Journal of Experimental Marine Biology and Ecology 287 (2003) 103 117 www.elsevier.

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Saline preferendum for the European sea bass, Dicentrarchus labrax, larvae and juveniles: effect of salinity on early development and sex determination
Eric Saillant a,b,*, Alexis Fostier c, Pierrick Haffray d, atrice Chatain a Bruno Menu a, Be
Laboratoire de Recherche en Pisciculture Marine, IFREMER, Chemin de Maguelonne 34250, Palavas-les-Flots, France b Ecloserie Marine de Gravelines, Voie des Enrochements, 59820 Gravelines, France c et Environnement, Station Commune de Recherches en Ichtyophysiologie, Biodiversite INRA, Campus de Beaulieu, 35042 Rennes, France d SYSAAF, Section aquacole, Station SCRIBE, Campus de Beaulieu, 35042 Rennes, France Received 22 August 2002; received in revised form 31 October 2002; accepted 5 November 2002
a

Abstract The sea bass, Dicentrarchus labrax, is an eurhyaline marine fish. Juveniles of this species are known to frequent estuaries and lagoons where salinity is lower than in the open sea. Sex determination occurs during this phase of fish life and has been shown labile and sensitive to environmental factors. In this work, the effect of rearing salinity on sex determination and early development of the sea bass was investigated. An excess of males (87%) was found in all groups and the salinity level [(natural sea water salinity, mean: 37x ) vs. (15x )], when maintained constant, had no effect on the sex-ratio. The transfer from low to high salinity at 93 days post-fertilization (p.f.) increased the percentage of males (93%) suggesting that sexual differentiation in this species may be influenced by such an osmotic stress. Growth was improved by a 15xsalinity at the beginning of larval rearing (14 days p.f.) and at the end of pre-growing (234 458 days p.f.), periods during which low temperatures were applied. Survival during larval rearing and nursery were also improved in the groups reared at low salinity and so was swimbladder inflation. These results show that sea bass juveniles have a low saline preferendum, a finding that corresponds to the conditions they actually

* Corresponding author. Present address: Department of Wildlife and Fisheries Sciences, Texas A&M University, TAMU 2258, College Station, TX 77843-2258, USA. Tel.: +1-979-845-1338; fax: +1-979-845-4096. E-mail address: esaillant@wfscgate.tamu.edu (E. Saillant). 0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 0 9 8 1 ( 0 2 ) 0 0 5 0 2 - 6

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encounter in the wild during their juvenile ecophase; sex determination is not directly modulated by the salinity level but seems to be subjected to complex environmental regulations. D 2002 Elsevier Science B.V. All rights reserved.
Keywords: Larval life; Sex determination; Dicentrarchus labrax; Salinity; Growth

1. Introduction The sea bass, Dicentrarchus labrax, is an euryhaline marine fish that frequents coastal waters of the Mediterranean sea and the north western Atlantic ocean. Juveniles of this species spend most of their first year of life in estuaries (Kelley, 1988) or lagoons , 1976), regions in which salinity is usually lower than in the open sea (10 20x (Barnabe vs. 30 40x ). Salinity may therefore influence fish metabolism and development and thus determine the survival of larvae and fitness of juveniles. Sex differentiation occurs during this ecophase for the sea bass and has been shown labile and sensitive to environmental factors, among which temperature appears to play an important role zquez et al., 1998; Pavlidis et al., 2000; Saillant et al., 2002). Salinity is another (Bla parameter worth to be explored as a putative active factor on sex determination in fish (Baroiller et al., 1999). Thus, the rearing salinity commonly applied to intensively cultured sea bass, i.e. high salinity (35 40x ) in most cases (Chatain, personal communication) as opposed to the low lagunar/estuarine salinity, may be involved in the systematic excess of males reported in aquaculture populations by Carrillo et al. (1995). A better knowledge of the effects of water salinity on sea bass early development should help to understand the life-history strategy of this species (Wootton, 1998). In the present work, the effect of rearing salinity on early development and sex-ratios of the sea bass was investigated in a long-term experiment, starting at fertilization and encompassing the labile period of sexual differentiation.

2. Materials and methods The experiment lasted 15 months. Fish were reared under different salinity conditions from the 4th day of life to the age of 465 days post-fertilization (p.f.). 2.1. Biological material, fertilization and incubation The study was carried out on a balanced mixture of the progeny of 10 sea bass breeders (three dams and seven sires) that had been caught along the West Mediterranean coast (south of France, areas of La-Ciotat and Palavas-les-Flots), with the exception of two females, the origin of which is unknown. Twenty-one sibs were produced by artificial insemination of the eggs from each of the three females by an excess of sperm of each male following the procedures described in Saillant et al. (2002). The 21 batches of eggs were incubated separately as described in Saillant et al. (2002). For each family, living

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eggs were separated from dead eggs by decantation at 48 h post-fertilization and used to constitute the experimental groups. 2.2. Experimental groups and salinity treatments At 48 h post-fertilization (p.f.), eight batches of eggs were constituted and put into separate tanks: each of these received an equal volume of living eggs (3 F 0.1 ml) measured with a graduated syringe, from each family. The eight batches were allocated to two treatments that were replicated four times. In the high salinity treatment (Hs), sea water was provided so that rearing salinity followed variations of the natural environment (coastal waters in the area of Palavas-les-Flots: 37xF 2.8 mean F standard deviation) throughout the experiment. In the low salinity treatment (Ls), sea water was mixed with fresh water of the urban network of Palavas-les-Flots in order to progressively lower rearing salinity from 40x(4 days p.f.) to 16x(11 days p.f.) and subsequently maintain it at 15 F 2xuntil the fish reached a mean Standard length of 138 mm (465 days p.f.). The timing of these salinity treatments included the period during which phenotypic sex can be influenced by zquez et al., 1995, 1998; Pavlidis et al., 2000). external factors in the sea bass (Bla At the age of 45 days p.f., half of each population from the four replicates of group Ls was transferred into four other tanks. These were then subsequently supplied with natural sea water (id group Hs) when the fish reached the age of 93 days p.f. (mean standard length of 44 mm) and until the end of the experiment (465 days p.f.), thus giving rise to another quadruplicated treatment: low-high salinity (LHs). Rearing conditions other than salinity were identical for all groups. 2.3. Larval rearing Larval rearing was performed in the facilities described in detail by Garcia de Leon et al. (1998) (500 l tanks connected to a recirculating system). Initial density was estimated from the volumes of eggs introduced in the tank following the method of Chatain (1994) (Table 1). Temperature was increased from 13.5 jC at hatching to 20 jC at 17 days p.f. and remained between 18.7 and 20.6 jC thereafter. Until the age of 13 days p.f., larvae were kept in the dark. At this stage, photophase was set at 12 h light. It was raised to 24 h at 19 days p.f. and maintained at this level until 33 days p.f. in order to solve a swimbladder overinflation problem. Finally, it was set at 16 h from 33 days p.f. to the end of larval rearing (45 days p.f.). Water hourly turnover was 20% of the volume until hatching. It was progressively increased in order to reach 50% h 1 at 27 days p.f. and maintained at this rate until the end of larval rearing so that oxygen level could be maintained beyond 5 mg l 1. Distribution of food, consisting of living Artemia nauplii started at 13 days p.f. The prey density was adjusted twice to seven times a day as a function of larvae consumption and the 4 tanks of a given treatment were fed equal quantities. Metanauplii were distributed from 25 to 49 days p.f. They were enriched with a lipid emulsion (Selco, INVE, Ghent, Belgium Customer: Les Salins du Midi, Le Grau du Roi, France) for 12 h before distribution (1 g per million artemia). From 45 days p.f., the amount of preys distributed was progressively lowered and totally replaced at 49 days p.f. by an artificial pellet to begin the weaning process.

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Table 1 Age, rearing density and survival rates of sea bass submitted to three salinity treatments (four replicates per treatment) during the labile period of sexual differentiation Rearing phase Larval rearing Group Age (days p.f.) Initial density (egg l 1) Survival rate (%) Age (days p.f.) Initial density (ind l 1) Survival rate (%) Age (days p.f.) Initial density (ind l 1) Survival rate (%) Age (days p.f.) Initial density (ind l 1) Survival rate (%) Age (days p.f.) Initial density (ind l 1) Survival rate (%) Hs Ls 2 45 114 F 4* 14 F 2.2b 51 92 4.3 62 F 3.7b 92 141 1.8 95 F 5.6 141 372 0.52 96 F 3.6 372 465 0.36 99 F 0.6 LHs

6.9 F 1.5a

Nursery

44 F 2.5a

Pre-growing

91 F 5.5

96 F 4.4

99 F 1.6

97 F 2.3y

97 F 1.4

99 F 1.8y

Hs = High salinity (natural salinity: 25 43x ), Ls = Low salinity (15 F 2x ), LHs = Low-high salinity (15 F 2x until 93 days p.f., natural salinity thereafter), p.f. = post-fertilization, mean values are given F standard deviation (n = 4 replicates per treatment) and groups with different letters (a,b) are significantly different, ind l 1 = number of individuals per litre. * Precision deduced from the precision of egg volume estimation. y Data from three replicates only (replicate LHs3 lost at 286 days p.f.).

2.4. Nursery and pre-growing The fish were kept in the facilities described above until 148 days p.f. At this stage, they were transferred to 1.4 m3 self-cleaning tanks until the end of the experiment (465 days p.f.). Input water was passed through a 50-Am sand filter (CHF, Le Canet, France) and the excess of soluble nitrogen was eliminated through a counter current air/water stripping system. All the surviving fish in each tank were counted at 45 days p.f. by a photographical method (Chatain et al., 1996) then at 92, 141, 372, and 465 days p.f. by manual counting under anaesthesia. At each step, densities were adjusted to match the lowest density level of all tanks by randomly discarding excess fish (Table 1). Water inflow to tanks was equalized and varied between 20% and 50% h 1 throughout the experiment. Artificial lighting reproduced the natural photoperiodic cycle (April 1996 May 1997). Temperature was maintained between 17 and 25 jC until 148 days p.f. and was natural thereafter (July 1996 May 1997). Commercial diets [Le Gouessant Marin Start (64% protein, 12% lipids, Le Gouessant, Lamballe, France) for particles of a mean diameter < 2.5 mm and Biomar Ecolife (45% crude proteins, 22% lipids, Biomar, Nersac, France) for larger pellets] were distributed from 45 days p.f. onwards. Feeding rates were adjusted according to the biomass (5 10%) until 90 days p.f. From that stage onwards, an on-demand feeding system was used. All tanks received equal quantities of food during this phase. All the fish from tank LHs3 (group LHs) were lost due to a technical failure at 286 days p.f. Subsequent results reported for the LHs treatment corresponds therefore to the data obtained in the three remaining replicates.

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2.5. Rearing parameters, sampling and measurements Water temperature was checked daily and salinity twice a day. Water quality was + controlled by a weekly check of pH and dissolved levels of NH4 + NH3. NO2 and NO3 levels were also measured in the recirculating systems. These last measurements allowed the proper working order of the biological nitrifying filters to be controlled. Larvae were randomly sampled in each tank at 3 (n = 10), 14 (n = 30), 23 and 49 days p.f. (n = 50). Total length and the presence of an inflated swimbladder were recorded as described in Garcia de Leon et al. (1998). At 88, 143, 234, 324, 408 and 458 days p.f., 50 fish were randomly selected from each tank under anesthesia. Their weight and Standard length were recorded to the nearest g and mm, respectively (length data are not available for 234 and 324 days p.f.). Survival rate in each tank was estimated at 45 days p.f., and was taken as being the ratio between the final larval counts and the number of eggs initially introduced in the tank. Survival rates were also calculated between 45 and 92, 92 and 141, 141 and 372, 372 and 465 days p.f. based on the ratios between the counts of living fish at the end and the beginning of each period. At 465 days p.f., 200 fish were sampled in each tank for assessment of sex-ratio and (1983), the phenotypic sex mean sexual dimorphism. According to Roblin and Brusle could not be unambiguously assessed for all the fish at this stage. The fish sampled were therefore kept for further growth under identical conditions (natural salinity, temperature and photoperiod) until the age of 533 days p.f. At this stage (mean Standard length of 160 mm), all the fish were killed by immersion in 400 ppm Phenoxy2ethanol, dissected, weighed and measured (Standard length). The phenotypic sex was identified according to (1976). The reliability of this sexing was checked by the criteria described by Barnabe analyzing the gonads of 150 randomly sampled fishes at the histological level. These gonads were fixed in Bouins fluid, dehydrated, paraffin embedded and longitudinally cut (5 Am) in order to take into account the longitudinal gradient of sexual differentiation (1983). The slides were stained with Erythrosin-Orange G reported by Roblin and Brusle and Toluidine Blue. The phenotypic sex was identified following the criteria described in (1983). Briefly, ovaries were recognized by the presence of an ovarian Roblin and Brusle cavity and ovarian lamellae filled with previtellogenic oocytes, whereas testes were recognized by the organization of the gonias in early peripheral cysts. Percentages of females and sex dimorphisms [(U mean weight h mean weight)/h mean weight] were estimated for each tank of the four groups. 2.6. Data analysis Survival, sex-ratio and sexual dimorphism data were Arcsine square root transformed before being used for ANOVA and all datasets were checked for variance homogeneity lie, 1975). and normality (Dagne Comparisons were made between the Hs and Ls groups and between the LHs and Ls groups, respectively. Survival rates, sex-ratios and sexual dimorphisms recorded at various stages of the experiment were compared using one-way ANOVAs. The homogeneity of the percentage

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of females intra-treatment was also tested by G tests (Sokal and Rohlf, 1981) in the Hs, Ls and LHs groups, respectively. Mean total lengths during larval rearing, standard lengths and weights during pregrowing were compared using two-ways ANOVAs: tank was treated as a random factor nested in the fixed factor salinity. Weights and lengths recorded at slaughtering were analyzed according to the previous model in which the sex factor was added as a fixed factor crossed with salinity.

3. Results 3.1. Larval rearing Water salinities and temperatures are indicated in Fig. 1a,b. Natural salinity range was 34 40xand the mean difference in salinity between the two treatments was 21x . Temperature was similar for the two salinity treatments. The pH range was 7.7 8.1 in the Hs group and 7.1 8.2 in the Ls group and the ammonia levels remained low in both + groups [ < 0.1 mg N (NH4 + NH3) l 1]. During larval rearing, mean larval length, which was 3.6 mm at hatching, reached 19.2 21.2 mm at 49 days p.f. Larvae of the Ls group were longer than those of the Hs group only after the first feeding (14 days p.f.) ( + 4.5%; F = 20.6, df = 1 and 6, P = 0.004). Larvae from both groups were of the same size at 23 days p.f., whereas the larvae of the Hs group were longer (20.8 vs. 19.8 mm) ( F = 9.4; df = 1 and 6; P = 0.02) at the end of larval rearing (Fig. 1c). Mean survival rate during larval rearing was higher in the Ls group (14%) than in the Hs group (7%) ( F = 27.8, df = 1 and 6, P = 0.0018). Eighty-seven percent of the larvae of the Ls group had developed a functional swimbladder at 14 days p.f. vs. 26% in the Hs group ( F = 17,2 ; df = 1 and 6; P = 0.0059). This percentage remained significantly higher in the Ls group until the end of larval rearing at which stage it reached 100% vs. 78% in the Hs group ( F = 80.0, df = 1 and 6, P = 0.0001). 3.2. Pre-growing and on-growing Rearing salinities and temperatures during this phase are shown in Fig. 2a,b. Natural salinity range was 25 43x and the mean difference in salinity between the two treatments was 22x . Temperature was similar for the two salinity treatments and ranged from 11 to 25 jC depending on the season (April 1998 April 1999). The pH range was 6.8 8.2 and showed similar evolution patterns among treatments (maximum difference: + 0.3 unit). Ammonia levels ranged between 0 and 2.9 mg N (NH4 + NH3) l 1. They were higher in the Hs treatment from 90 to 130 days p.f. [mean levels of 1.1 and 0.2 mg N + (NH4 + NH3) l 1 in the Hs and Ls groups, respectively], whereas they were higher in the LS group from 193 to 231 days p.f. (mean levels of 0.7 and 2.3 mg N l 1 in the Hs and Ls groups, respectively). The on-growing period lasted 1 year. The length of the fish, that was 19.2 21.2 mm at the end of larval rearing, reached 121 139 mm at 458 days p.f. (Fig. 2c).

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Fig. 1. Growth of sea bass reared at natural (34 40x , group Hs) and low (15 F 2x , group Ls) salinity during larval rearing (2 51 days post-fertilization). (a) Water salinity recorded in group Hs () and group Ls (- - -), (b) water temperature recorded in group Hs () and group Ls ( - - - ), (c) mean total length F standard deviation (n = 4 replicates per treatment) recorded in group Hs (in black) and group Ls (in white).

The three groups (Ls, Hs, LHs) had the same mean weight until 143 days p.f. (Fig. 2d). At the age of 234 days p.f., the fish from the Ls group were heavier than those of the groups reared at high salinity (Fig. 2d) (Ls>Hs: 27.8 vs. 24.4 g; F = 12, df = 1 and 6, P =0.01), (Ls>LHs: 27.8 vs. 22.0 g; F = 23, df = 1 and 6, P = 0.003). This pattern was maintained until the end of the experiment (458 days p.f.), although the differences could not be detected at 408 days p.f. due to a strong tank effect. At 234 days p.f., the relative advantage for the Ls group was 14% and 26% when compared to the Hs group and LHs groups, respectively. This gap increased to 28% and 32% at 458 days p.f. The same pattern was observed for lengths (Fig. 2c) except that the difference between the Ls and LHs groups at 408 days p.f. was found to be significant.

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Fig. 2. Growth of sea bass submitted to three salinity treatments [group Hs: natural salinity (25 43x ); group Ls: 15 F 2x ; group LHs: 15 F 2xuntil 91 days p.f. and natural salinity thereafter] during juvenile development (51 465 days post-fertilization). (a) Water salinity recorded in group Hs (), group Ls ( - - - ), group LHs: see group Hs from 91 days p.f. (b) Water temperature recorded in group Hs (), group Ls (- - - ), group LHs: see group Hs from 91 days p.f. (c) Mean standard length F standard deviation (n = 4 replicates per treatment) recorded in group Hs (in black), group Ls (in white) and group LHs (in grey). (d) Mean weight F SD recorded in group Hs (in black), group Ls (in white) and group LHs (in grey).

A significant tank effect was detected at 143 days p.f. during the comparison of groups Ls and LHs ( F = 7.6, df = 6 and 392, P < 0.0001 for weight, F = 6, df = 6 and 392, P < 0.0001 for length). This tank effect was attributable to tank LHs2 in which the fish were shorter (mean

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length 69 vs. 74 mm) and lighter (mean weight 6.8 vs. 9.2 g) than in the other tanks. The tank effect was also significant for both weight and length at 408 days p.f. (Ls vs. Hs: F = 5.1, df = 6 and 392, P < 0.0001 for weight, F = 4.9, df = 6 and 391, P < 0.0001 for length) (Ls vs. LHs: F = 6.5, df = 5 and 343, P < 0.0001 for weight, F = 6.6, df = 5 and 342, P < 0.0001 for length). At this stage, the tank effect was due to the heterogeneous growth recorded in group Ls: the fish sampled from the Ls1 and Ls4 tanks belonging to this group were longer (mean length 133 vs. 123 mm) and heavier (44.1 vs. 35.2 g) than those sampled from tanks Ls2 and Ls3. Survival rates during the nursery phase (45 92 days p.f.) were lower in the Hs group (44%) than in the Ls group (62%) ( F = 37.6, df = 1 and 6, P < 0.0001). After this stage, and until the end of the treatment period, survival was no longer influenced by rearing salinity (mean survival rate: 90%; F < 0.3, df = 1 and 6 or 1 and 5, P>0.6) (Table 1).

Fig. 3. Sex ratios and sexual dimorphism of sea bass subjected, in quadruplicate, to three salinity treatments [group Hs: natural salinity (25 43x , mean 37x ); group Ls: 15 F 2x ; group LHs: 15 F 1xuntil 91 days p.f. and natural salinity thereafter] during the labile period of sex differentiation. (a) Percentages of females recorded at 533 days p.f. (mean length 160 mm) in group Hs (n), group Ls (5) and group LHs ( ). (b) Sexual dimorphism [100 (U mean weight h mean weight)/(h mean weight)] recorded at a mean standard length of 160 mm in group Hs (n), group Ls (5) and group LHs ( ).

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3.3. Sex-ratios and sexual dimorphism The percentage of females was very low in all groups (mean: 13%). The Ls and Hs groups had the same percentage of females (15%; F = 0.02, df = 1 and 6, P = 0.9), these values being significantly higher than that recorded in the LHs group (8%) ( F = 76, df = 1 and 5, P = 0.0003) (Fig. 3a). Sex-ratios were homogenous in the Ls (Gadj = 0.4, df = 3, P = 0.9) and LHs (Gadj = 0.9, df = 2, P = 0.6) groups, although they varied significantly among rearing tanks in the Hs group (Gadj = 11.8, df = 3, P = 0.008). These differences in sex-ratio had no apparent relationship with either the mortality rates recorded at various stages of the experiment or the sizes reached at each step. At slaughtering, both weight ( F = 44.8, df = 1 and 2189, P < 0.0001) and length ( F=58.2, df = 1 and 2189, P < 0.0001) were strongly influenced by the sex. The amplitude of the sexual dimorphism ranged between 4.3% and 25.8% (mean 13%, Fig. 3b) and was not significantly influenced by rearing salinity (Ls = Hs: 11% vs. 13%; F = 0.2, df = 1 and 6, P = 0.66) (Ls = LHs: 11% vs. 16%; F = 2, df = 1 and 5, P = 0.21).

4. Discussion 4.1. Sex determination In this work, two rearing salinity levels were tested for their effects on sex determination. The salinity of coastal sea-waters which is commonly applied in intensive culture of the sea bass (25 43x ), was compared to a low salinity (15x ), this latter value being closer to that encountered by the larvae and juveniles of this species in estuaries and lagoons. The salinity level, when maintained constant, had no effect on sex-ratio. Since the treatments were applied during the entire labile period, these results suggest that this level is not involved in sex determination of the sea bass and in the massive masculinization that is usually observed in intensive culture of this species, a masculinization that was reproduced in the present work where all groups showed a large excess of males (87%). The only difference in sex-ratio was generated when the fish were transferred from low to high salinity in the middle of the labile period (at 93 days p.f., 44 mm). Indeed, there were significantly less females in this group than in the one kept at low salinity. It seems that the already strong masculinizing effect observed in the other groups was strengthened. This result may be linked to the osmotic stress generated by the transfer and could therefore involve the physiology of osmoregulation and stress. To our knowledge, the relationship between sex-differentiation and these physiological mechanisms has not been explored in gonochoric fish species but Godwin and Thomas (1993) observed that, in the protandrous Amphiprion melanopusto, the levels of Cortisol exhibited an increase during sex change. No conclusion could be drawn about the implication of this hormone in the prevention of sex change in this work but the authors suggested that the high levels recorded could be involved in the mobilization of energy reserve during ovarian differentiation. According to our results, it would be interesting to study the

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occurrence of such relationships during sex differentiation of the sea bass. In such a case, the osmotic stress involved in the present study was light: no significant growth depression was induced since no significant difference in growth was detected between the two groups (Ls and LHs) before 234 days p.f. (i.e. 141 days after the transfer). The environmental component of sex determination in sea bass appears to be very complex. Three studies showed that temperature plays an important role in sex differ zquez et al., 1998; Pavlidis et al., 2000; Saillant et al., 2002). A detailed entiation (Bla examination of these results revealed that no constant temperature pattern could lead to high percentages of females (Saillant et al., 2002) but suggested that only an increasing pattern of temperature, probably promoting fast growth during the labile phase of sexdifferentiation, could lead to feminization. The present work showed that a stress during the labile period may also affect sex determination. The process of sex determination in sea bass appears therefore to be linked to a complex combination of factors. Among these, the level of stress and the growth rates, two parameters that characterize rearing conditions as compared to natural conditions for the fish, may be two major inductors and they may be involved in the massive masculinization reported in aquaculture. The percentage of females also varied significantly among the four replicates reared at high salinity. These sex-ratios showed no apparent relationship with either the mortality rates or the mean sizes recorded at the various steps of the experiment. Thus, based on the information obtained in this study, no clear hypotheses can be drawn to explain the variability recorded in this group (range: 10% to 22% females). If stress is involved in sex determination as discussed above, this result could reflect a higher level of stress in the replicates that showed lower percentages of females. If such is the case, this could not be detected during the experiment and the involved differences between replicates would therefore be very small. Some families giving high percentages of females may also have been selected in some tanks during the early life stages where mortality was high. Indeed, parental effects on sex-ratios have already been reported in the sea bass and they were zquez et al., 1999; Saillant et al., 2002), some families showing highly significant (Bla higher percentages of females than the others. The overall sex-ratio may have been increased in some tanks if the progenies that gave the highest percentages of females were selected. 4.2. Growth Growth was influenced by rearing salinity in this study but not in a constant manner. The larvae reared at low salinity grew faster until their first feeding (14 days p.f.), as already reported by Marangos (1985). Then, those reared at high salinity grew faster until 49 days p.f., following which stage no difference was recorded between the two groups until they reached about 8 months of age (234 days p.f.). Between 8 and 15 months of age, i.e. the end of the experiment, the fish reared at low salinity became heavier and longer than the others. This growth improvement may be due to the water temperature. Indeed, the faster growth in the low salinity group was only observed over the period during which temperature was low (mean 15 jC, Winter 1997) and thus corresponded to the temperature salinity interaction described by Alliot et al. (1983). These authors reported that juveniles of sea bass grew faster at low salinity (11x , close to our Ls treatment), in

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comparison to high salinities (37x , close to our Hs treatment) when temperature was low (15 jC), but at high temperatures (22 jC), an inverse tendency was observed during the major part of the experiment, no difference being detected at the end of the treatments. The long treatments applied in the present study allow to clarifying this pattern at high temperature. The juveniles reared at various salinity during the first 5 months of the experiment (from 23 to 143 days p.f., mean rearing temperature: 21.5 jC), grew faster at high salinity at the beginning of this rearing phase (23 49 days p.f.), a result in accordance with the work of Alliot et al. (1983), but this advantage was not further observed and the fish reared at various salinity kept the same growth thereafter and until 143 days p.f. These results suggest that long-term growth of sea bass juveniles is not influenced by salinity when temperature is high (around 22 jC). Conversely, a long-term growth advantage was observed at low salinity and low temperature (from 8 to 15 months p.f.). This advantage corresponded to a higher food conversion rate since both treatments received equal quantities of food. This effect may have been due to an energy gain generated by a lower need for osmoregulation, as already reported in other marine species such as Gadus morua (Lambert et al., 1994), Takifugu rubripes (Han et al., 1995) and Scophthalmus maximus (Gaumet et al., 1995). Indeed, a salinity of 15x is close to the osmolarity of sea bass blood (Pavlidis et al., 1997). Salinity may also have improved the food conversion rate by directly acting on digestive enzymes, as described in Oreochromis niloticus (Woo et al., 1997), although a preliminary study conducted in sea bass did not reveal a significant effect of salinity on trypsin activity at low temperature (Alliot et al., 1983). Salinity may also have influenced physiological mechanisms involved in growth such as the secretion of growth hormone or prolactin. These hormones were shown to be influenced by rearing salinity and may be involved in the effect of salinity on growth in Oreochromis mossambicus (Shepherd et al., 1997). The secretion of thyroxin, which is involved in growth regulation, was also proved to be stimulated by isotonic conditions in O. niloticus (Woo et al., 1997). Energy saving at low salinity may also involve behavioral mechanisms since Johnson and Katavic (1986) reported that swimming activity during larval rearing is easier and global activity is lower in such conditions. From our point of view, the degradation in water quality observed in the high salinity group between the 3rd and 5th months, and in the low salinity group between the 6th and 8th months, could not have influenced the growth results. Indeed, the ammonia levels + recorded (less than 3 mg N NH4 + NH3 l 1) were far too low to induce any growth , G., personal communication). depression in the sea bass (Lemarie The variations in optimum salinity as a function of temperature evoked above may correspond to different ecophases for wild sea bass, as has been reported in another marine fish, the fugu T. rubripes. Indeed, in this last species, this phenomenon was related to changes in life habitat during the life cycle (Han et al., 1995). 4.3. Larval rearing: survival and swimbladder inflation Salinity influenced survival rates in this work. During larval rearing and nursery, periods during which most of the mortalities occurred, survival was doubled at low salinity. Such a result was also obtained in sea bass by Johnson and Katavic (1986) when

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testing a comparable rearing salinity range. The survival rates during larval rearing were globally low (range 5 15%). Such high mortality rates in larval rearing are often encountered in marine species and in sea bass. In the present study, these mortality rates were homogeneous within treatment and they were not correlated to the other studied variables. In the present work, the rate of normal swimbladder development was also higher at and Guissi (1993) and Johnson and Katavic low salinity, as already reported by Barnabe (1986). This was observed as early as the stage of inflation (i.e. 14 days p.f.), suggesting that the differences recorded at the end of larval rearing were not due to a selective mortality of the larvae with non-functional swimbladders but rather to an improved efficiency in the process of inflation. Indeed, this phenomenon, which has been studied in details by Chatain (1994), appears to be linked to hydrological factors, among which salinity may be a key factor. Thus, sea bass larval performances appear to be significantly improved under isotonic conditions, as has also been reported in the rearing of other marine species such as Sparus aurata (Tandler et al., 1995) and Mugil cephalus (Harel et al., 1998). The decreased performances recorded at high salinity during early development may be linked to limits in the osmoregulation capacity during early larval development. Indeed, according to Stamatis (2001), the full osmoregulation capacity only appears at the end of this phase. In conclusion, this work shows that salinity is not directly involved in the sexual differentiation of sea bass, but that the osmotic stress generated by a salinity shock during the labile period of sexual differentiation may increase a masculinizing effect attributable to others environmental factors. Survival rate and swimbladder inflation are improved at low salinity, and so are growth rate and conversion rate when temperature is low. Sea bass larvae therefore appear to show a low saline preference, which may correspond to the conditions they actually encounter in their juvenile ecophase.

Acknowledgements We would like to thank Marie Odile Vidal, Sylvain Rols, Patrik Legall, Christian ` re for their help in the zootechnical phases and Dr. M. Fauvel and Jean-Claude Falguie Johnson for proof-reading the English. Eric Saillant was supported by the French diterrane e Pisciculhatcheries Ecloserie Marine de Gravelines (Gravelines, France), Me ture (Salses, France), GAEC Poissons du Soleil (Balaruc les Bains, France) and the National Association for Technical Research (Grant CIFRE no. 96581). This study was carried out with financial support from the French Ministry of Education, Research and Technology and the French Ministry of Agriculture: Agriculture Demain no. 95.G.0108 contributions are also supported by OFIMER. [SS]

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