Osmoregulatory response of Senegalese sole (Solea senegalensis) to changes in environmental salinity

The osmoregulatory response of Senegalese sole (Solea senegalensis, Kaup 1858) to 14-day exposure and throughout 17-day exposure to different environmental salinities was investigated. A linear relationship was observed between environmental salinity and gill Na(+),K(+)-ATPase activity whereas kidney Na(+),K(+)-ATPase activity was unaffected. Two osmoregulatory periods could be distinguished according to variations in plasma osmolality: an adjustment period and a chronic regulatory period. No major changes in plasma osmolality and ions levels were registered at the end of the 14- to 17-day exposure period, indicating an efficient adaptation of the osmoregulatory system. Plasma levels of glucose and lactate were elevated in hypersaline water, indicating the importance of these energy substrates in these environments. Glucose was increased during hyper-osmoregulation but only in the adjustment period. Cortisol proved to be a good indicator of chronic stress and stress induced by transfer to the different osmotic conditions. This work shows that S. senegalensis is able to acclimate to different osmotic conditions during short-term exposure.     

Cloning and expression analysis of Mx cDNA from Senegalese sole (Solea senegalensis)

Senegalese sole (Solea senegalensis) is a promising fish species of growing interest in European aquaculture. In fish farming, viral infections are a constant threat therefore, understanding fish defence mechanisms is a main priority to avoid economic losses. Mx proteins are involved in the innate antiviral response of fish. They are induced by type I interferons (alpha and beta) and are essential to investigate viral defence mechanisms in fish, due to the difficulty in tracking interferon activity in these species. In this study a full-length Senegalese sole Mx cDNA has been RT-PCR cloned, resulting in 2322bp coding for 623 amino acids. The sequence accounts for the main characteristics of Mx proteins but lacking nuclear localisation signal (NLS), which suggests cytoplasmic localisation. The alignments of Senegalese sole Mx sequence showed the highest identity with the flatfish species, 80.1% identity with flounder and 78.9% with halibut. The spatial and temporal expression pattern has been analysed in control and challenged fish by RT-PCR. In control fish a constitutive level of sole Mx expression has been obtained and a clear induction was observed after treatment with Poly[I:C], which supports a putative role for the Mx in Senegalese sole viral defence. These findings contribute to increasing the knowledge of the role of interferon pathway in fish innate immunity and to develop new tools to fight virus infections in the culture of this species.     

Olfactory transduction pathways in the Senegalese sole Solea senegalensis

This study tested whether differences in sensitivity between the upper and lower olfactory epithelia of Solea senegalensis are associated with different odorant receptors and transduction pathways, using the electro‐olfactogram. Receptor mechanisms were assessed by cross‐adaptation with amino acids (L‐cysteine, L‐phenylalanine and 1‐methyl‐L‐tryptophan) and bile acids (taurocholic acid and cholic acid). This suggested that relatively specific receptors exist for 1‐methyl‐L‐tryptophan and L‐phenylalanine (food‐related odorants) in the lower epithelium, and for taurocholic acid (conspecific‐derived odorant) in the upper. Inhibition by U73122 [a phospholipase C (PLC) inhibitor] suggested that olfactory responses to amino acids were mediated mostly, but not entirely, by PLC‐mediated transduction (IC₅₀; 15–55 nM), whereas bile acid responses were mediated by both PLC and adenylate cyclase–cyclic adenosine monophosphate (AC–cAMP) (using SQ‐22536; an AC inhibitor). Simultaneous application of both drugs rarely inhibited responses completely, suggesting possible involvement of non‐PLC and non‐AC mediated mechanisms. For aromatic amino acids and bile acids, there were differences in the contribution of each transduction pathway (PLC, AC and non‐PLC and non‐AC) between the two epithelia. These results suggest that differences in sensitivity of the two epithelia are associated with differences in odorant receptors and transduction mechanisms.     

c-Lysozyme from Senegalese sole (Solea senegalensis): cDNA cloning and expression pattern

Lysozymes are key molecules of innate immunity and proved high bactericidal activity in fish, thus becoming attractive as tools for enhancing fish defences. In this study, a full-length c-type lysozyme cDNA from Senegalese sole (Solea senegalensis) has been cloned and characterized. The cDNA sequence was inferred from two overlapping fragments obtained by RACE-PCR and consisting on 631bp coding for 143 aminoacids. Catalytic and other conserved residues required for lysozyme activity were identified. Pair wise alignments showed the higher identities with c-type lysozyme from other flatfish. Expression patterns under various conditions showed a basal level and a clear upregulation mostly in hematopoietic organs after stimulation with LPS or infection with Photobacterium damselae. This study represents a first step on the genetics and function of the c-lysozyme of Senegalese sole, though disclosing g-DNA structure, allelic variability and antibacterial activity must be requirements prior its immunological properties might have biotechnological applications.     

Food deprivation induces chronic stress and affects thyroid hormone metabolism in Senegalese sole (Solea senegalensis) post-larvae

In vertebrates, stress and thyroid systems interact closely, most likely because of the involvement of both systems in energy metabolism. However, studies on these interactions, especially during larval development, are scarce. Recently, cDNAs coding for corticotropin-releasing hormone (CRH) and CRH-binding protein (CRH-BP), two key players in the regulation of the neuroendocrine stress response, were characterized for the flatfish Senegalese sole (Solea senegalensis). To investigate the involvement of stress and thyroid systems in this species, the effects of food deprivation during early development of S. senegalensis were assessed. Growth was arrested in food-deprived post-larvae, which was also reflected by decreased carbon and nitrogen contents, indicating increased catabolism. Food deprivation induces chronic stress, as illustrated by enhanced whole-body cortisol levels, as well as an up regulation of crh and a decrease of crh-bp expression levels. Furthermore, whole-body total T3 concentrations of food-deprived post-larvae were reduced, although tshβ subunit expression levels remained unaffected. Our results show that food deprivation is a chronic stressor that induces energy-releasing catabolic processes that compensate for the reduced energy intake, and inhibits anabolic processes via the peripheral thyroid system.     

Characterization of Vibrio harveyi strains recovered from diseased farmed Senegalese sole (Solea senegalensis)

Aim:  To characterize 16 Vibrio harveyi strains isolated from different epizootic outbreaks affecting farmed Senegalese sole.
Materials and Results:  The Vibrio harveyi strains tested have broad phenotypic diversity based on their biochemical and exoenzymatic patterns, outer membrane proteins (OMP), extracellular product (ECP) patterns and presence of prophages. Lethal dose 50 (LD₅₀) of the strains and in vitro antagonism tests with two probiotic strains were also determined. The OMP analysis revealed three different patterns (A, M and V). The electrophoretic analysis of the ECP showed two different groups. All strains considered virulent based on their LD₅₀ exhibited the same protein pattern in their ECP (pattern I), while all nonvirulent strains showed a different profile (pattern II). About 32% of the tested strains were positive for prophages, although a clear relationship between virulence and the presence of prophages has not been established.
Conclusions:  The results obtained have shown differences between virulent and avirulent strains isolated from diseased farmed Senegalese sole based on the protein patterns of their ECP. However, a clear relationship between virulence and presence of prophages has not been established.
Significance and Impact of the Study:  The differences observed between virulent and nonvirulent strains could be used to design prophylactic strategies against diseases caused by V. harveyi in farmed Senegalese sole.     

Microalgal biomasses have potential as ingredients in microdiets for Senegalese sole (Solea senegalensis) post-larvae

Senegalese sole (Solea senegalensis) production presents several nutritional challenges, making this species a good candidate to study the dietary potential of bioactive compounds. Since proper nutrition plays a fundamental role in fish biology, it is necessary to further investigate species-specific and well-balanced diets in order to improve Senegalese sole juvenile farming. Algae have antioxidant properties, high-quality dietary protein, and are a source of bioactive compounds. This study evaluates the effects of dietary microalgal inclusion in both health status and growth performance of Senegalese sole post-larvae. Individuals 41 days after hatching (DAH) were randomly distributed among 12 tanks and four experimental diets were randomly distributed by triplicate groups. A basal diet served as CTRL and the experimental diets were formulated to include 3% of each of the algal biomass (CHLO, Chlorella sp. from heterotrophic production; PHAEO, Phaeodactylum sp.; and NANNO, Nannochloropsis sp.). At 50 DAH, 20 post-larvae/tank were collected and homogenized for analysis of immune and oxidative status, and at 61 DAH the total length, dry weight, and survival were assessed. No changes were observed in survival and total length of individuals, post-larvae fed NANNO, and CHLO dietary treatments increased dry weight at 61 DAH compared with those fed CTRL. While post-larvae immune status was apparently not altered by dietary treatments at 50 DAH, the total glutathione content decreased in fish fed PHAEO and CHLO dietary treatments compared to control diet. The observed results on improvement of growth performance without adverse effects on the immune status and decrease of endogenous total glutathione point to the fact that Nannochloropsis sp., Phaeodactylum sp., and Chlorella sp. could work as potential candidates for inclusion in microdiets for Senegalese sole.

     

Evidence for functional asymmetry in the olfactory system of the Senegalese sole (Solea senegalensis)

The two olfactory epithelia of flatfish of the family Soleidae are essentially in contact with two distinct environments; the upper (right) side samples open water while the lower (left) side samples interstitial water. This study assessed whether there are differences in the responsiveness of the two epithelia by use of the electro-olfactogram in the Senegalese sole (Solea senegalensis). The upper epithelium was significantly more responsive to the basic amino acids (L-lysine and L-arginine), glycine, and L-threonine than the lower epithelium. The lower epithelium was significantly more responsive to aromatic amino acids (L-tryptophan, L-tyrosine, L-DOPA, and L-phenylalanine), L-leucine, and L-asparagine than the upper. Both epithelia had similar responsiveness to the sulphur-containing amino acids (L-cysteine and L-methionine), L-alanine, L-serine, and L-glutamine. Neither side was responsive to the acidic amino acids (L-aspartate and L-glutamate) or the D-isomers of any amino acid tested. The upper olfactory organ was much more responsive to conspecific-derived stimuli (bile and intestinal fluid) than the lower organ. We suggest that these differences in responsiveness may be related to different functional roles of the upper and lower epithelia in feeding and chemical communication.     

Phaeodactylum tricornutum biomass in microdiets enhances Senegalese sole (Solea senegalensis) larval growth performance during weaning

Journal of Applied Phycology - Microalgae are one of the most promising groups of functional ingredients for inclusion in microdiets for Senegalese sole (Solea senegalensis) larvae, supplying...     

Micro‐anatomical characterization of vertebral curvatures in Senegalese sole Solea senegalensis

The micro‐anatomical changes associated with lordotic and kyphotic vertebral curvatures (VC) in juvenile and adult Senegalese sole Solea senegalensis are described. In addition, it is demonstrated that the tissue and cellular structures of individual vertebrae can be severely affected. Two main conformations were found in deformed juvenile specimens: flattened vertebrae with dorso‐ventral compression and trapezoidal vertebrae forming concave and convex sides under compressive and tensile stresses. Histological analyses revealed the occurrence of an ectopic cartilaginous tissue within the acellular bone, both in juveniles and adults, possibly to cope with altered mechanical stress in deformed vertebrae. The results suggest that the alteration in loading to which curved vertebral columns are subjected might trigger vertebral reshaping and differentiation of cells towards this ectopic tissue. In addition, mesenchymal cells appear to play an important role in its formation. It is here proposed that the acellular bone of S. senegalensis is capable of adaptively responding to altered loading regimes at the structural level by reshaping vertebrae and at the micro‐anatomical level by recruiting chondrocyte‐like cells to areas of altered mechanical stress.     

An immunocytochemical study of pituitary cells of the Senegalese sole, Solea senegalensis (Kaup 1858)

Different antisera directed against mammalian and piscine pituitary hormones, as well as a battery of various conventional histochemical techniques (PAS, Alcian Blue pH 2.5, Bromophenol Blue) and lectins, were used to identify the different hormonal cell types in the pituitary of the Senegalese sole, Solea senegalensis. Prolactin and adrenocorticotrophic cells were located in the rostral pars distalis of the pituitary. Gonadotrophic, thyrotrophic and growth hormone cells were distributed in the proximal pars distalis, but gonadotrophic cells appear also at the border of the pars intermedia. Somatolactin cells, as well as α-melanotrophic cells were located in the pars intermedia of the Solea senegalensis pituitary. The PAS reaction was positive in somatolactin cells, which were unreactive with the lead--Haematoxylin technique, whereas melanotrophic cells were positive. Glycoproteins containing mannose and/or glucose, as well as N-acetyl-glucosamine and sialic acid sugar residues, are synthesized and secreted by gonadotrophic, thyrotrophic and somatolactin cells. Adrenocor ticotrophic cells and, especially, the amphiphilic somatolactin and acidophilic growth hormone cells were stained with the Bromophenol Blue technique that identifies proteins in general, but adrenocorticotrophic and growth hormone cells were unreactive towards PAS, Alcian Blue pH 2.5 and lectins (Con A and WGA)     

An immunocytochemical study of pituitary cells of the Senegalese sole, Solea senegalensis (Kaup 1858)

Different antisera directed against mammalian and piscine pituitary hormones, as well as a battery of various conventional histochemical techniques (PAS, Alcian Blue pH 2.5, Bromophenol Blue) and lectins, were used to identify the different hormonal cell types in the pituitary of the Senegalese sole, Solea senegalensis. Prolactin and adrenocorticotrophic cells were located in the rostral pars distalis of the pituitary. Gonadotrophic, thyrotrophic and growth hormone cells were distributed in the proximal pars distalis, but gonadotrophic cells appear also at the border of the pars intermedia. Somatolactin cells, as well as α-melanotrophic cells were located in the pars intermedia of the Solea senegalensis pituitary. The PAS reaction was positive in somatolactin cells, which were unreactive with the lead--Haematoxylin technique, whereas melanotrophic cells were positive. Glycoproteins containing mannose and/or glucose, as well as N-acetyl-glucosamine and sialic acid sugar residues, are synthesized and secreted by gonadotrophic, thyrotrophic and somatolactin cells. Adrenocor ticotrophic cells and, especially, the amphiphilic somatolactin and acidophilic growth hormone cells were stained with the Bromophenol Blue technique that identifies proteins in general, but adrenocorticotrophic and growth hormone cells were unreactive towards PAS, Alcian Blue pH 2.5 and lectins (Con A and WGA) This revised version was published online in November 2006 with corrections to the Cover Date.     

Ovarian development and plasma sex steroid levels in cultured female Senegalese sole Solea senegalensis

Ovarian development was studied in cultured female Senegalese sole Solea senegalensis. Females with regressed ovaries, mainly occupied by perinucleolar oocytes, predominated throughout summer exhibiting low condition factor (K), gonadosomatic index (I(G)), and plasma 17beta-estradiol and testosterone levels. Throughout autumn and winter (ovaries at early and intermediate maturation), oocytes progressed to cortical alveoli and vitellogenic stages accompanied by increasing K, I(G), and plasma 17beta-estradiol and testosterone levels. At late winter/early spring, ovarian development reached its maximum with the predominance of females at intermediate and final maturation (the latter occupied by late vitellogenic oocytes and few early maturation oocytes) and peak values of K, I(G), and 17beta-estradiol and testosterone concentrations. Steroid levels were lower (especially testosterone) than those for naturally-spawning females, which might cause extensive atresia without final oocyte maturation (no spawning was observed). This degenerative process reduced de size of the ovary (initial and intermediate phases of regression) in association with declining K, I(G), and plasma 17beta-estradiol and testosterone levels and increasing proportions of perinucleolar oocytes. The circulating 17,20beta-dihydroxy-4-pregnen-3-one levels, the proposed maturation-inducing steroid, remained relatively constant throughout the experimental period, suggesting that oocytes were unable to respond adequately to its stimulation. We propose the inadequate seasonal thermal regime as the main cause of such dysfunction.     

Development and characterization of microsatellites from Senegal sole (Solea senegalensis)

To assist in the domestication of the sole fish Solea senegalensis, 15 microsatellites were developed from its genome (13 di‐ and two trinucleotide). These loci, along with three from S. solea that cross‐amplified, were characterized on a sample of 20 individuals of S. senegalensis from a hatchery stock. All loci were polymorphic displaying from two to 15 alleles/locus (mean 6.5). The results obtained from the hatchery population screening indicate that they might be useful for studies of genetic structuring and/or kinship analysis in S. senegalensis.     

Treatment of GnRHa-implanted Senegalese sole (Solea senegalensis) with 11-ketoandrostenedione stimulates spermatogenesis and increases sperm motility

The effect of 11-ketoandrostenedione (OA) on plasma concentrations of sexual steroids and spermatogenesis of Senegalese sole (Solea senegalensis) implanted with gonadotropin-releasing hormone agonist (GnRHa) was investigated. Males were treated with saline (control) or with GnRHa implants (50 mug kg(-1)) in the presence or absence of OA (2 or 7 mg kg(-1)) during twenty eight days. Treatment with GnRHa alone slightly stimulated spermatogenesis and milt production with respect to controls, and this was associated with a transient elevation of plasma 11-ketotestosterone (11-KT) at day seven and an increase of 5beta-reduced metabolite(s) of 17,20beta-dihydroxy-pregn-4-en-3-one (17,20betaP) at day twenty eight. However, treatment with GnRHa+OA increased plasma concentrations of 11-KT and free+sulphated 5beta-reduced metabolites of 17,20betaP at days seven, fourteen and twenty one. After twenty eight days, the testis of GnRHa+OA-treated fish showed a lower number of spermatogonia B and spermatocytes I, and a higher number of spermatids, than fish treated with GnRHa alone. In addition, the motility of spermatozoa produced by GnRHa+OA males was enhanced by 2-fold with respect to controls or GnRHa males. These results suggest that treatment of Senegalese sole with GnRHa+OA stimulates spermatogenesis resulting in more motile sperm. Such effects could be mediated by an increased synthesis of 11-KT and/or 17,20betaP in the testis but further studies will be required to elucidate the specific mechanism involved.     

Cloning,tissue and ontogenetic expression of the taurine transporter in the flatfish Senegalese sole (Solea senegalensis)

Flatfish species seem to require dietary taurine for normal growth and development. Although dietary taurine supplementation has been recommended for flatfish, little is known about the mechanisms of taurine absorption in the digestive tract of flatfish throughout ontogeny. This study described the cloning and ontogenetic expression of the taurine transporter (TauT) in the flatfish Senegalese sole (Solea senegalensis). Results showed a high similarity between TauT in Senegalese sole and other vertebrates, but a change in TauT amino acid sequences indicates that taurine transport may differ between mammals and fish, reptiles or birds. Moreover, results showed that Senegalese sole metamorphosis is an important developmental trigger to promote taurine transport in larvae, especially in muscle tissues, which may be important for larval growth. Results also indicated that the capacity to uptake dietary taurine in the digestive tract is already established in larvae at the onset of metamorphosis. In Senegalese sole juveniles, TauT expression was highest in brain, heart and eye. These are organs where taurine is usually found in high concentrations and is believed to play important biological roles. In the digestive tract of juveniles, TauT was more expressed in stomach and hindgut, indicating that dietary taurine is quickly absorbed when digestion begins and taurine endogenously used for bile salt conjugation may be recycled at the posterior end of the digestive tract. Therefore, these results suggest an enterohepatic recycling pathway for taurine in Senegalese sole, a process that may be important for maintenance of the taurine body levels in flatfish species.