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.     

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.     

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.     

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.     

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.     

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)     

2‐D DIGE analysis of Senegalese sole (Solea senegalensis) testis proteome in wild‐caught and hormone‐treated F1 fish

In the farmed flatfish Senegalese sole, F1 males reared in captivity often show lower sperm production and fertilization capacity than wild‐caught males. To gain insights into the molecular mechanisms that may be altered in the F1 testis, we used 2‐D DIGE to compare the protein profiling of the testis of wild‐caught males at the spermiation stage with that of F1 males showing different stages of germ cell development after hormone treatment in vivo. The abundance of 58 out of 1014 protein spots was found to differ significantly between the groups. De novo identification of these proteins by MS/MS revealed that proteins implicated in oxidoreductase activity, protein catabolism, formation of the zona pellucida receptor, cytoskeleton organization, and lipid binding and metabolism, were regulated in the F1 testes as germ cell development progressed. However, distinct isoforms or PTMs of some of these proteins, as well as of proteins involved in iron and glucose metabolism and ATP production, were expressed at lower levels in the testes of F1 males than in wild fish regardless of the hormone treatment. These results contribute to identifying proteins associated with spermatogenesis not previously described in teleosts, and suggest potential mechanisms that may be involved in the poor reproductive performance of Senegalese sole F1 males.     

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.     

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.     

Effects of temperature and dietary protein level on hepatic oxidative status of Senegalese sole juveniles (Solea senegalensis)

Effects of 55 and 45% dietary protein levels (55P and 45P diets, respectively) and temperature (12 and 18°C) on hepatic activity of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase (GR), glucose-6-phosphate dehydrogenase and lipid peroxidation (LPO) levels of Solea senegalensis juveniles were studied. Further, effects of acute thermal shocks provoked by a drop (18°C to 12°C) or a rise (12°C to 18°C) of water temperature on sole oxidative state was also evaluated. Dietary protein reduction increased LPO levels though no major alterations were found on antioxidant enzyme activities between dietary treatments. At 12°C GR activity was higher and SOD activity was lower than 18°C but LPO levels were not affected. In both thermal shock cases, LPO levels increased in 55P group, probably due to insufficient antioxidant enzyme activation. In contrast, fish of 45P group under acute exposition to warmer and colder temperature exhibited no substantial changes and a significant decrease on LPO levels, respectively, along with no major changes in antioxidant enzymes. Overall, results suggest that independently of rearing temperatures 45P group was more susceptible to oxidative stress than 55P group. Thermal shock either due to rise or drop of temperature seemed to induce oxidative stress in 55P group.     

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.     

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.     

Molecular regulation of both dietary vitamin A and fatty acid absorption and metabolism associated with larval morphogenesis of Senegalese sole (Solea senegalensis)

The present study aimed to deepen the understanding of molecular mechanisms governing the absorption and metabolism of some nutrients, growth and development in larvae of Senegalese sole (Solea senegalensis) fed with Artemia enriched with Easy Selco© (ES, INVE) or Aquagrow Gold© (AGG, ABN), which mainly differed in their vitamin A (VA) content and fatty acid composition. The expression profile of genes involved in VA metabolism (crbp2, rbp, crabp1), lipid transport (i-fabp, l-fabp), nuclear receptors for VA and fatty acids (rarα1, rxrα, pparβ), growth (igf1, igf2 and their receptor igf1r) and development (bgp) was analyzed at 22, 30 and 38 days post hatching. The main results suggested that the amount of VA absorbed by larvae is controlled at the intestinal level by crbp2 in both groups, preventing excessive accumulation of this vitamin in the target tissues. The stable expression of i-fabp in the ES group with age could cause an excessive fat accumulation in the intestine inducing, in turn, the steatosis found in the liver and vascular system of these specimens. In liver, the regulation of rbp and fabp expression reflected the status of the physiological functions demanding VA and lipids. The findings revealed that dietary composition induced different strategies for VA and lipid absorption and metabolism affecting, in turn, larval development, growth and health.     

Validation and comparison of microsatellite markers derived from Senegalese sole (Solea senegalensis, Kaup) genomic and expressed sequence tags libraries

In this work, we tested 100 potential new microsatellites (SSRs) equally derived from expressed sequence tag (EST) and enriched genomic-DNA libraries from Senegalese sole (Solea senegalensis, Kaup), a valuable cultured flatfish species. A final set of 69 new polymorphic microsatellites were validated after a population analysis, 37 of which corresponded to the first EST library constructed for Senegalese sole (EST-SSR). Although differences were not significant, EST sequences provided a higher proportion of quality markers (74%) than anonymous ones (64%). Most of the rejected anonymous SSRs (17 loci) were discarded because they did not generate PCR products; only one was monomorphic. On the contrary, all EST-SSRs gave PCR products, although monomorphism was more frequent (26%). Altogether, the number of alleles per locus was fairly similar in both SSR types, ranging from 2 to 19. The observed and expected heterozygosities varied from 0.105 to 1 and from 0.108 to 0.937, respectively. The main difference between the two sets was the percentage of annotated loci, being higher in EST-SSRs, as expected. Within the EST-SSRs, 46% of them showed flanking regions that significantly matched with EST sequences from other three flatfish species; however, the microsatellite itself was present only on half of these cases. These two new SSR sets constitute a suitable tool for fingerprinting, gene flow, genetic diversity, genome mapping studies and molecular-assisted breeding in this species.     

The Circadian Clock Machinery During Early Development of Senegalese sole (Solea senegalensis): Effects of Constant Light and Dark Conditions

Circadian rhythms are established very early during vertebrate development. In fish, environmental cues can influence the initiation and synchronization of different rhythmic processes. Previous studies in zebrafish and rainbow trout have shown that circadian oscillation of clock genes represents one of the earliest detectable rhythms in the developing embryo, suggesting their significance in regulating the coordination of developmental processes. In this study, we analyzed the daily expression of the core clock components Per1, Per2, Per3, and Clock during the first several days of Senegalese sole development (0–4 d post fertilization or dpf) under different lighting regimes, with the aim of addressing when the molecular clock first emerges in this species and how it is affected by different photoperiods. Rhythmic expression of the above genes was detected from 0 to 1 dpf, being markedly affected in the next few days by both constant light (LL) and dark (DD) conditions. A gradual entrainment of the clock machinery was observed only under light-dark (LD) cycles, and robust rhythms with increased amplitudes were established by 4 dpf for all clock genes currently studied. Our results show the existence of an embryonic molecular clock from the 1st d of development in Senegalese sole and emphasize the significance of cycling LD conditions when raising embryos and early larvae. (Author correspondence: munoz.cueto@uca.es; carlos.pendon@uca.es)