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Title: Growth performance and osmoregulation in the shi drum (Umbrina cirrosa) adapted to different environmental salinities
Article Type: Research Paper
Keywords: shi drum; Umbrina; osmoregulation; growth; chloride cells; gill Na+/K+-ATPase; chloride cells
Corresponding Author: Dr. Constantinos Chrysanthos Mylonas, Ph.D.
Corresponding Author's Institution: Hellenic Center for Marine Research
First Author: Constantinos C Mylonas, Ph.D.
Order of Authors: Constantinos C Mylonas, Ph.D.; Michalis Pavlidis; Nikos Papandroulakis; Mario M Zaiss; Dimitris Tsafarakis; Stamatis Varsamos
Abstract: In order to investigate the ability of shi drum (Umbrina cirrosa) to be reared at diverse locations, growth and osmoregulatory performance were assessed at full-strength seawater (40 psu), nearly iso- osmotic water (10 psu) and low salinity, hypo-osmotic water (4 psu). At the end of the 84-day experimental period, fish reared at 4 psu 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 reflected by changes in plasma triglycerides and cholesterol, with fish reared at 4 psu exhibiting the lowest mean concentrations, while there was no significant difference in mean plasma glucose concentrations among treatments. Plasma osmolality was lower at 4 psu from day 42 onwards, while there was no significant difference in mean plasma K+ and Cl- concentrations. Plasma sodium and 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. The total number of chloride cells did not differ between the three salinities, however differences in their location were found. Our results indicate that shi drum reared from full-strength seawater to nearly iso-osmotic salinity do not face any osmoregulatory imbalance, while fish reared in hypo-osmotic water displayed osmoregulatory impairment and low growth performance.
Suggested Reviewers: Gert Flik Ph.D. Head of Department, Animal Physiology, University of Nijmegen [email protected]
Steven McCormick Ph.D. Section leader, Physiology, U.S. Geological Survery, Conte Anadromous Fish Research Center [email protected]
Opposed Reviewers: Manuscript
2
Growth performance and osmoregulation in the shi drum (Umbrina cirrosa)
4 adapted to different environmental salinities
6 Constantinos C. Mylonas1,* Michalis Pavlidis2, Nikos Papandroulakis1, Mario M. Zaiss1,
Dimitris Tsafarakis1,2 and Stamatis Varsamos3, #
8
10 1 Institute of Aquaculture, Hellenic Center for Marine Research, P.O. Box 2214, Iraklion,
Crete 71003, Greece
12 2 Department of Biology, University of Crete, P.O. Box 2208, Iraklion, Crete 71409,
Greece
14 3 Université Montpellier II, SkuldTech, CC 091, Place Eugène Bataillon, 34095,
Montpellier, France
16
18
20 * Corresponding author: tel +30 2810 337878, fax +30 2810 7825,
22 # Current address: European Commission, DG Fisheries and Maritime Affairs, Unit A3.
Research, Data Collection and Scientific Advice, Rue Joseph II 79, 2/34, B 1049 Comment [C1]: Check if OK 24 Brussels.
1 Abstract
26 In order to investigate the ability of shi drum (Umbrina cirrosa) to be reared at
diverse locations, growth and osmoregulatory performance were assessed at full-strength
28 seawater (40 psu), nearly iso-osmotic water (10 psu) and low salinity, hypo-osmotic
water (4 psu). At the end of the 84-day experimental period, fish reared at 4 psu
30 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
32 salinity on growth performance was also reflected by changes in plasma triglycerides and
cholesterol, with fish reared at 4 psu exhibiting the lowest mean concentrations, while
34 there was no significant difference in mean plasma glucose concentrations among
treatments. Plasma osmolality was lower at 4 psu from day 42 onwards, while there was
36 no significant difference in mean plasma K+ and Cl- concentrations. Plasma sodium and
gill Na+/K+-ATPase activity showed minimum values on day 42 at 4 psu, but at the end
38 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
40 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. The
42 total number of chloride cells did not differ between the three salinities, however
differences in their location were found. Our results indicate that shi drum reared from
44 full-strength seawater to nearly iso-osmotic salinity do not face any osmoregulatory
imbalance, while fish reared in hypo-osmotic water displayed osmoregulatory
46 impairment and low growth performance.
48 Key words: shi drum, Umbrina, osmoregulation, growth, chloride cells, gill Na+/K+-
ATPase, chloride cells
2 50 Introduction
Euryhaline fishes exhibit the ability to maintain relatively constant ionic
52 composition of plasma, lymph and interstitial fluid, in a broad range of environmental
salinities (Marshall and Grosell, 2006). This is achieved through morphological, cellular,
54 physiological and endocrine adaptations (McCormick and Bradshaw, 2006; Sakamoto
and McCormick, 2006; Shane et al., 2006). The strategy of ion exchange with the
56 external medium is mediated mainly through the gastrointestinal epithelium, kidney and
gills (Marshall and Grosell, 2006). In adult teleost fish, the gills are recognized as the
58 main site for ion exchange (Wilson and Laurent, 2002) mediated by different
transporters, mainly Na+/K+-ATPase (McCormick, 1995; McCormick, 2001), and ion
60 channels located in the chloride cells, which are also called mitochondria-rich cells
(MRCs) or more generally ionocytes (Pisam and Rambourg, 1991; Hirose et al., 2003;
62 Marshall and Grosell, 2006).
Adaptation of euryhaline fish to different salinities involves changes in ion
64 transport mechanisms that generally induce changes in oxygen consumption, suggesting
variations in the energetic demands for osmoregulation (Morgan and Iwama, 1991).
66 Several scenarios have been suggested to describe changes in oxygen metabolism in fish
following salinity challenge that, in some cases, can result in altered or enhanced growth
68 (Boeuf and Payan, 2001). The relationship between osmoregulation and growth
performance in marine fish has stimulated a large amount of research and, so far, it
70 seems to be species-specific (Boeuf and Payan, 2001). Each fish species seems to have
its own salinity optimum for growth, and osmoregulatory imbalance is often associated
72 with whole animal-level stress changes, including decreased growth performance (De
Silva and Perera, 1976; Boeuf and Payan, 2001; Imsland et al., 2001).
3 74 Some members of the family Sciaenidae (Nelson, 2006) display a great potential
for aquaculture, and some have been reared in commercial facilities for some time.
76 These include the red drum (Sciaenops ocellatus) in the United States (Miranda and A.J.,
1985; Thomas et al., 1995; Gardes et al., 2000), the mulloway (Argyrosomus japonicus)
78 in Australia (Battaglene and Talbot, 1994; Fielder and Bardsley, 1999), and the maigre
(Argyrosomus regius) in Europe (Pastor Garcia et al., 2002; Poli et al., 2003). Another
80 Sciaenid species of interest in the eastern part of the Mediterranean Sea is the shi drum
(Umbrina cirrosa) (Cardellini et al., 1999; Barbaro et al., 2002; Mylonas et al., 2004;
82 Zaiss et al., 2006). Sciaenid fishes are considered as euryhaline species (Miranda and
A.J., 1985; Fielder and Bardsley, 1999; Doroudi et al., 2006), therefore they could be
84 considered for aquaculture diversification in coastal lagoons with brackish water and
areas near the mouth of large rivers, where the commonly employed marine species are
86 not suitable. In the case of the shi drum, as well as some of the other sciaenids with
aquaculture potential, there is a complete lack of knowledge on its adaptation potential
88 and growth performance in different salinities.
The objective of the present work was to study the osmoregulatory capacity and the
90 interaction between salinity adaptation and growth performance in shi drum, in order to
provide scientific background for the development of optimized husbandry practices for
92 this species. For this purpose, fish were reared for three months at different salinities
representing full-strength seawater (40 psu), nearly iso-osmotic water (10 psu) and hypo-
94 osmotic water (4 psu), and changes in gill histology, osmoregulation, growth and
energetic metabolism related parameters were evaluated.
96
98 2. Material and Methods 2.1. Animal husbandry and experimental design
4 100 Experiments were conducted using shi drum juveniles at the facilities of the
Institute of Aquaculture, Hellenic Centre for Marine Research (Crete, Greece). Fish
102 were produced in July 2004 from wild-caught, captive-reared broodstock, after hormonal
induction of spawning with gonadotropin-releasing hormone agoinst (GnRHa) implants
104 (Mylonas et al., 2004). Fish with mean ± S.D. total length (TL) of 11.8 ± 0.8 cm and
body weight (BW) of 22.8 ± 4.5 g were reared in three independent recirculating
106 aquaculture systems (RAS) at different salinities (40, 10 and 4 psu). Each RAS was
fitted with mechanical and biological filters, and supported two replicated 500-l
108 cylindroconical polyester tanks containing the experimental fish (n =30). The
recirculated water was maintained at 19 ± 1ºC and >80% oxygen saturation throughout
110 the course of the experiment (84 days), and new water exchange rate was 30% every 2-3
d. Fish were fed ad libitum with commercial feed (1st period EXCEL, SKRETTING) by
112 the use of a demand-feeder and the photoperiod was fixed at 24L:0D throughout the
experiment.
114 After randomly allocating the fish in their tanks (n = 30) at 40 psu, two replicated
groups were acclimated gradually to the lower experimental salinities over the course of
116 6 d. Total length and BW measurements were taken from all fish in each tank at 0, 14,
28, 42, 56, 70 and 84 days from the start of the experiment. Blood samples were taken
118 initially (day 0) from ten fish from the stock population and then from five fish from each
tank at 46 and 84 d. Blood was collected from the caudal vusculature with heparinized
120 syringes, centrifuged at 5,000 rpm for 15 min at 4C, and the plasma was stored at -80C
until further analysis. Five fish from each treatment group were also sacrificed at 46 and
122 84 days, the second gill arch was excised for histological analysis and the third gill arch
was quickly stored at -80°C for determination of Na+/K+-ATPase activity (see later for
124 method). For all measurements, fish were first anaesthetized in a solution of 0.4 ml L-1
5 of clove oil (Mylonas et al., 2005), dissolved first 1:10 v/v in 96% EtOH. Fish handling
126 was carried out according to the European Union Directive (86/609EEC) for the
protection of animals used for experimental and other scientific purposes (EEC, 1986)
128 and the "Guidelines for the treatment of animals in behavioural research and teaching"
(Anonymous, 1998).
130
2.2. Biochemical analyses
132 Plasma metabolites were determined by enzymatic colorimetric procedures
(glucose GOD/PAP; cholesterol PAP; triglycerides GPO/PAP) using commercial kits
134 (Biosis, Greece). Osmolality was measured using a cryoscopic osmometer (Gonotec,
Osmomat 030). Plasma samples were diluted 1:600 with lithium nitrate 15 mmol L-1 and
136 electrolytes (K+, Na+) were determined by flame emission spectrophotometry. Chloride
ions were determined by spectrophotometric method based on the reaction of Cl- with
138 mercuric thiocyanate.
Gill Na+/K+-ATPase activity was determined according to Varsamos et al. (2004).
140 Briefly, gill arches were homogenized with an IKA disperser (IKA®Yellowline, USA), at
9,500 rpm for 5 sec, with 1 ml of buffer containing: 250 mM sucrose, 10 mM Hepes, 5
142 mM MgCl2, 0.1 mM Na2-EDTA, dH2O (pH 7.4). The homogenate was centrifuged
(4,000 rpm, 5 min, 4C) and the supernatant used for assays. Homogenate protein
144 content was determined in triplicate as detailed by Bradford (1976), using BSA (Sigma,
A-4503) as standard. The Na+/K+-ATPase activity was assessed as the difference of total
146 ATP hydrolysis (in presence of Na+, K+, Mg2+ and ATP) and that in absence of K+ but in
presence of an optimal concentration of the specific inhibitor ouabain (1 mg ml-1). The
148 amount of released phosphate was assessed by comparison with commercial reference
standards (Calimat, BioMérieux, France). The Na+/K+-ATPase specific activity was
6 150 expressed in μmol Pi h-1 mg-1 protein. The total activity was calculated as the product of
the specific activity and the total protein amount of the sample and expressed in μmol Pi
152 h-1.
154 2.5. Histological analyses
A section of the second gill arch was fixed in 4% formaldehyde: 1% gluteraldehyde
156 buffered saline (McDowell and Trump, 1976) for at least 24 h. Before embedding in a
methacrylate resin (Technovit 7100®, Heraeus Kulzer, Germany) they were dehydrated
158 through a series of increasing concentrations of ethanol (70-96%). The mounted gills
were cut in 3-μm thick sections on a microtome (RM 2245, Leica, Germany), stained
160 with Methylene Blue (Sigma, Germany)/Azure II (Sigma, Germany)/Basic Fuchsin
(Polysciences, USA)(Bennett et al., 1976) and examined under a light microscope (BH-2,
162 Olympus, Japan). In addition, four sections per gill sample were stained with the
combined Alcian blue - periodic acid Schiff (AB-PAS) technique for neutral and acid
164 mucins (Mowry, 1956) which stains mucus cells specifically and allows identification of
different types of mucins (Shephard, 1994).
166 Chloride cell counts were made on histological sections of primary lamellae, where
both the cartilaginous vestige of the gill septum was visible and the primary lamella was
168 sectioned in the middle, along its whole longitudinal axis. Chloride cell counts were
made along 1 mm length of three different primary lamellae (starting from the base of
170 each primary lamella on the gill arch) and the average value was used to represent each
fish sampled. It was not possible to make reliable mucus cell counts, because different
172 sections of the gill filament contained markedly different numbers.
174 2.2. Estimation of growth parameters and statistical analyses
7 Comparisons of TL and BW between groups reared at different salinities were
176 made using regression analysis (Sokal and Rohlf, 1981). The general model used was of
the form: Y a0 a1 t a2 D a3 t D , where Y is the dependent variable, t the time,
178 D a dummy variable with values 0 and 1 for each condition tested and αi (I = 1, 2 or 3)
constants. This method tests the hypothesis that the constants α2 and α3 are zero. Time
180 series have the same slope when constant α3 is zero, and the same initial value when α2 is
zero. When both constants are zero, time series describe similar dependent variables.
182 Specific growth rate (SGR) was estimated as the slope of the above model. Food
conversion ratio (FCR) was estimated as FCR = F / (Bf – Bi), where F = consumed food
184 (kg), Bf = final biomass (kg), Bi = initial biomass (kg).
To evaluate differences in FCR, plasma metabolites, electrolytes, osmolality, gill
186 Na+/K+-ATPase activity and chloride cell density, data were subjected to 2-way Analysis
of Variance (ANOVA) (time x salinity), followed by Duncan’s New Multiple Range
188 (DNMR) test on main effects, and mean comparisons for interactions. Statistical
analysis was performed using linear statistics software (SuperAnova, Abacus Concepts
190 or SigmaStat, Systat Software, Inc.) at a minimum significance level of P < 0.05. All
results are presented as mean ± SEM.
192
194 3. Results 3.1. Growth performance of shi drum in different salinities
196 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
198 or appetite were observed. However, salinity did have a statistically significant effect on
growth (P < 0.05), with fish reared at 4 psu having significantly smaller TL and BW than
200 the fish reared at 10 or 40 psu, whereas there were no differences between fish reared at
8 10 or 40 psu salinity (Fig. 1). The final mean BW of shi drum reared at the three
202 salinities were 63.3 ± 3.2, 67.5 ± 2.1 and 44.5 ± 2.2g for 40, 10 and 4 psu, respectively.
As expected from the above measurements, SGR over the course of the study was also
204 affected by environmental salinity, and in fish reared at 4 psu was 0.265 d-1, significantly
lower (P < 0.05) than of fish adapted to 10 or 40 psu, which was the same at 0.513 d-1.
206 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
208 not be explained by the experimental design. Nevertheless, the statistical analysis over
the course of the study indicated the existence of a significant difference (P < 0.05)
210 between the FCR of fish reared at 4 psu and the FCR of fish reared at 10 or 40 psu,
(Table 1). The differences were due to higher values of the 4 psu fish during the first 56
212 days, when FCR ranged between 1.36 ± 0.14 and 4.71 ± 2.2, compared to a range of 1.06
± 0.05 and 1.78 ± 0.07 of fish reared at 10 or 40 psu salinity. During the period between
214 57 and 84 days after the onset of the experiment no significant difference in FCR was
found among the three salinities.
216
3.2. Plasma metabolites, osmolality, electrolytes and gill Na+/K+-ATPase activity
218 There were no significant differences in mean plasma glucose concentration among
fish reared at the different salinities and fish reared in all salinities had significantly (P <
220 0.05) higher glucose levels at the onset of the experiment (Table 2). In all groups,
increased triglycerides concentrations were found at 42 d and reached a maximum at 84
222 d. Maximum cholesterol concentrations at 10 and 40 psu salinity were found at 42 d,
while at 4 psu salinity at 0 d and 42 d (Table 2). Plasma triglycerides and cholesterol
224 concentrations were significantly lower (P < 0.05) in fish reared at 4 psu than in the other
two groups at both sampling points (Table 2).
9 226 Plasma osmolality was significantly lower (P < 0.05) in fish reared at 4 psu than in
the other two groups at 42 d after the start of the experiment, but not at the end of the
228 experiment, after 84 d (Table 3). There also appeared to be a gradual increase in plasma
osmolality over the course of the experiment, regardless of environmental salinity. In
230 terms of plasma electrolytes, there was no difference in plasma K+ or Cl- concentrations
among fish reared at different salinities, in either sampling times, though some variation
232 existed (Table 3). On the contrary, significantly (P < 0.05) lower plasma Na+
concentrations were found 42 d after the start of the experiment in fish reared at 4 psu
234 compared to fish reared at 10 or 40 psu. However, by the end of the experimental period
there was no difference in plasma Na+ concentrations among the three groups.
236 Gill Na+/K+-ATPase specific activity 42 d after the onset of the experiment was
significantly lower (P < 0.05) in fish reared at 4 psu salinity, compared to that measured
238 either in 10 or 40 psu (Figure 2). However, at the end of the experimental period a
significant increase in the gill Na+/K+-ATPase activity was recorded in fish reared at 4
240 psu salinity and no difference in this parameter was found at that sampling point among
fish reared at different salinities.
242
3.3. Gill morphology in different salinities
244 Pavement cells, mucus cells and chloride cells were identified on the gill epithelium
of fish from all salinities (Fig. 3). In fish reared at 40 psu, most mucus cells contained
246 mostly acid mucins, which stained blue-purple with AB-PAS, whereas in fish reared at 4
psu the mucus cells contained mostly neutral mucins, which stained red (data not shown).
248 At the near-isoosmotic salinity of 10 psu there did not seem to be a dominance of any of
the two types of mucins. Mucus cells were observed both on the primary and secondary
250 lamellae of the gill filaments, as well as the gill arch epithelium itself.
10 The total number of chloride cells did not differ significantly between the three
252 experimental salinities, even though at 42 d a slight reduction (P = 0.10) was observed in
the fish reared at 4 psu (Fig. 3). However, there was a significant increase (P < 0.01)
254 over the course of the study, regardless of treatment (Fig. 3). Chloride cells were most
frequently observed on the primary lamellae of the gill filaments (Fig. 4). Although not
256 often, in fish reared at 4 psu, chloride cells were also found on the epithelium of the
secondary lamellae. In fish reared at 40 psu salinity, chloride cells appeared to be greater
258 in size compared to fish adapted to 4 or 10 psu (dat not shown). In fish reared at 4 psu, a
thickening of the primary and secondary lamellar epithelium was observed compared to
260 both fish reared at 10 psu and 40 psu at both sampling times (Fig. 4C).
262
4. Discussion
264 It has been shown that several euryhaline teleosts display a two-stage adaptation
pattern following salinity challenge, consisting of an adaptive period when changes in
266 osmotic and metabolic parameters occur, and of a regulatory period which generally
leads to a new steady state (Jensen et al., 1998; Sangiao-Alvarellos et al., 2005). Our
268 data have clearly showed that shi drum juveniles are able to maintain constant plasma
osmolality and electrolyte levels over a relatively range of environmental salinities and
270 during a long period. However, it appears that rearing at very low salinities (4 psu)
affects important physiological functions such as osmoregulation and growth. Thus,
272 body size, SGR and FCR were lower in shi drum exposed to 4 psu salinity compared to
those reared at 10 or 40 psu salinity. The blood osmolality of teleosts is around 12 psu,
274 i.e. approximately one third of that of sea water (~38 psu) and more than 50-fold higher
than the salinity of freshwater (~0,2 psu). It is generally admitted that marine fish
11 276 achieve higher growth rates at lower salinity (Boeuf and Payan, 2001) and that each
species has its own salinity optimum for growth, under a certain water temperature and
278 ontogenetic phase, although existing data are sometimes contradictory. For instance, the
best salinity conditions for growth in gilthead sea bream (Sparus aurata) have been
280 reported at 12 psu (Laiz-Carrión et al., 2005) or 28 psu (Klaoudatos and Conides, 1996),
in grey mullet (Mugil cephalus) at 20 psu (De Silva and Perera, 1976), in turbot
282 (Scopththalmus maxumus) at 15 psu (Imsland et al., 2001) and in European sea bass
(Dicentrarhus labrax) at 15 psu (Saillant et al., 2003) or 28-30 psu (Dendrinos and
284 Thorpe, 1985; Conides and Glamuzina, 2006). The present results indicate that salinity
affects FCR and growth in shi drum juveniles adapted to low salinity. The metabolic and
286 energetic cost of osmoregulation should, at least partly, reflect the effect of salinity on
growth, however other possibilities should be taken into account. This may include the
288 effect of salinity on feeding behavior, appetite, or stimulation/inactivation of other
metabolic and endocrine pathways (Boeuf and Payan, 2001; McCormick, 2001).
290 To our knowledge this is the first report on plasma cholesterol and triglyceride
concentrations for shi drum, and the values reported are within the range measured in
292 other marine species (Kavadias et al., 2003). Interestingly, both parameters seemed to
reflect metabolic effects of long term salinity adaptation in this species, whereas plasma
294 glucose levels where unaffected by salinity treatments. Thus, the low growth
performance of shi drum reared at 4 psu was concomitant with lower plasma cholesterol
296 and triglyceride concentrations compared to those measured in fish reared at 10 or 40
psu, suggesting a negative effect of low salinity on energy metabolism. It has been
298 already suggested that changes in plasma metabolites during the first days of salinity
challenge are related to modifications in energy supplies in osmoregulatory tissues
300 (Sangiao-Alvarellos et al., 2005). For example, in gilthead sea bream plasma triglyceride
12 did not show significant changes following acclimation to different salinities (i.e., 6, 12,
302 38 psu) for 100 d, although growth and SGR were correlated to salinity (12>38>6 psu)
(Laiz-Carrión et al., 2005). However, in the same species, a 20 and 25% decrease in
304 plasma glucose and triglyceride levels, respectively, was observed after adaptation to 6
psu salinity, which was then followed by full recovery within 7 d (Sangiao-Alvarellos et
306 al., 2005). The low cholesterol levels recorded in shi drum adapted to 4 psu salinity
could be explained partly by differences in feeding rate compared to the other
308 experimental salinities. That kind of correlation between the feeding rate and plasma
cholesterol levels has been reported in European seabass (Lemaire et al., 1991) as well as
310 in Eurasian perch (Perca fluviatilis) (Vellas et al., 1994). The absence of differences in
glucose levels among the salinity treatments in the present experiments seems surprising,
312 since the role of glucose as fuel for tissues during osmotic challenge or stress conditions,
in general, is well known (Wendelaar Bonga, 1997). However, in long term adaptation,
314 such as in the present study, it seems that the involvement of this metabolite is less
discriminating, and similar results have been obtained in gilthead sea bream (Laiz-
316 Carrión et al., 2005).
The results obtained in the present experiment indicate that shi drum reared at 40 or
318 10 psu salinity for 84 days did not phase major osmoregulatory imbalance, based on the
fact that there were no significant differences in plasma osmoregulatory parameters. On
320 the contrary, fish reared at 4 psu displayed osmoregulatory difficulties, as indicated by
the observed lower plasma Na+ after 42 d and osmolality after 42 and 84 d. Low Na+
322 levels have also been reported in the gilthead sea bream after 100 d rearing at 6 psu
salinity (Laiz-Carrión et al., 2005) and in European sea bass 24 h after transfer to
324 salinities less than 20 psu (Venturini et al., 1992). Difficulties in maintaining
homeostasis in low salinities have also been reported in other euryhaline teleost species,
13 326 such as the sheepshead minnow (Cyprinodon variegates) (Nordlie, 1985) and flounder
(Paralichthys orbignyanus) (Sampaio and Bianchini, 2002). Franklin et al. (1992)
328 suggested that such osmoregulatory impairments indicate salinity stress, whereas the
negative effect of stress on growth is well documented in teleosts (Wendelaar Bonga,
330 1997).
It is generally admitted that gills are the organs that consume most energy during
332 adaptation of teleosts in different salinities, since they must maintain differential
regulation of intracellular and extracellular fluids. Moreover, in case of transition from
334 hypoosmotic to hyperosmotic 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+-
336 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
338 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
340 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
342 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
344 direct relationship between salinity and Na+/K+-ATPase activity has been shown in some
teleosts (McCormick, 1995; McCormick, 2001; Marshall, 2002). Nevertheless, at the
346 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
348 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
350 d at low salinity corresponds to low osmoregulatory cost. Rather, a transient alteration in
14 cellular metabolism and in the function of membrane exchangers/transporters could be
352 involved, due to salinity-induced stress. Hence, unlike other euryhaline teleosts, it
appears that the adaptation period following low salinity acclimation in shi drum
354 juveniles is much longer and displays a different pattern. Moreover, other transporters,
such as H+-ATPase (Lin and Randall, 1995) or Ca+-ATPase (Marshall, 2002), could be
356 involved in low salinity adaptation of shi drum.
In fish, the gill epithelium consists mainly of three types of cells, namely the
358 pavements cells (representing 90–95% of the total epithelial area), the chloride cells and
the mucus cells (Shephard, 1994; Wilson and Laurent, 2002). In shi drum reared at full
360 seawater, most mucus cells of the gills stained purple indicating a mixture of acid and
neutral mucins. Similar results were reported in other species, such as the gilthead sea
362 bream, Senegal sole (Solea senegalensis) and Siberian sturgeon (Acipenser barei)
(Sarasquete et al., 2001). On the contrary, most mucus cells of shi drum exposed to
364 hypo-osmotic environment stained reddish, indicating that they consisted mainly of
neutral mucins, a result similar to that documented for species maintained in freshwater,
366 such as the rainbow trout (Oncorhynchus mykiss) (Ferguson et al., 1992) and Atlantic
salmon (Salmo salar) (Roberts and Powell, 2003). Our findings suggest that changes in
368 the mucin production of gill mucus cells, could be part of the salinity adaptation process
in the shi drum.
370 Gill chloride cells are recognized as the main site for ion exchange mediated by
different transporters and ion channels (Lin and Randall, 1995; Marshall, 2002; Marshall
372 and Singer, 2002; McCormick and Bradshaw, 2006). Changes in the number and/or size
of these cells have been related to salinity acclimation in several teleost species
374 throughout the postembryonic development (Pisam and Rambourg, 1991; Varsamos et
al., 2002). In the present study there was no significant difference in the chloride cell
15 376 density among shi drum reared at the three different salinities. This is in accordance with
findings from studies in total chloride cells counts in Adriatic sturgeon (A. naccarii)
378 (Cataldi et al., 1995; McKenzie et al., 1999) or sea trout (Salmo trutta) (Brown, 1992)
after acclimation from freshwater to saltwater. Future ultrastructural and molecular
380 studies are necessary to study more thoroughly the role of these cells in shi drum
adaptation to different salinities.
382 In conclusion, the present study indicates that shi drum acclimated to full seawater or
nearly isoosmotic water do not phase any osmoregulatory imbalance, while fish reared at
384 4 psu displayed osmoregulatory difficulties and low growth performance. The ability of
shi drum to utilize environments of lower salinity, up the iso-osmotic point, opens new
386 possibilities in the development of shi drum aquaculture in coastal lagoons and estuaries,
thus further contributing to the sustainable diversification of the aquaculture industry.
388
390 5. Acknowledgements
Financial support for this study has been provided by a grant to CCM under the
392 2003-2005 Programme for Research and Technology Cooperation between the Republics
of Greece and Cyprus (Project “ICHTHIONIMFI”, Protocol number 2206; 26 February,
394 2004), from the General Secretariat for Research and Technology, Greece and the
Foundation for Promotion of Research, Cyprus.
16 396
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24 590
Table 1. Mean (± SEM) food conversion ratio (FCR = F / [Bf – Bi], where F =
592 consumed food, Bf = final biomass, Bi = initial biomass) of shi drum reared at different
salinities (4, 10 and 40 psu) in duplicated tanks (i.e., n = 2). Significant differences (P <
594 0.05) at different sampling points and over the whole course of the study between the 4
psu, and the 10 or 40 psu group are indicated by different letter superscripts next to the
596 mean values. Within each salinity treatment, there were no differences in FCR between
different sampling times (P = 0.19)
598 FCR
Salinity 4 psu 10 psu 40 psu
600 Time period (d)
0-14 3.39 ± 1.89a 1.78 ± 0.05a,b 1.29 ± 0.14b
602 15-28 3.32 ± 0.22a 1.13 ± 0.08b 1.06 ±0.05b
29-42 1.36 ± 0.14 1.21 ± 0.14 1.25 ± 0.02
604 43-56 4.71 ± 2.20a 1.68 ± 0.26b 1.42 ± 0.02b
57-70 1.28 ± 0.04 1.31 ± 0.21 1.12 ± 0.21
606 70-84 1.54 ± 0.26 1.54 ± 0.40 1.58 ± 0.68
608 Whole duration (d)
0-84 2.60 ± 0.53 a 1.44 ± 0.10 b 1.28 ± 0.10 b
610
25 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). Different small letter superscripts indicate significant differences (P < 0.05) between different sampling times within salinity treatments, while capital letter superscripts indicate 615 significant differences between different salinities within sampling time.
4 psu 10 psu 40 psu
0 42 84 0 42 84 0 42 84
Glu 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 (mmol l-1)
Chol 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 (mmol l-1)
Trig 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 (mmol l-1)
26 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). Different small letter superscripts indicate 620 significant differences (P < 0.05) between different sampling times within salinity treatments, while capital letter superscripts indicate significant differences between different salinities within sampling time.
4 psu 10 psu 40 psu
0 42 84 0 42 84 0 42 84
Osmolality 350 ± 3a 338 ± 2aA 410 ± 7b 350 ± 3a 355 ± 2aB 398 ± 5b 350 ± 3a 353 ± 1aB 409 ± 2b (mOsm kg-1)
Na+ 196 ± 8 181 ± 5A 179 ± 5 196 ± 8 201 ± 5B 192 ± 3 196 ± 8 205 ± 8B 188 ± 6 (mmol l-1)
K+ 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 (mmol l-1)
Cl- 161 ± 7a 145 ± 1a 197 ± 5b 161 ± 7a 158 ± 2a 188 ± 3a 161 ± 7a 156 ± 1a 196 ± 1b (mmol l-1)
27 625 Figure legends
Figure 1. 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 84 days. Reduction of salinity to 4 psu had a statistically significant effect on
growth over the whole course of the study (regression analysis, P < 0.05), as indicated by
630 different letter superscripts next to the growth curves.
Figure 2. Changes in mean (± SEM) gill Na+/K+ -ATPase specific activity in shi drum
(n = 10) reared at different salinities during a period of 84 days. Different small letter
superscripts indicate significant differences (P < 0.05) between different salinities within
635 a sampling date. There were no significant differences between sampling times.
Figure 3. Number of chloride cells (mm-1 of gill filament) in shi drum (n = 5) reared at
different salinities (4, 10 and 40 psu) during a period of 84 days. Different capital letter
superscripts indicate significant differences (P < 0.05) between sampling times,
640 regardless of salinity. There were no significant differences between salinity treatments,
either at 42 or 84 days.
Figure 4. Histological sections of gill filaments (consisting of primary and secondary
lamellae) showing structural changes in the epithelium of shi drum reared at different
645 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 lamella, sl = secondary lamella. Bars
represent 50 µm.
28 Figure(s)
18
17 A a a 16 ) m c ( 15 b h t g
n 14 e L
l a
t 13 o T 12
11
10 0 20 40 60 80 100
70 a a 60 B ) g (
50 t h g
i b e 40 W
y d o 30 B 4 psu 20 10 psu 40 psu 10 0 20 40 60 80 100 Time (days)
Figure 1 Mylonas et al. 4 psu 10 psu 40 psu
2 b b 1.6
1.2
0.8 a
0.4
0 0 42 84 Time (days)
Figure 2 Mylonas et al. 4 psu 10 psu 40 psu
40 35 C B 30 25 20 15 A 10 5 0 0 42 84 Time (days)
Figure 3 Mylonas et al. AA BB CC sl sl sl pc cc cc
cc cc pl pl pl cc cc mc
Figure 4 Mylonas et al.