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Aquaculture 287 (2009) 203–210

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Aquaculture

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Growth performance and osmoregulation in the shi drum (Umbrina cirrosa) adapted to different environmental salinities

Constantinos C. Mylonas a,⁎, Michalis Pavlidis b, Nikos Papandroulakis a, Mario M. Zaiss a, Dimitris Tsafarakis a,b, Ioannis E. Papadakis a, Stamatis Varsamos c,1 a Institute of Aquaculture, Hellenic Center for Marine Research, P.O. Box 2214, Iraklion, Crete 71003, Greece b Department of Biology, University of Crete, P.O. Box 2208, Iraklion, Crete 71409, Greece c Université Montpellier II, SkuldTech, CC 091, Place Eugène Bataillon, 34095, Montpellier, France a r t i c l e i n f o a b s t r a c t

Article history: In order to investigate the ability of shi drum (Umbrina cirrosa) to be reared at diverse locations, growth and Received 30 November 2007 osmoregulatory performance were assessed at full-strength seawater (40 psu), nearly iso-osmotic water Received in revised form 12 October 2008 (10 psu) and low salinity water (4 psu). At the end of the 84-day experimental period, fish reared at 4 psu Accepted 15 October 2008 displayed shorter mean fork length, lower mean body weight, lower specific growth rate and higher food conversion efficiency than fish reared at 10 or 40 psu. The effect of salinity on growth performance was also Keywords: reflected by changes in plasma triglycerides and cholesterol, with fish reared at 4 psu exhibiting the lowest Shi drum fi Umbrina mean concentrations, while there was no signi cant difference in mean plasma glucose concentrations Osmoregulation among treatments. Plasma osmolality was lower at 4 psu from day 42 onwards, while there was no + + + Chloride cells significant difference in mean plasma K and Cl− concentrations. Plasma sodium and gill Na /K -ATPase Gill Na+/K+-ATPase activity showed minimum values on day 42 at 4 psu, but at the end of the experiment there was no difference among groups. Pavement cells, mucus cells and chloride cells were identified by histology on the gill epithelium. In shi drum reared at full seawater, mucus cells contained a mixture of acid and neutral mucins, whereas in fish adapted to hypo-osmotic environment neutral mucins were mainly observed. There was a significant increase in chloride cell number over the course of the study in all fish, but there was no difference among the three experimental salinities. Finally, in fish reared at 40 psu salinity, chloride cells increased in size significantly compared to fish adapted to 4 psu, whereas at 10 psu after 42 d there was a significant reduction in chloride cell size. These results indicate that shi drum reared from full-strength seawater to iso-osmotic salinity do not face any osmoregulatory imbalance, while fish reared in hypo- osmotic water displayed osmoregulatory impairment and low growth performance. © 2008 Elsevier B.V. All rights reserved.

1. Introduction cells (MRCs) or more generally ionocytes (Pisam and Rambourg, 1991; Hirose et al., 2003; Marshall and Grosell, 2006). Euryhaline fishes exhibit the ability to maintain relatively constant Adaptation of euryhaline fish to different salinities involves ionic composition of plasma, lymph and interstitial fluid, in a broad changes in ion transport mechanisms that generally induce changes range of environmental salinities (Marshall and Grosell, 2006). This is in oxygen consumption, suggesting variations in the energetic de- achieved through morphological, cellular, physiological and endocrine mands for osmoregulation (Morgan and Iwama, 1991). Several sce- adaptations (McCormick and Bradshaw, 2006; Sakamoto and McCor- narios have been suggested to describe changes in oxygen metabolism mick, 2006; Shane et al., 2006). The strategy of ion exchange with the in fish following salinity challenge that, in some cases, can result in external medium is mediated mainly through the gastrointestinal altered or enhanced growth (Boeuf and Payan, 2001). The relationship epithelium, kidney and gills (Marshall and Grosell, 2006). In adult between osmoregulation and growth performance in marine fish has teleost fish, the gills are recognized as the main site for ion exchange stimulated a large amount of research and, so far, it seems to be (Wilson and Laurent, 2002) mediated by different transporters, species-specific (Boeuf and Payan, 2001). Each fish species seems to mainly Na+/K+-ATPase (McCormick, 1995, 2001), and ion channels have its own salinity optimum for growth, and osmoregulatory im- located in the chloride cells, which are also called mitochondria-rich balance is often associated with whole animal-level stress changes, including decreased growth performance (De Silva and Perera, 1976; Boeuf and Payan, 2001; Imsland et al., 2001). ⁎ Corresponding author. Tel.: +30 2810 337878; fax: +30 2810 337875. Some members of the family Sciaenidae (Nelson, 2006) display a E-mail address: [email protected] (C.C. Mylonas). 1 Current address: European Commission, DG MARE, Unit A3. Research, Data great potential for aquaculture, and some have been reared in com- Collection and Scientific Advice, Rue Joseph II 79, 2/34, B 1049 Brussels. mercial facilities for some time. These include the red drum (Sciaenops

204 C.C. Mylonas et al. / Aquaculture 287 (2009) 203–210 ocellatus) in the United States (Miranda and A.J., 1985; Thomas et al., 2.2. Biochemical analyses 1995; Gardes et al., 2000), the mulloway (Argyrosomus japonicus) in Australia (Battaglene and Talbot, 1994; Fielder and Bardsley, 1999), and Plasma metabolites were determined by enzymatic colorimetric the maigre (Argyrosomus regius) in Europe (Pastor Garcia et al., 2002; procedures (glucose GOD/PAP; cholesterol PAP; triglycerides GPO/PAP) Poli et al., 2003). Another Sciaenid species of interest in the eastern part using commercial kits (Biosis, Greece). Osmolality was measured using a of the Mediterranean Sea is the shi drum (Umbrina cirrosa) (Cardellini cryoscopic osmometer (Gonotec, Osmomat 030). Plasma samples were et al., 1999; Barbaro et al., 2002; Mylonas et al., 2004; Zaiss et al., 2006). diluted 1:600 with lithium nitrate 15 mmol L−1 and electrolytes (K+, Na+) Sciaenid fishes are considered as euryhaline species (Miranda and A.J., were determined by flame emission spectrophotometry. Chloride ions 1985; Fielder and Bardsley, 1999; Doroudi et al., 2006), therefore they were determined by spectrophotometric method based on the reaction could be considered for aquaculture diversification in coastal lagoons of Cl− with mercuric thiocyanate. with brackish water and areas near the mouth of large rivers, where the Gill Na+/K+-ATPase activity was determined according to Varsamos commonly employed marine species are not suitable. In the case of the et al. (2004). Briefly, gill arches were homogenized with an IKA disperser shi drum, as well as some of the other sciaenids with aquaculture (IKA®Yellowline, USA), at 9,500 rpm for 5 sec, with 1 ml of buffer potential, there is a complete lack of knowledge on its adaptation containing: 250 mM sucrose, 10 mM Hepes, 5 mM MgCl2, 0.1 mM Na2- potential and growth performance in different salinities. EDTA, dH2O (pH 7.4). The homogenate was centrifuged (4000 rpm, The objective of the present work was to study the osmoregulatory 5 min, 4 °C) and the supernatant used for assays. Homogenate protein capacity and the interaction between salinity adaptation and growth content was determined in triplicate as detailed by Bradford (1976), performance in shi drum, in order to provide scientific background for using BSA (Sigma, A-4503) as standard. The Na+/K+-ATPase activity was the development of optimized husbandry practices for this species. assessed as the difference of total ATP hydrolysis (in presence of Na+, K+, For this purpose, fish were reared for three months at different Mg2+ and ATP) and that in absence of K+ but in presence of an optimal salinities representing full-strength seawater (40 psu), nearly iso- concentration of the specific inhibitor ouabain (1 mg ml−1). The amount osmotic water (10 psu) and hypo-osmotic water (4 psu), and changes of released phosphate was assessed by comparison with commercial in gill histology, osmoregulation, growth and energetic metabolism reference standards (Calimat, BioMérieux, France). The Na+/K+-ATPase related parameters were evaluated. specific activity was expressed in μmol Pi h−1 mg−1 protein. The total activity was calculated as the product of the specific activity and the total 2. Material and methods protein amount of the sample and expressed in μmol Pi h−1.

2.1. Animal husbandry and experimental design 2.3. Histological analyses

Experiments were conducted using shi drum juveniles at the facilities A section of the second gill arch was fixed in 4% formaldehyde: 1% of the Institute of Aquaculture, Hellenic Centre for Marine Research gluteraldehyde buffered saline (McDowell and Trump, 1976) for at least (Crete, Greece). Fish were produced in July 2004 from wild-caught, 24 h. Before embedding in a methacrylate resin (Technovit 7100®, captive-reared broodstock, after hormonal induction of spawning with Heraeus Kulzer, Germany), gill samples were dehydrated through a gonadotropin-releasing hormone agonist (GnRHa) implants (Mylonas series of increasing concentrations of ethanol (70–96%). The mounted et al., 2004). Fish with mean±S.D. total length (TL) of 11.8±0.8 cm and gills were cut in 3-μm thick sections on a microtome (RM 2245, Leica, body weight (BW) of 22.8±4.5 g were reared in three independent Germany), stained with Methylene Blue (Sigma, Germany)/Azure II recirculating aquaculture systems (RAS) at different salinities (40,10 and (Sigma, Germany)/Basic Fuchsin (Polysciences, USA) (Bennett et al., 4 psu). Each RAS was fitted with mechanical and biological filters, and 1976) and examined under a light microscope (BH-2, Olympus, Japan). In supported two replicated 500-l cylindroconical polyester tanks contain- addition, four sections per gill sample were stained with the combined ing the experimental fish (n=30). The recirculated water was maintained Alcian blue — periodic acid Schiff (AB-PAS) technique for neutral and at 19±1°C and N80% oxygen saturation throughout the course of acid mucins (Mowry, 1956) which stains mucus cells specifically and the experiment (84 days), and new water exchange rate was 30% every allows identification of different types of mucins (Shephard, 1994). 2–3 d. Fish were fed ad libitum with commercial feed (1st period EXCEL, Chloride cells were located along the primary lamellae, in the area SKRETTING) by the use of a demand-feeder, and photoperiod was 24L:0D between the bases of the secondary lamellae (Fig. 1). They contained a two months prior to and throughout the salinity manipulation. A large, but lighter staining nucleus than the pavement cells or red blood cells constant light photoperiod was used due to mortalities from jumping out visible in the sections. As reported earlier (McCormick, 2001), chloride cells of the tanks during lights-on and off. in fish reared in sea water contain a deep apical crypt and are larger than in After randomly allocating the fish in their tanks (n=30) at 40 psu, two freshwater fish, where an apical surface substitutes the apical crypt (Fig.1). replicated groups were acclimated gradually to the lower experimental Chloride cell counts were made on histological sections of primary salinities over the course of 6 d. Total length and BW measurements lamellae, where both the cartilaginous vestige of the gill septumwas visible were taken from all fish in each tank at 0, 14, 28, 42, 56, 70 and 84 days and the primary lamella was sectioned in the middle, along its whole from the start of the experiment. Blood samples were taken initially longitudinal axis. Chloride cell counts were made along 1 mm length of (day 0) from ten fish from the stock population and then from five fish three different primary lamellae (starting from the base of each primary from each tank at 46 and 84 d. Blood was collected from the caudal lamella on the gill arch) and the average value was used to represent each vasculature with heparinized syringes, centrifuged at 5000 rpm for fish sampled. Size estimation of the chloride cells was made using the 15 min at 4 °C, and the plasma was stored at −80 °C until further analysis. average of the maximum (D) and minimum diameter (d). All chloride cells Five fish from each treatment group were also sacrificed at 46 and sectioned through the nucleus (n=13–42), along the length of a primary 84 days, the second gill arch was excised for histological analysis and the filament containing three secondary lamellae were measured for three third gill arch was quickly stored at −80 °C for determination of Na+/K+- different areas and the average was used to represent each fish sampled. It ATPase activity (see later for method). For all measurements, fish were was not possible to make reliable mucus cell counts, because different first anaesthetized in a solution of 0.4 ml L−1 of clove oil (Mylonas et al., sections of the gill filament contained markedly different numbers. 2005), dissolved first 1:10 v/v in 96% EtOH. Fish handling was carried out according to the European Union Directive (86/609EEC) for the pro- 2.4. Estimation of growth parameters and statistical analyses tection of animals used for experimental and other scientific purposes (EEC, 1986) and the “Guidelines for the treatment of animals in be- Comparisons of TL and BW between groups reared at different sa- havioural research and teaching” (Anonymous, 1998). linities were made using regression analysis (Sokal and Rohlf,1981). The Author's personal copy

C.C. Mylonas et al. / Aquaculture 287 (2009) 203–210 205

Fig. 1. Microphotograph of primary lamella from shi drum reared at different salinities. (A) 40 psu. Note the larger size of chloride cells and the presence of a prominent apical crypt. (B) 4 psu. The chloride cells are smaller and an apical surface has substituted the apical crypt. pc = pavement cells, ac = apical crypt, as = apical surface, cc = chloride cells. Bar represents 25 µm. general model used was of the form: Y = a + a t + a D + a t D, fish reared at 10 or 40 psu salinity. During the period between 57 and 0 1 Á 2 Á 3Á Á where Y is the dependent variable, t the time, D a dummy variable with 84 days after the onset of the experiment no significant difference in values 0 and 1 for each condition tested and αi (I=1, 2 or 3) constants. FCR was found among the three salinities. This method tests the hypothesis that the constants α2 and α3 are zero. + + Time series have the same slope when constant α3 is zero, and the same 3.2. Plasma metabolites, osmolality, electrolytes and gill Na /K -ATPase initial value when α2 is zero. When both constants are zero, time series activity describe similar dependent variables. Specific growth rate (SGR) was estimated as the slope of the above model. Food conversion ratio (FCR) There were no significant differences in mean plasma glucose was estimated as FCR=F/(Bf −Bi), where F = consumed food (kg), Bf = concentration among fish reared at the different salinities and fish final biomass (kg), Bi = initial biomass (kg). To evaluate differences in FCR, plasma metabolites, electrolytes, osmolality, gill Na+/K+-ATPase activity and chloride cell density or size, data were subjected to 2-way Analysis of Variance (ANOVA) (time x salinity), followed by Duncan's New Multiple Range (DNMR) test on main effects, and mean comparisons for interactions. Statistical analysis was performed using linear statistics software (SuperAnova, Abacus Concepts or SigmaStat, Systat Software, Inc.) at a minimum significance level of Pb0.05. All results are presented as mean±SEM.

3. Results

3.1. Growth performance of shi drum in different salinities

Survival was unaffected by salinity and at the end of the 84-day experimental period it was greater than 97% in all salinities. Also, no differences in feeding behaviour or appetite were observed. However, salinity did have a statistically significant effect on growth (Pb0.05), with fish reared at 4 psu having significantly smaller TL and BW than the fish reared at 10 or 40 psu, whereas there were no differences between fish reared at 10 or 40 psu salinity (Fig. 2). The final mean BW of shi drum reared at the three salinities were 63.3±3.2, 67.5±2.1 and 44.5±2.2 g for 40, 10 and 4 psu, respectively. As expected from the above measurements, SGR over the course of the study was also affected by environmental salinity, and in fish reared at 4 psu was 0.265 d− 1, significantly lower (Pb0.05) than of fish adapted to 10 or 40 psu, which was the same at 0.513 d− 1. There was large variation in the FCR between the two replicates of the 4 psu treatment at different times, as indicated by the large SEM values (Table 1), which could not be explained by the experimental design. Nevertheless, the statistical analysis over the course of the study indicated the existence of a significant difference (Pb0.05) between the FCR of fish reared at 4 psu and the FCR of fish reared at 10 Fig. 2. Changes in mean (±SEM) total length (A) and body weight (B) of shi drum (n=25– 30) reared in duplicated tanks at different salinities (4, 10 and 40 psu) during a period of or 40 psu (Table 1). The differences were due to higher values of the 84 days. Reduction of salinity to 4 psu had a statistically significant effect on growth fi fi 4 psu sh during the rst 56 days, when FCR ranged between 1.36± over the whole course of the study (regression analysis, Pb0.05), as indicated by 0.14 and 4.71±2.2, compared to a range of 1.06±0.05 and 1.78±0.07 of different letter superscripts next to the growth curves. Author's personal copy

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Table 1 10 psu and 40 psu at both sampling times. In fish reared at 40 psu, fi Mean (±SEM) food conversion ratio (FCR=F/[Bf −Bi], where F = consumed food, Bf = nal most mucus cells contained mostly acid mucins, which stained blue– biomass, B = initial biomass) of shi drum reared at different salinities (4, 10 and 40 psu) i purple with AB-PAS, whereas in fish reared at 4 psu the mucus cells in duplicated tanks (i.e., n=2) contained mostly neutral mucins, which stained red (data not shown). FCR At 10 psu there did not seem to be a dominance of any of the two types Salinity 4 psu 10 psu 40 psu of mucins. Mucus cells were observed both on the primary filament Time period (d) and secondary lamellae of the main gill structure, as well as the gill a a,b b 0–14 3.39±1.89 1.78±0.05 1.29±0.14 arch epithelium itself. 15–28 3.32±0.22a 1.13±0.08b 1.06 ±0.05b fi Pb 29–42 1.36±0.14 1.21±0.14 1.25±0.02 There was a signi cant increase ( 0.001) in chloride cell number 43–56 4.71±2.20a 1.68±0.26b 1.42±0.02b over the course of the study in fish exposed to all three salinities 57–70 1.28±0.04 1.31±0.21 1.12±0.21 (Fig. 5A). However, the total number of chloride cells did not differ 70–84 1.54±0.26 1.54±0.40 1.58±0.68 significantly between the three experimental salinities, even though at 42 d a slight reduction (P=0.10) was observed in the fish reared at Whole duration (d) fi 0–84 2.60±0.53a 1.44±0.10b 1.28±0.10 b 4 psu. In sh reared at 40 psu salinity, chloride cells increased in size during the course of the study, and were significantly (Pb0.001) fi Signi cant differences (Pb0.05) at different sampling points and over the whole course fi of the study between the 4 psu, and the 10 or 40 psu group are indicated by different greater in size compared to sh adapted to 4, both at 42 and 84 days letter superscripts next to the mean values. Within each salinity treatment, there were after the onset of the experiment (Fig. 5B). On the contrary, in fish no differences in FCR between different sampling times (P=0.19). reared at 10 psu after 42 d there was a significant reduction in chloride cell size. reared in all salinities had significantly (Pb0.05) higher glucose levels at the onset of the experiment (Table 2). In all groups, increased 4. Discussion triglyceride concentrations were found at 42 d and reached a maximum at 84 d. Maximum cholesterol concentrations at 10 and 40 psu It has been shown that several euryhaline teleosts display a two- salinity were found at 42 d, while at 4 psu salinity at 0 d and 42 d stage adaptation pattern following salinity challenge, consisting of an (Table 2). Plasma triglycerides and cholesterol concentrations were adaptive period when changes in osmotic and metabolic parameters significantly lower (Pb0.05) in fish reared at 4 psu than in the other occur, and of a regulatory period which generally leads to a new steady two groups at both sampling points (Table 2). state (Jensen et al., 1998; Sangiao-Alvarellos et al., 2005). Our data have Plasma osmolality was significantly lower (Pb0.05) in fish reared clearly shown that shi drum juveniles are able to maintain constant at 4 psu than in the other two groups at 42 d after the start of the plasma osmolality and electrolyte levels over a relatively broad range of experiment, but not at the end of the experiment, after 84 d (Table 3). environmental salinities and during a long period. However, it appears There also appeared to be a gradual increase in plasma osmolality over that rearing at very low salinities (4 psu) affects osmoregulation and the course of the experiment, regardless of environmental salinity. In growth. Thus, body size, SGR and FCR were lower in shi drum exposed to terms of plasma electrolytes, there was no difference in plasma K+ or 4 psu salinity compared to those reared at 10 or 40 psu salinity. The Cl− concentrations among fish reared at different salinities, in either blood osmolality of teleosts is around 12 psu, i.e. approximately one sampling times, though some variation existed (Table 3). On the third of that of sea water (~38 psu) and more than 50-fold higher than contrary, significantly (Pb0.05) lower plasma Na+ concentrations were the salinity of freshwater (~0.2 psu). Consensus exists that marine fish found 42 d after the start of the experiment in fish reared at 4 psu achieve higher growth rates at lower salinity (Boeuf and Payan, 2001) compared to fish reared at 10 or 40 psu. However, by the end of the and that each species has its own salinity optimum for growth, under a experimental period there was no difference in plasma Na+ concen- certain water temperature and ontogenetic phase, although existing trations among the three groups. data are sometimes contradictory. For instance, the best salinity Gill Na+/K+-ATPase specific activity 42 d after the onset of the conditions for growth in gilthead sea bream (Sparus aurata) have been experiment was significantly lower (Pb0.05) in fish reared at 4 psu reported at 12 psu (Laiz-Carrión et al., 2005b) or 28 psu (Klaoudatos and salinity, compared to that measured either in 10 or 40 psu (Fig. 3). Conides, 1996), in grey mullet (Mugil cephalus) at 20 psu (De Silva and However, at the end of the experimental period a significant increase Perera, 1976), in turbot (Scopththalmus maximus) at 15 psu (Imsland in the gill Na+/K+-ATPase activity was recorded in fish reared at 4 psu et al., 2001) and in European sea bass (Dicentrarhus labrax) at 15 psu salinity and no difference in this parameter was found at that sam- (Saillant et al., 2003) or 28–30 psu (Dendrinos and Thorpe, 1985; pling point among fish reared at different salinities. Conides and Glamuzina, 2006). The present results indicate that salinity affects FCR and growth in shi drum juveniles adapted to low salinity. The 3.3. Gill morphology in different salinities metabolic and energetic cost of osmoregulation should, at least partly, reflect the effect of salinity on growth, however other possibilities With a very few exceptions, chloride cells were located exclusively should be taken into account. This may include the effect of salinity on on the primary filament of the main gill structure (Fig. 4). In fish feeding behavior, appetite, or stimulation/inactivation of other meta- reared at 4 psu, a thickening of the primary filament and secondary bolic and endocrine pathways (Boeuf and Payan, 2001; McCormick, lamellar epithelium was observed compared to both fish reared at 2001).

Table 2 Mean (±SEM) plasma glucose (Glu), cholesterol (Chol) and triglycerides (Trig) of shi drum (n=10) reared at different salinities (4, 10 and 40 psu), and sampled at different times after the onset of the experiment (0, 42 and 84 days)

4 psu 10 psu 40 psu 0 42 84 0 42 84 0 42 84 Glu (mmol l− 1) 7.8±0.7a 3.5±0.2b 3.6±0.1b 7.8±0.7a 3.7±0.2b 3.6±0.2b 7.8±0.7a 3.2±0.1b 3.1±0.1b Chol (mmol l− 1) 3.3±0.2a 3.1±0.2abA 2.6±0.1bA 3.3±0.2b 4.0±0.1aB 3.2±0.1bB 3.3±0.2b 4.7±0.4aB 3.3±0.2aB Trig (mmol l− 1) 2.0±0.1b 3.1±0.3abA 4.3±0.4aA 2.1±0.1c 7.1±1.1bC 10.4±1.0aB 2.1±0.1c 4.7±0.5bB 9.5±0.8aB

Different small letter superscripts indicate signiﬁcant differences (Pb0.05) between different sampling times within salinity treatments, while capital letter superscripts indicate signiﬁcant differences between different salinities within sampling time. Author's personal copy

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Table 3 Mean (± SEM) plasma osmolality, sodium (Na+), potassium (K+) and chloride (Cl−) of shi drum (n=50–60) reared at different salinities, and sampled at different times after the onset of the experiment (0, 42 and 84 days)

4 psu 10 psu 40 psu 0 42 84 0 42 84 0 42 84 Osmolality (mOsm kg− 1) 350±3a 338±2aA 410±7b 350±3a 355±2aB 398±5b 350±3a 353±1aB 409±2b Na+ (mmol l− 1) 196±8 181±5A 179±5 196±8 201±5B 192±3 196±8 205±8B 188±6 K+ (mmol l− 1) 8.0±0.3a 6.0±0.2b 8.3±0.3a 8.0±0.3a 6.2±0.3b 8.1±0.4a 8.0±0.3a 6.5±0.3b 8.0±0.2a Cl− (mmol l− 1) 161±7a 145±1a 197±5b 161±7a 158±2a 188±3a 161±7a 156±1a 196±1b

To our knowledge this is the first report on plasma cholesterol and since the role of glucose as fuel for tissues during osmotic challenge or triglyceride concentrations for shi drum, and the values reported are stress conditions, in general, is well known (Wendelaar Bonga, 1997). within the range measured in other marine species (Kavadias et al., However, in long term adaptation, such as in the present study, it 2003). Interestingly, both parameters seemed to reflect metabolic seems that the involvement of this metabolite is less discriminating, effects of long-term salinity adaptation in this species, whereas and similar results have been obtained in gilthead sea bream (Laiz- plasma glucose levels where unaffected by salinity treatments. Thus, Carrión et al., 2005b). the low growth performance of shi drum reared at 4 psu was The results obtained in the present experiment indicate that shi concomitant with lower plasma cholesterol and triglyceride concen- drum reared at 40 or 10 psu salinity for 84 days did not face major trations compared to those measured in fish reared at 10 or 40 psu, osmoregulatory imbalance, based on the fact that there were no suggesting a negative effect of low salinity on energy metabolism. It significant differences in plasma osmoregulatory parameters. On the has been already suggested that changes in plasma metabolites during contrary, fish reared at 4 psu displayed osmoregulatory difficulties, as the first days of salinity challenge are related to modifications in indicated by the observed lower plasma Na+ after 42 d and osmolality energy supplies in osmoregulatory tissues (Sangiao-Alvarellos et al., after 42 and 84 d. Low Na+ levels have also been reported in the 2005). For example, in gilthead sea bream plasma triglycerides did not gilthead sea bream after 100 d rearing at 6 psu salinity (Laiz-Carrión show significant changes following acclimation to different salinities et al., 2005b) and in European sea bass 24 h after transfer to salinities (i.e., 6, 12, 38 psu) for 100 d, although growth and SGR were correlated less than 20 psu (Venturini et al., 1992). Difficulties in homeostasis in to salinity (12N38N6 psu) (Laiz-Carrión et al., 2005b). However, in the low salinities have also been reported in other euryhaline teleost same species, a 20 and 25% decrease in plasma glucose and species, such as the sheepshead minnow (Cyprinodon variegates) triglyceride levels, respectively, was observed after adaptation to (Nordlie, 1985) and flounder (Paralichthys orbignyanus) (Sampaio and 6 psu salinity, which was then followed by full recovery within 7 d Bianchini, 2002). Franklin et al. (1992) suggested that such osmor- (Sangiao-Alvarellos et al., 2005). The low cholesterol levels recorded in egulatory impairments indicate salinity stress, whereas the negative shi drum adapted to 4 psu salinity could be explained partly by effect of stress on growth is well documented in teleosts (Wendelaar differences in feeding rate compared to the other experimental Bonga, 1997). salinities. That kind of correlation between the feeding rate and It is well recognized that gills are the organs that consume most plasma cholesterol levels has been reported in European sea bass energy during adaptation of teleosts in different salinities, since they (Lemaire et al., 1991) as well as in Eurasian perch (Perca fluviatilis) must maintain differential regulation of intracellular and extracellular (Vellas et al., 1994). The absence of differences in glucose levels among fluids. Moreover, in case of transition from hypoosmotic to hyper- the salinity treatments in the present experiments seems surprising, osmotic conditions (or vice versa), Na+ and Cl− fluxes across the gill epithelia must switch from ion uptake to ion excretion (and vice versa). The Na+/K+-ATPase plays a pivotal role in these processes and, thus, its activity could be related to some extent to the energetic cost of osmoregulation (McCormick, 1995). In our experiments the lowest activity for this enzyme was recorded in shi drum juveniles reared at low salinity after 42 d, and could suggest, at first sight, a lower energy cost for osmoregulation at that salinity. Our results are in contrast with the U-shaped variations in gill Na+/K+-ATPase activity reported for euryhaline fish, with high levels in freshwater or very low salinity and seawater, and lower levels in fish adapted to brackish water (10– 12 psu) (Jensen et al., 1998; Boeuf and Payan, 2001; Varsamos et al., 2002). However, a direct relationship between salinity and Na+/K+- ATPase activity has been shown in some teleosts (McCormick, 1995, 2001; Marshall, 2002). Nevertheless, at the end of the present experiment there was no significant difference in Na+/K+-ATPase activity among the experimental groups, indicating that fish reached a steady state within 84 d after the challenge. These observations, together with the data mentioned above, suggest that it is rather unlikely that the low enzyme activity observed in the gills after 42 d at low salinity corresponds to low osmoregulatory cost. Rather, a transient alteration in cellular metabolism and in the function of membrane exchangers/transporters could be involved, due to salinity- Fig. 3. Changes in mean (±SEM) gill Na+/K+-ATPase specific activity in shi drum (n=10) induced stress. Hence, unlike other euryhaline teleosts, it appears that reared at different salinities during a period of 84 days. Different small letter superscripts indicate significant differences (Pb0.05) between different salinities the adaptation period following low salinity acclimation in shi drum within a sampling date. There were no significant differences between sampling times. juveniles is much longer and displays a different pattern. Moreover, Author's personal copy

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Fig. 4. Histological sections of gill filaments (consisting of primary filament and secondary lamellae) showing structural changes in the epithelium of shi drum reared at different salinities. A: Gill filament of shi drum at the beginning of the experiment (reared at 38 psu). B: Gill filament of shi drum control fish reared 40 psu for 84 days. C: Gill filament of shi drum reared at 4 psu for 84 days. Abbreviations: cc = chloride cell, mc = mucus cell, pc = pavement cell, pl = primary filament, sl = secondary lamella. Bars represent 50 µm. other transporters, such as H+-ATPase (Lin and Randall, 1995) or Ca+- barei) (Sarasquete et al., 2001). On the contrary, most mucus cells of shi ATPase (Marshall, 2002), could be involved in low salinity adaptation of drum exposed to hypo-osmotic environment stained reddish, indicating shi drum. In terms of the absolute values of mean ATPase activity in shi that they consisted mainly of neutral mucins, a result similar to that drum in the present study in comparison with other fishes, it was documented for species maintained in freshwater, such as the rainbow found that it was lower than in saltwater adapted Japanese eel trout (Oncorhynchus mykiss) (Ferguson et al., 1992) and Atlantic salmon (Anguilla japonica) (Utida et al., 1971), chum salmon (O. keta) (Uchida (Salmo salar) (Roberts and Powell, 2003). Our findings suggest that et al., 1996), silver perch (Bidyanus bidyanus) and golden perch changes in the mucin production of gill mucus cells, could be part of the (Macquaria ambigua) (Alam and Frankel, 2006), and was an order of salinity adaptation process in the shi drum. magnitude lower than larval European sea bass (Varsamos et al., 2004). Gill chloride cells are recognized as the main site for ion exchange, In fish, the gill epithelium consists mainly of three types of cells, mediated by different transporters and ion channels (Lin and Randall, namely the pavements cells (representing 90–95% of the total epithelial 1995; Marshall, 2002; Marshall and Singer, 2002; McCormick and area), the chloride cells and the mucus cells (Shephard,1994; Wilson and Bradshaw, 2006). Changes in the number and/or size of these cells have Laurent, 2002). In shi drum reared at full seawater, most mucus cells of been related to salinity acclimation in several teleost species throughout the gills stained purple indicating a mixture of acid and neutral mucins. post-embryonic development (Karnaky et al., 1976a; Pisam and Similar results were reported in other species, such as the gilthead sea Rambourg, 1991; Zydlewski and McCormick, 2001; Varsamos et al., bream, Senegal sole (Solea senegalensis) and Siberian sturgeon (Acipenser 2002; Hiroi and McCormick, 2007). In the present study there was a

Fig. 5. Mean (±SEM) number of chloride cells (mm− 1 of gill filament) (A) and cell diameter ([max diameter, D+min diameter, d]/2) of chloride cells (µm) in shi drum (n=5) reared at different salinities (4, 10 and 40 psu) during a period of 84 days. The results of the two-way ANOVA are indicated on the graphs. Different capital letter superscripts indicate significant differences (Pb0.001) between sampling times, regardless of salinity; whereas lower letter superscripts indicate significant differences among salinity treatments, within the specific sampling time. n/a=not available. Author's personal copy

C.C. Mylonas et al. / Aquaculture 287 (2009) 203–210 209 significant increase in chloride cell numbers related to age, but there was Brown, P., 1992. Gill chloride cell surface-area is greater in freshwater-adapted adult sea fi trout (Salmo trutta, L.) than those adapted to seawater. Journal of Fish Biology 40, no signi cant difference among shi drum reared at the three different 481–484. salinities. These results are in accordance with studies in Adriatic Cardellini, P., Franceson, A., Zanella, S., Bozzato, G., Benedetti, P., Borgoni, N., Barbaro, A., sturgeon (A. naccarii) (Cataldi et al., 1995; McKenzie et al., 1999) or sea 1999. Captive rearing of shi drum, Umbrina cirrosa (L.), in different thermal – Salmo trutta conditions. Biologia Marina Mediterranea 6, 287 290. trout ( ) (Brown, 1992) acclimated to saltwater, but are Carmona, R., García-Gallego, M., Sanz, A., Domezaín, A., Ostos-Garrido, M.V., 2004. Chloride contrary to what has been reported in other fishes (McCormick et al., cells and pavement cells in gill epithelia of Acipenser naccarii: ultrastructural 2003; Carmona et al., 2004). More intuitive, however, were the observed modifications in seawater-acclimated specimens. Journal of Fish Biology 64, 553–566. increases in chloride cell size in shi drum adapted to 40 psu compared to Cataldi, E., Ciccotti, E., Di Marco, P., Di Santo, O., Bronzi, P., Cataudella, S., 1995. Acclimation trials of juvenile Italian sturgeon to different salinities: morpho-physiological 4 psu, as reported also in the guppy (Poecilia reticulata) (Shikano and descriptors. Journal of Fish Biology 47, 609–618. Fujio, 1999), Mozambique tilapia (Oreochromis mossambicus) (Uchida Conides, A.J., Glamuzina, B., 2006. Laboratory simulation of the effects of environmental et al., 2000), killifish (Fundulus heteroclitus) (Karnaky et al., 1976b) and salinity on acclimation, feeding and growth of wild-caught juveniles of European sea bass Dicentrarchus labrax and gilthead seabream Sparus aurata. Aquaculture salmonids (Uchida et al., 1996; Hiroi and McCormick, 2007). Such 256, 235–245. increases in chloride cell size are attributed to the considerable De Silva, S.S., Perera, P.A.B., 1976. Studies on the young grey mulet, Mugil cephalus L. I. enlargement of the basolateral tubular system, which is related to an Effects of salinity on food intake, growth and food conversion. Aquaculture 7, 327–338. + + Dendrinos, P., Thorpe, J.P.,1985. Effects of reduced salinity on growth and bodycomposition increased activity of Na /K -ATPase (Karnaky et al.,1976a,b; Laiz-Carrión in the European bass Dicentrarchus labrax (L.). Aquaculture 49, 333–358. et al., 2005a). Furthermore, the presence of a distinct apical crypt in shi Doroudi, M.S., Fielder, D.S., Allan, G.L., Webster, G.K., 2006. Combined effects of salinity drum reared in 40 psu in the present study has been described as a and potassium concenration on juvenile mulloway (Argyrosomus japonicus, Temmicnck and Schlegel) in inland saline groundwater. Aquaculture Research 37, distinctive feature of chloride cells of marine teleosts, or as a structural 1034–1039. change that occurs in this cell when a euryhaline species passes from EEC, 1986. Council Directive 86/609 EEC for the protection of animals used for fresh water to saltwater (Hossler et al., 1979a,b; Foskett et al., 1983). As experimental and other scientific purposes. Official Journal L358, 1–28. Ferguson, H.W., Morrison, D., Ostland, V.E., Lumsden, J., Bryne, P., 1992. Responses of expected, shi drum reared in 4 psu water showed no apical crypt and had mucus-producing cells in gill disease of rainbow trout (Oncorhynchus mykiss). smaller chloride cells. On the contrary, shi drum reared at 10 psu for Journal of Comparative Pathology 106, 255–265. 42 days had significantly smaller chloride cells compared to both fish Fielder, D.S., Bardsley, W., 1999. A preliminary study on the effects of salinity on growth reared at 4 and 40 psu, similar to what has been reported in gilthead sea and survival of mulloway Argyrosomus japonicus larvae and juveniles. Journal of the World Aquaculture Society 30, 380–387. bream (Laiz-Carrión et al., 2005a). It has been speculated, that this could Foskett, J.K., Bern, H.A., Machen, T.E., Conner, M., 1983. Chloride cells and the hormonal be due to a lower need for ion pumps required in fish reared at iso- control of teleost fish osmoregulation. Journal of Experimental Biology 106, 255–281. osmotic media (Laiz-Carrión et al., 2005a). Overall, in view of some of the Franklin, C.E., Forster, M.E., Davison, W., 1992. Plasma cortisol and osmoregulatory changes in sockeye salmon transferred to sea water: comparison between discrepancies observed in the results of the present study, further successful and unsuccessful adaptation. Journal of Fish Biology 41, 113–122. physiological, ultrastructural and molecular studies are necessary to Gardes, L., Villanove, P., Buchet, V., Fauvel, C., 2000. Induced spawning of red drum, study more thoroughly the role of chloride cells in shi drum adaptation Sciaenops ocellatus: use of multivariate and univariate analysis methods in the search for side effects of LH-RHa treatments and ovarian development state upon to different salinities. spawn quality. Aquatic Living Resources 13, 19–27. In conclusion, the present study indicates that shi drum acclimated Hiroi, J., McCormick, S.D., 2007. Variation in salinity tolerance, gill Na+/K+-ATPase, Na+/K+/ − to full seawater or nearly isoosmotic water do not phase any 2Cl cotransporter and mitochondria rich cell distribution in three salmonids Salveli- fi nus namycush, Salvelinus fontinalis, and Salmo salar. The Journal of Experimental osmoregulatory imbalance, while sh reared at 4 psu displayed Biology 210, 1015–1024. osmoregulatory difficulties and low growth performance. The ability Hirose, S., Kaneko, T., Naito, N., Takei, Y., 2003. Molecular biology of major components of shi drum to utilize environments of lower salinity, up the iso- of chloride cells. Comparative Biochemistry and Physiology 136B, 593–620. Hossler, F.E., Ruby, J.R., McIlwain, T.D., 1979a. The gill arch of the mullet Mugil cephalus I. osmotic point, opens new possibilities in the development of shi drum surface ultrastructure. Journal of Experimental Zoology 208, 379–398. aquaculture in coastal lagoons and estuaries, thus further contributing Hossler, F.E., Ruby, J.R., McIlwain, T.D., 1979b. The gill arch of the mullet Mugil cephalus to the sustainable diversification of the aquaculture industry. II. modifications in surface ultrastructure and Na.K-ATPase content during adaptation to various salinities. Journal of Experimental Zoology 208, 399–406. Imsland, A.K., Foss, A., Gunnarsson, S., Berntssen, M.H.G., FitzGerald, R., Bonga, S.W., von Acknowledgements Ham, E., Nævdal, G., Stefansson, S.O., 2001. The interaction of temperature and salinity on growth and food conversion in juvenile turbot (Scophthalmus maximus). Financial support for this study has been provided by a grant to Aquaculture 198, 353–367. Jensen, M.K., Madsen, S.S., Kristiansen, K., 1998. Osmoregulation and salinity effects on CCM under the 2003–2005 Programme for Research and Technology the expression and activity of Na+/K+ ATPase in the gills of the European sea bass Cooperation between the Republics of Greece and Cyprus (Project Dicentrarchus labrax (L.). Journal of Experimental Zoology 282, 290–300. fi “ICHTHIONIMFI”, Protocol number 2206; 26 February, 2004), from the Karnaky, K.J., Ernst, S.A., Philpott, C.W., 1976a. Teleost chloride cell I. Response of pup sh Cyprinodon variegatus gill Na+, K+-ATPase and chloride cell fine structure to various General Secretariat for Research and Technology, Greece and the high salinity environments. Journal of Cell Biology 70, 157–177. Foundation for Promotion of Research, Cyprus. Karnaky, K.J., Kinter, L.B., Kinter, W.B., Stirling, C.E., 1976b. Teleost chloride cell II. Autoradiographic localization of gill Na+,K+-ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments. 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