Cellular and molecular immune responses of the sea bass (Dicentrarchus labrax) experimentally infected with betanodavirus

Naïve sea bass juveniles (38.4 ± 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-β, TCRβ, CD4, CD8α, IgM, by using a quantitative PCR array system developed for sea bass.The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable “in vitro” increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-β and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.     

Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae.

Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE).     

A microsatellite linkage map of the European sea bass Dicentrarchus labrax L

A genetic linkage map of the European sea bass (Dicentrarchus labrax) was constructed from 174 microsatellite markers, including 145 new markers reported in this study. The mapping panel was derived from farmed sea bass from the North Adriatic Sea and consisted of a single family including both parents and 50 full-sib progeny (biparental diploids). A total of 162 microsatellites were mapped in 25 linkage groups. Eleven loci represent type I (coding) markers; 2 loci are located within the peptide Y (linkage group 1) and cytochrome P450 aromatase (linkage group 6) genes. The sex-averaged map spans 814.5 cM of the sea bass genome. The female map covers 905.9 cM, whereas the male map covers only 567.4 cM. The constructed map represents the first linkage map of European sea bass, one of the most important aquaculture species in Europe.     

Embryonic development of the sea bass Dicentrarchus labrax

The embryonic development of the sea bass Dicentrarchus labrax during the endotrophic period is discussed. An 8 cells stage, not reported for other studied species, results from two rapid successive cleavages. Blastula occurs at the eighth division when the embryo is made of 128 cells. During gastrulation, the infolded blastoderm creates the endomesoblastic layer. The Kupffer??s vesicle is reported to drive the left/right patterning of brain, heart and digestive tract. Heart formation starts at 8 pairs of somites, differentiation of myotomes and sclerotomes starts at the stage 18 pairs of somites; main parts of the digestive tract are entirely formed at 25 pairs of somites. At 28 pairs of somites, a rectal region is detected, however, the digestive tube is closed at both ends, the jaw appears the fourth day after hatching, but the mouth is not opened before the fifth day. Although cardiac beating and blood circulation are observed, gills are not reported in newly hatched individuals; eye melanization appears concomitant with exotrophic behavior.     

Ultrasound measurement of reproductive organs in juvenile European sea bass Dicentrarchus labrax

In this study, ultrasonographic examination was performed thrice, 15 days apart, on juvenile European sea bass Dicentrarchus labrax, from 330 to 360 days of age, to assess the size and the morphology of male and female. Results have proved this method as a suitable and non‐invasive procedure to assess sexual differentiation.     

Range‐wide population structure of European sea bass Dicentrarchus labrax

The euryhaline European sea bass Dicentrarchus labrax L., inhabiting the coasts of the eastern Atlantic Ocean and Mediterranean Sea, has had many opportunities for differentiation throughout its large natural range. However, evidence for this has been incompletely documented geographically and with an insufficient number of markers. Therefore, its full range was sampled at 22 sites and individuals were genotyped with a suite of mapped markers, including 14 microsatellite loci (N = 536) and 46 neutral or gene‐linked single nucleotide polymorphisms (SNPs; N = 644). We confirm that the Atlantic and Mediterranean basins harbour two distinct lineages. Within the Atlantic Ocean no pattern was obvious based on the microsatellite and SNP genotypes, except for a subtle difference between South‐eastern and North‐eastern Atlantic sea bass attributed to limited introgression of alleles of Mediterranean origin. SNP genotypes of the Mediterranean lineage differentiated into three groups, probably under the influence of geographical isolation. The Western Mediterranean group showed genetic homogeneity without evidence for outlier loci. The Adriatic group appeared as a distinct unit. The Eastern Mediterranean group showed a longitudinal gradient of genotypes and most interestingly an outlier locus linked to the somatolactin gene. Overall, the spatial pattern fits those observed with other taxa of between‐basin segregation and within‐basin connectivity, which concurs well with the swimming capabilities of European sea bass. Evidence from a few outlier loci in this and other studies encourages further exploration of its regional connectivity and adaptive evolution.     

A polygenic hypothesis for sex determination in the European sea bass Dicentrarchus labrax

Polygenic sex determination, although suspected in several species, is thought to be evolutionarily unstable and has been proven in very few cases. In the European sea bass, temperature is known to influence the sex ratio. We set up a factorial mating, producing 5.893 individuals from 253 full-sib families, all reared in a single batch to avoid any between-families environmental effects. The proportion of females in the offspring was 18.3%, with a large variation between families. Interpreting sex as a threshold trait, the heritability estimate was 0.62 +/- 0.12. The observed distribution of family sex ratios was in accordance with a polygenic model or with a four-sex-factors system with environmental variance and could not be explained by any genetic model without environmental variance. We showed that there was a positive genetic correlation between weight and sex (r(A) = 0.50 +/- 0.09), apart from the phenotypic sex dimorphism in favor of females. This supports the hypothesis that a minimum size is required for sea bass juveniles to differentiate as females. An evolution of sex ratio by frequency-dependent selection is expected during the domestication process of Dicentrarchus labrax populations, raising concern about the release of such fish in the wild.     

Not so much a sea bass: Divergent European sea bass (Dicentrarchus labrax L.) freshwater incursions

European sea bass (Dicentrarchus labrax [Linnaeus, 1758]) is a euryhaline marine migrant fish highly valuable for fisheries and aquaculture. Although juveniles are known to use estuaries and occasionally move to freshwater environments, these freshwater incursions had not been reported for adults. Recently, this behavior was observed in the Tagus River (Portugal) for adults occurring up to 150 km from the river mouth, about 80 km upstream from the tidal influence, suggesting the existence of a freshwater contingent. Fisheries management of sea bass should consider the putative existence of a freshwater contingent.     

Melatonin binding sites in the brain of European sea bass (Dicentrarchus labrax)

Characteristics, day-night changes, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) modulation, and localization of melatonin binding sites in the brain of a marine teleost, European sea bass Dicentrarchus labrax, were studied by radioreceptor assay using 2-[(125)I]iodomelatonin as a radioligand. The specific binding to the sea bass brain membranes was rapid, stable, saturable and reversible. The radioligand binds to a single class of receptor site with the affinity (Kd) of 9.3 +/-0.6 pM and total binding capacity (Bmax) of 39.08 +/-0.86 fmol/mg protein (mean+/-SEM, n=4) at mid-light under light-dark (LD) cycles of 12:12. Day-night changes were observed neither in the Kd nor in the Bmax under LD 12:12. Treatment with GTPgammaS significantly increased the Kd and decreased the Bmax both at mid-light and mid-dark. The binding sites were highly specific for 2-phenylmelatonin, 2-iodomelatonin, melatonin, and 6-chloromelatonin. Distribution of melatonin binding sites in the sea bass brain was uneven: The Bmax was determined to be highest in mesencephalic optic tectum-tegmentum and hypothalamus, intermediate in telencephalon, cerebellum-vestibulolateral lobe and medulla oblongata-spinal cord, and lowest in olfactory bulbs with the Kd in the low picomolar range. These results indicate that melatonin released from the pineal organ and/or retina plays neuromodulatory roles in the sea bass brain via G protein-coupled melatonin receptors.     

QTL for body weight,morphometric traits and stress response in European sea bass Dicentrarchus labrax

Natural mating and mass spawning in the European sea bass (Dicentrarchus labrax L., Moronidae, Teleostei) complicate genetic studies and the implementation of selective breeding schemes. We utilized a two‐step experimental design for detecting QTL in mass‐spawning species: 2122 offspring from natural mating between 57 parents (22 males, 34 females and one missing) phenotyped for body weight, eight morphometric traits and cortisol levels, had been previously assigned to parents based on genotypes of 31 DNA microsatellite markers. Five large full‐sib families (five sires and two dams) were selected from the offspring (570 animals), which were genotyped with 67 additional markers. A new genetic map was compiled, specific to our population, but based on the previously published map. QTL mapping was performed with two methods: half‐sib regression analysis (paternal and maternal) and variance component analysis accounting for all family relationships. Two significant QTL were found for body weight on linkage group 4 and 6, six significant QTL for morphometric traits on linkage groups 1B, 4, 6, 7, 15 and 23 and three suggestive QTL for stress response on linkage groups 3, 14 and 23. The QTL explained between 8% and 38% of phenotypic variance. The results are the first step towards identifying genes involved in economically important traits like body weight and stress response in European sea bass.     

More polymorphic microsatellite markers in the European sea bass (Dicentrarchus labrax L.)

We provide details of five microsatellite loci screened in 163 individual sea bass. Large numbers of alleles were observed at three loci (20–25) and heterozygosities ranged from 0.52 to 0.86. These loci should prove useful for population genetic studies and for the pedigree analysis and genetic management of this species in aquaculture.     

Sexual differentiation and juvenile intersexuality in the European sea bass (Dicentrarchus labrax)

Sexual differentiation was studied at the histological level using a mixture of 30 families of sea bass Dicentrarchus labrax . Most of the fish (93%) differentiated into males as usually observed in farmed populations. All testes were differentiated when the males reached 12 cm and no more undifferentiated fish were found from 419 days post-fertilization (p.f.). In 28% of the males, among the biggest, sexual differentiation had already begun at 168 days p.f. (8.3–9.5 cm) and these fish started spermatogenesis in their first year of life. The other males differentiated later and remained immature at the end of their first year of life. Ovaries could be identified at the histological level from the age of 168 days p.f. (7.9–9.0 cm) and the females became significantly longer than the males from the age of 191 days p.f., i.e. during the process of ovarian differentiation. In the studied group, 62% of the males developed intratesticular oocytes. Such intersexuality had no consequence on growth rate. Intratesticular oocytes were also recorded in testes of wild males originating from Atlantic (Britain and Gulf of Gascogne) and West Mediterranean showing that juvenile intersexuality is not restricted to farmed populations but is a widespread phenomenon in sea bass.     

Embryonic occurrence of ionocytes in the sea bass Dicentrarchus labrax

Because of the permeability of the chorion, sea bass embryos are exposed to seawater before hatching and hence require precocious osmoregulatory processes. Several studies of other species have demonstrated the existence of ion-transporting cells located on the yolk sac membrane of embryos. In these cells, called ionocytes, ion movements are controlled by a pool of transmembrane proteins. Among them, the Na⁺/K⁺-ATPase, an abundant driving enzyme, has been used to reveal the presence or absence of ionocytes. We have immunostained the Na⁺/K⁺-ATPase in sea-bass embryos and shown the presence of the first ionocytes on the yolk sac membrane at stage 12 somites and the occurrence of ionocytes at other sites before hatching. Ionocytes located on the first gill slits have been identified at stage 14 somites. Primitive enteric ionocytes have also been detected at stage 14 somites in the mid and posterior gut. The presence of these cells might be related to the early opening of the gut to perivitelline fluids, both anteriorly by the gill slits and posteriorly by the anus. The role of embryonic ionocytes in osmoregulation before hatching is discussed.     

Seasonal effects on hematological and innate immune parameters in sea bass Dicentrarchus labrax

The temperate aquatic environment is affected by two primary components of season, temperature and photoperiod, during the annual cycle. Many organisms respond to seasonal change physiologically, behaviorally or both. The aim of this study was to investigate the effect of seasonality on cortisol, hematological and innate immune parameters in European sea bass reared under traditional semi-intensive aquaculture. Sea bass (Dicentrarchus labrax) were reared in an outdoor pond and serum cortisol, hematocrit, leucocrit, serum lysozyme activity and total glutathione were bimonthly monitored over a 14-months period. The effect of seasonality was observed for all parameters carried out, with generally higher values in summer and lower in winter. These results could improve the understanding of the influence of seasonal cues on the immune system and hematological parameters in fish in order to optimize the husbandry practices.     

Older triploid fish retain impaired reproductive endocrinology in the European sea bass Dicentrarchus labrax

This paper reports on an evaluation of growth, gonadal development and reproductive endocrinology of older triploid (3n) European sea bass Dicentrarchus labrax in comparison with their diploid (2n) counterparts throughout their fifth and seventh annual cycle of life. While older triploids retained impaired reproductive endocrinology, a sexually related dimorphic growth was observed with 3n females attaining the largest sizes. Comparisons of some body indexes showed that 3n females had a significantly lower hepato‐somatic index (I_H) than 2n females but a significantly higher viscero‐somatic index (I_F). In contrast, both male and female triploids showed significantly lower gonado‐somatic index (I_G) than diploids. Accordingly, diploids produced mature gametes but triploids did not, demonstrating that despite the longer time given to triploids for gonadal development, they could not reproduce. Furthermore, older triploids had lower levels of plasma sex steroids (testosterone, T; 11‐ketotestosterone, 11‐KT and oestradiol‐17β, E₂) and luteinizing hormone (LH) than their 2n counterparts with 3n females showing drastic effects of triploidization on their reproductive endocrinology. Vitellogenin (VTG) was undetectable in 3n females. Gonadal content of steroid hormones and Sparus aurata‐type gonadotropin‐releasing hormone (sbGnRH) in the brain and pituitary were also lower in triploids compared with diploids. These results suggest that older 3n D. labrax retain functional sterility in both sexes, and 3n females might reach larger sizes than 3n males and their 2n counterparts in this species.     

Steroid metabolism by ovarian follicles of the sea bass Dicentrarchus labrax

Ovarian samples from fear sea bass, Dicentrarchus labrax, were collected for the in vitro incubations during the spawning period. Follicles with fully developed vitellogenic oocytes showing central germinal vesicle (stage I follicles) and follicles with oocytes showing initial germinal vesicle migration (stage II follicles) were treated with either (1) 20 μg sea bass hypophysis plus 50 ng 17-hydroxyprogesterone (17-P), (2) 20 μg hypophysis alone, (3) 50 ng 17-P alone and (4) media alone. Structure-activity experiments used stage II follicles treated with several dosages (0.1, 1.0 and 10.0 ng/ml) of either 17-P, 17,20β-P, or 17,20β,21-P. Free and conjugated (sulfates and glucuronides) levels of the established teleost oocyte maturation inducing steroids (MIS), i.e. 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) were measured in the incubation media by high performance liquid chromatography. Our results show that the synthesis of free and conjugated 17,20β-P is constant (0.1–0.2 ng/ml) in all incubates. In contrast, the synthesis of free and conjugated 17,20β,21-P is higher in incubates containing stage II follicles (up to 5 ng/ml) than in those having stage I follicles (up to 3 ng/ml; P<0.01). Structure-activity data reveal that 17-P is not effective at inducing in vitro germinal vesicle breakdown whereas both 17,20β-P and 17,20β,21-P are equally potent and highly effective. These results demonstrate that 17,20β-P and 17,20β,21-P are synthesized in vitro by follicles of sea bass and that sulfation is the main route for the metabolism of the C₂₁-steroids in riper follicles. The highest levels of 17,20β,21-P, found in incubates containing stage II follicles, points at 17,20β,21-P, rather than 17,20β-P, as the most probable MIS in sea bass, nonetheless, this hypothesis requires further confirmation.     

Thermally induced phenotypic plasticity of swimming performance in European sea bass Dicentrarchus labrax juveniles

The vulnerability of embryonic and larval stages of European sea bass Dicentrarchus labrax to environmental temperature and the longer-term consequences for the early juveniles was demonstrated. This phenotypic plasticity was highlighted by subjecting D. labrax at 15·2 ± 0·3 or 20·0 ± 0·4° C (mean ± s . d .) up to metamorphosis and then at the same temperature (18·5 ± 0·7° C). After 4–6 weeks at the same temperature, the measurement of critical swimming speed at four exercise temperatures (15, 20, 25 and 28° C) showed a significantly higher swimming capacity in the fish initially reared at 15° C than for fish initially reared at 20° C. This performance was correlated with significant differences in the phenotype of red muscle. Thermally induced phenotypic plasticity was clearly demonstrated as an important mechanism controlling swimming performance in early juveniles of D. labrax .