand 28x(14x>28x),

Fig. 3. Adjustments (0984 h) of plasmatic ion concentrations (mM/l; Na+, Cl and K+) and plasma osmolality

(mOsm kg 1) in Atlantic wolffish directly transferred to four salinities (7x, 14x, 21xand 28x) and FW.



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and at last sampling (130 days), mean length of the groups held at 14xwas significantly

higher than the fish held at 28x(14x: 18.64F1.32 compared to 28x: 17.85F1.12 mm,

p = 0.006) (Fig. 4A). No significant differences in mean mass were observed between the

different experimental salinities at the last sampling (130 days) (7x: 51.80F13.04, 14x:

55.91F15.57, 21x: 56.54F 15.67 and 28x: 50.42F 11.1; p = 0.077). But significant

differences were observed at days 70 and 80 (Fig. 4A). The general tendency indicates

from day 70 and the subsequent days a slightly higher mean mass at 14xand 21x

compared to 7xand 28x(a recurrent 10% higher mean mass) (Fig. 4B). The slopes

calculated for the growth trajectories in weight and length of the groups held at 14xand

28xare significantly different (n = 200, weight14x:y = 0.206 days + 28.02, r2 = 0.42

and 28x

: y = 0.158

days + 26.85, r

2

= 0.46; length14x

: y = 0.032

days + 14.55,r2 = 0.59 and 28x: 0.026 days + 14.53, r2 = 0.61). These linear relations indicate a

significantly higher weight and length for a given day (after 70 days) for the fish held at

14xcompared to the group at 28x, a discrepancy that seems to increase with time. The

length weight relation for Atlantic wolffish juveniles is expressed by the following

formulae: weight = 0.015 length0.790 (r2 = 0.908) (n = 800).

At week 8, mean lipid and protein content of the experimental groups were 12.30F

2.83% and 22.04F 3.01%, respectively, without any significant differences observed

among the different salinities. Water content was, however, significantly higher at 7x

compared to the fish at higher salinities at weeks 8 and 19. At the 19th week, the fish at

lowest salinity had a mean water content of 77.42 F 1.31% compared to the fish held at14xand higher (75.51F1.12%) indicating muscle hydration.

No differences in trypsin activity levels among the experimental salinities were detected

after 19 weeks of exposure (p = 0.200, mean value: 93.865F 14.304 U/mg protein). In

addition, intestinal transit monitoring revealed a longer transit time for the fish held at

14xcompared with 7xand 28x(results not shown) (c 50 h at 14xcompared to 15

and 31 h for 28xand 7x, respectively).

3.5. Gill enzymatic adjustments

3.5.1. Branchial Na+

K+

ATPase and metabolic enzyme activitiesOn an overall basis, from time 0 to the 19th week inclusive, Na+K+ATPase activity

was significantly higher in groups held at 28xthan all the other salinities. The first

Table 2

0+ Atlantic wolffish final growth characteristics (length, weight, dry weight), water and protein content after the

20-day growth trial at different salinities

Salinity Length (mm) Weight 1

(g) Dry weight (g) Water (%) Protein (mg/g)7x 35.8F 2.2a 0.399F 0.086a 0.051F 0.007 86.4F 0.5a 97.3F 8.1a

14x 34.7F 2.7ab 0.359F 0.081ab 0.058F 0.011 85.0F 0.7b 97.7F 8.8a

21x 35.2F 2.4ab 0.350F 0.063ab 0.051F 0.009 84.9F 0.7b 88.4F 12.5b

28x 34.2F 2.8ab 0.331F 0.072b 0.049F 0.012 84.3F 0.8b 76.6F 6.1c

35x 33.6F 3.0b 0.323F 0.07b 0.046F 0.015 84.6F 0.8b 74.1F 5.4c

p 0.0421 0.0041 0.2070 0.0000 0.0000

Different superscript letters indicate significantly different values for a given parameter (vertical axis).1 Initial mean weight = 0.1674 g, wet weight (p =0.132) 7=14=21=28=35x.



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Fig. 4. Growth in weight (A) and length (B) and calculated growth trajectories for Atlantic wolffish reared at

four salinities (7x, 14x, 21xand 28x) throughout the study (130 days). Results are given in meanF s.d.,

n = 25/replicate/salinity.



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strong indication of a significant reduction in the level of activity of Na+K+ATPase at

near isoosmotic salinity compared to full-strength SW (salinity 28) was noticeable

after 14 weeks of exposure. At this time, the fish held at salinity 14xand 21x

presented significantly lower Na+K+ATPase activity than the fish held at 28xand 7x(14x: 5.67F 1.41; 21x: 5.50F 0.94 and 28x: 7.27F 1.23 U/mg protein). At week

19, fish held at near isoosmotic salinity (14x) presented significantly lower levels of

Na+K+ATPase activity than the group at 28x (5.44F 0.86 and 6.86F 1.34 U/mg

protein) (Table 3).

Metabolic enzyme activities had changed after 14 weeks of exposure (Table 3).

Cytochrome c oxidase activity of the group held at 7xdisplayed significantly lower

values than all other experimental salinities (Table 3). At that time, PK and CS did not

show significant differences among treatments. LDH was, however, significantly lower at

14xthan at 28x(12.46F 1.34 and 17.17F 2.67 U/mg protein, respectively).

Lactate dehydrogenase (LDH) seemed to gradually mimic the U-shaped model of

enzyme adjustments in relation to salinity of marine fish species (Table 3). After 19 weeks,

this trend was confirmed; LDH activity was found significantly lower at isoosmotic

salinity (14x) than at 28x. COX activity maintained a relatively linear response in

relation to elevation in salinity; the fish held at 7xpresented significantly lower COX

activity than the groups held at 28x(1.65F 0.79 compared to mean activity of

3.55F 0.66 U/mg protein) with a positive relationship with increasing salinity (Table 3).

After 14 weeks, Na+K+ATPase and LDH activity were, respectively, 12% and 16%

lower in the group held at 14xcompared to 28x. After 19 weeks, the level of activity of

Na

+

K

+

ATPase and LDH activity of the fish held at 14x

(near isoosmotic value) were,respectively, 20% and 27.4% lower than the fish held at 28xindicating the persistence of

a decreasing trend at isoosmotic salinity in comparison with full-strength salinity. A strong

Table 3

Branchial Na+K+ATPase and metabolic enzymes activities (COX, CS, PK and LDH in U/mg protein) of Atlantic

wolffish after 14 and 19 weeks of exposure to four salinities (7x, 14x, 21xand 28x) (n =10)1

Week Enzyme Experimental salinities

(U/mg protein) activity7x 14x 21x 28x P2 P3

14 Na+K+ATPase 5.9F 1.47ab 5.67F 1.41a 5.5F 0.94a 7.27F 1.23b 0.047 0.050

COX 1.65F 0.79a

3.58F 1.52ab

3.98F 0.98b

3.92F 0.43b

0.026 0.463CS 7.72F 2.76 7.85F 2.01 7.97F 1.85 7.52F 0.95 0.951 0.716

PK 13.98F 7.97 11.04F 6.7 11.39F 4.79 13.35F 7.43 0.790 0.481

LDH 14.44F 5.09 10.4F 2.14 12.38F 4.12 12.39F 2.02 0.075 0.026

19 Na+K+ATPase 6.45F 1.34ab 5.44F 0.86a 5.67F 1.22ab 6.86F 1.34b 0.034 0.067

COX 2.46F 0.78a 3.44F 1.01ab 3.57F 0.99ab 3.96F 0.76b 0.041 0.365

CS 7.55F 1.77 7.46F 1.29 7.3F 1.47 8.52F 1.84 0.192 0.186

PK 10.9F 2.28 10.14F 2.15 10.82F 1.75 11.72F 2.41 0.478 0.548

LDH 16.05F 3.42ab 12.46F 1.34a 14.33F 2.53ab 17.17F 2.67b 0.037 0.008

1 Significant differences among the four experimental salinities are identified with different superscript letters,

while values in bold indicate significant differences after statistical comparison between 14xand 28x.2 Probabilities after conducting a nested ANOVA using salinity and replicate (nested) as factors on all

experimental groups (7, 14, 21, 28).3 Probabilities after conducting a nested ANOVA using salinity and replicate (nested) as factors for a 14 vs. 28

of salinity comparison.



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positive linear relationship between Na+K+ATPase activity, LDH and PK activity was

found, and it is expressed using the formulas: [log y = 1.107log LDH 0.914, p = 0.0000,

r2 = 0.46 (n = 33) at weeks 14 and 19 and with PK (n = 18) at week 19 log y = 1.143log

PK 0.595 p = 0.001 r2 = 0.497]. This indicates that from week 14 onward, the Na+K+AT-Pase activity adjustments in relation to salinity are associated with adjustments in LDH

and after 19 weeks with PK activity.

4. Discussion

In general, growth performances in weight or length display their highest values nearest

to the isoosmotic salinity while the reverse is true for Na+K+ATPase and LDH activities

(lowest values near the isoosmotic point).

Le Francois and Blier (2003) recently provided evidence of the uncoupled nature of

Na+K+ATPase activity and key metabolic enzymes (COX, CS, PK and LDH) following

SW exposures in a salmonid species. In the present study, alterations in the activities of

LDH and PK coupled to Na+K+ATPase adjustments in relation to salinity have been

observed as proposed by Gaumet et al. (1995) and Jensen et al. (1998) for marine fish

species. A strong linear relationship was observed between Na+K+ATPase activity and PK

or LDH. However, despite the absence of a linear relationship between Na+K+ATPase

activity and LDH during SW acclimation in brook charr reported in Le Francois and Blier

(2003), sterile nonmaturing fish, free from the detrimental effects of sexual maturation on

seawater adaptability (Le Francois et al., 1997; Persson et al., 1998), displayed higherNa+K+ATPase and LDH activities than maturing fish during the spawning season. This

observation and our present results on Atlantic wolffish support our assumptions for the

existence of a functional relationship at the gill level between energy-demanding

osmoregulatory function (Na+K+ATPase) and lactate oxidation (as reflected by LDH

activity levels) in both a salmonid and a marine fish species. Given that gill tissue relies on

glucose and lactate obtained either through gluconeogenesis or via the bloodstream as their

main fuel to perform osmoregulatory tasks (Perry and Walsh, 1989; Soengas et al., 1995),

our results indicate that the capacity of gills to mobilize and oxidize lactate is, given

appropriate SW exposure time, tailored to the Na+K+ATPase energy needs.

The absence of clear adjustments of COX in relation to Na+

K+

ATPase adjustments andsalinity suggests, as several other studies reported previously (Thibault et al., 1997; Belanger

et al., 2002; Le Francois and Blier, 2003), that this mitochondrial enzyme might be regulated

differently. As suggested in two studies, COX activity could be maintained in excess in gill

(McCormick et al., 1989) or muscle (Blier and Lemieux, 2001) tissue.

Our results suggest a reduction in the metabolic work done by the gill at isoosmotic

salinity and that this energy is likely available for somatic growth. Imsland et al. (2002)

reported enhanced growth of turbot (Scophthalmus maximus) at intermediate salinities

using ARN/ADN ratios as growth indicators. The time course needed to detect significant

differences in the level of activity of Na+K+ATPase and LDH at isoosmotic salinity reflects

a long adjustment period, and accordingly, growth benefits were slow to appear. In thisregard, temperature has been shown to have a significant effect on growth and osmoreg-

ulatory processes (Handeland et al., 2000; Imsland et al., in press). Thus, the potential



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growth benefits originating from the reduced metabolic costs or other effective agents can

be further improved given appropriate tailoring of the external environment (optimal

salinitytemperature combination).

In accordance with the work of Foss et al. (2001) on a closely related species (spottedwolffish; A. minor), efficient homeostatic control at salinities lower than 28 was observed.

Na+ and Cl levels either remained stable at intermediate salinities or slightly declined

after acclimation to the lowest experimental salinity. Further evidence is provided by stable

moisture content at intermediate salinities, moderate tissue hydration and absence of

mortality at 7x. Evidence of the haloplasticity and efficient osmoregulatory strategy of 0+

and 1+ Atlantic wolffish in a salinity range of 735xis provided.

A recent review by Boeuf and Payan (2001) attributed the growth enhancement at

isoosmotic salinities for stenohaline and euryhaline fish species to multiple causality, i.e.

reduction of energetic costs (ionic and osmotic regulation), increased food intake, food

conversion and hormonal stimulation. Foss et al. (2001), working on the spotted wolffish

(A. minor), did not report any effect of salinity on feeding rate, food conversion or protein

efficiency ratio, although indications of enhanced growth at isoosmotic salinity are

discussed. Despite better growth and food conversion at intermediate salinity in juvenile

cod (G. morhua), Dutil et al. (1997) reported similar standard metabolic rate and protein

digestibility among a range of experimental salinities. However, our indications of a

slower intestinal transit at the intermediate salinities for the Atlantic wolffish could

suggest, as reported by Ferrarris et al. (1986), a prolonged digestion time for nutrient

absorption that could translate into better digestibility and increased energy for growth

allocation.Given that there are also direct energy costs of ion transport in other osmoregulatory

organs such as the kidney and intestine and that Na+K+ATPase in those tissues has been

shown to be reduced at intermediate salinities (Kelly et al., 1999b), we propose that the

reduced gill Na+K+ATPase and LDH activities observed at isoosmotic salinity in the

Atlantic wolffish are indications that the reduction of energy expenditures in the

osmoregulatory organs as a whole contributes significantly to improvements in growth

performance. Interestingly, Lambert et al. (1994) stated that a reduction of the metabolic

rate in the order of 15% at isoosmotic salinities is insufficient to explain the difference in

growth observed in Atlantic cod at intermediate salinities. They suggested that additional

mechanisms, mainly reductions in spontaneous activity or swimming performance, could be involved. Since the Atlantic wolffish displays low swimming activities (sedentary

habits and sheltered behaviour), we can tentatively suggest that these behavioural

characteristics likely allow a larger proportion of energy savings to be allocated to somatic

growth. Sedentary species such as Atlantic wolffish and Atlantic halibut (Hippoglossus

hippoglossus; Fraser et al., 1998) display higher white muscle protein synthesis retention

efficiency (92% and 77%, respectively) than more active species such as salmonids (52

73% as reported in McCarthy et al., 1999).

The physiological significance of a U-shaped salinity dependence of branchial

Na+K+ATPase and a key-metabolic enzyme, LDH, indicates that marine fish species that

live under natural conditions exposed to varying estuarine conditions display efficientenzymatic adjustment mechanisms to lower their energetic costs. Juvenile stage residency

and long-incubation period in estuarine coastal waters during the life history of the



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Atlantic wolffish make this adaptation particularly appropriate in terms of available energy

for growth and/or maintenance of homeostasis over a wide range of environmental

salinities. In a mariculture context, our results suggest that Atlantic wolffish cultivation

(early juveniles to commercial size) may be extended to estuarine coastal environmentswith potential positive effects on productivity.

Acknowledgements

The authors would like to express their gratitude to Mrs. K. Lord, Mr. J.-C. Blais and B.

Archer for their technical assistance. This research was funded by a grant from the

Ministere de lagriculture, des pecheries et de lalimentation du Quebec to NRLF and PB.

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