Crustacean amphipods from marsh ponds: a nutritious feed resource with potential for application in Integrated Multi-Trophic Aquaculture.

Coastal protection, nutrient cycling, erosion control, water purification, and carbon sequestration are ecosystem services provided by salt marshes. Additionally, salt ponds offer coastal breeding and a nursery habitat for fishes and they provide abundant invertebrates, such as amphipods, which are potentially useful as a resource in aquaculture. Fishmeal and fish oil are necessary food resources to support aquaculture of carnivorous species due to their omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Currently, aquaculture depends on limited fisheries and feed with elevated n-3 LC-PUFA levels, but the development of more sustainable food sources is necessary. Amphipods appear to be a potential high quality alternative feed resource for aquaculture. Hence, a nutritional study was carried out for several main amphipod species-Microdeutopus gryllotalpa, Monocorophium acherusicum, Gammarus insensibilis, Melita palmata and Cymadusa filosa-in terrestrial ponds in the South of Spain. These species showed high protein content (up to 40%), high n-3 PUFA and phospholipid levels, and high levels of phophatidylcholine (PC), phosphatidylethanolamine (PE) and triacylglycerols (TAG), the latter being significantly high for M. acherusicum. M. gryllotalpa and M. acherusicum showed the highest proportion of lipids (19.15% and 18.35%, respectively). Isoleucine, glycine and alanine were the dominant amino acids in all species. In addition, amphipods collected from ponds showed low levels of heavy metals. Furthermore, the biochemical profiles of the five species of amphipods have been compared with other studied alternative prey. Therefore, pond amphipods are good candidates to be used as feed, and are proposed as a new sustainable economic resource to be used in aquaculture. G. insensibilis may be the best for intensive culture as an alternative feed resource because it shows: (1) adequate n-3 PUFA and PL composition; (2) high levels of glycine, alanine, tyrosine, isoleucine and lysine; (3) high natural densities; (4) large body size (≥1 cm), and (5) high concentration of calcium. Moreover, a combined culture of amphipods and fishes in these marsh ponds seems a promising and environmentally sustainable way to develop Integrate Multi-Trophic Aquaculture (IMTA) in these ecosystems.

Common and Species-Specific Effects of Phosphate on Marine Microalgae Fatty Acids Shape Their Function in Phytoplankton Trophic Ecology.

The use of fatty acids (FA) to infer structure of phytoplankton assemblages and as indicators of microalgae nutritional value is acquiring relevance in modern phytoplankton ecology and new advances concerning factors influencing FA variability among microalgae are demanded. In this regard, the relationship between phosphorus and FA remains particularly little studied in marine phytoplankton. In the present study, we focus on phosphate effects on FA from a diversified set of marine microalgae and provide new insights into the applicability of FA in phytoplankton trophic ecology. Phosphate deprivation mainly induced monounsaturated FA production in eight out of nine microalgae and their changes were species-specific, with palmitoleic acid exhibiting extreme variation and discriminating between haptophyte classes. The important phosphate-induced and interspecific variability found for oleic acid was perceived as a concern for the current application of this FA as a trophic position indicator in grazers. Chloroplast C-16 and C-18 polyunsaturated FA were more affected by phosphate than C-20 and C-22 highly unsaturated FA (HUFA). The relative stability of stearidonic acid to phosphate in cryptophytes and haptophytes pinpointed this FA as a suited marker for both microalgae groups. Taken all species together, phosphate deprivation and taxonomy accounted for 20.8 and 50.7% of total FA variation, respectively. HUFA were minimally affected by phosphate indicating their suitability as indicators of phytoplankton trophic value. The asymptotic relationship between HUFA and phosphorus cell content suggested mineral composition (phosphorus) could be more important than HUFA content as attribute of marine microalgae nutritional value at the species level.

Aspects of phosphorus physiology associated with phosphate-induced polar lipid remodelling in marine microalgae.

Marine microalgae exhibit a diversified phosphorus physiology and have also been recently found to show high inter-taxa variability in their phosphate induced-polar lipids' remodelling. Identification of phosphorus physiology aspects that are more related to lipid remodelling can contribute to better understanding of such intricate phytoplankton lipid metabolism. Therefore, some aspects of phosphorus physiology related to its uptake, storage and use were evaluated in a taxonomically diversified group of nine marine microalgae that was arranged into three subgroups, each of them including species showing similar polar lipid responses to phosphate. Luxury phosphate uptake (PU) was the physiological aspect best associated to microalgal polar lipid metabolism as it was maximal in species (Picochlorum atomus, Tetraselmis suecica and Nannochloropsis gaditana) that were able to counterbalance between phospholipids (PL) and betaine lipids (BL). Cryptophytes (Rhodomonas baltica, Chroomonas placoidea), characterized by their constitutive BL and flexible PL contents in response to phosphate, had almost no luxury PU and showed higher phosphorus cell quota (QP) under phosphate deprivation. Haptophyes (Isochrysis galbana, Diacronema vlkianum), with constitutive BL contents and permanently minimal PL contents, showed the lowest QP when deprived of phosphate while their luxury PU was below that for green microalgae. Induction of alkaline phosphatase activity following phosphate depletion was maximal in diatoms (Phaeodactylum tricornutum, Chaetoceros gracilis) and I. galbana but it was unrelated to lipid remodelling. Despite strong influence of taxonomy, polar lipid remodelling accounted for 38.8% of total variation when microalgae were ordinated using their physiological responses to phosphorus as descriptive variables.

Polar Lipids Analysis of Cultured Phytoplankton Reveals Significant Inter-taxa Changes, Low Influence of Growth Stage, and Usefulness in Chemotaxonomy.

The high lipid diversity of microalgae has been used to taxonomically differentiate phytoplankton taxa at the class level. However, important lipids such as phospholipids (PL) and betaine lipids (BL) with potential chemotaxonomy application in phytoplankton ecology have been scarcely studied. The chemotaxonomy value of PL and BL depends on their intraspecific extent of variation as microalgae respond to external changing factors. To determine such effects, lipid class changes occurring at different growth stages in 15 microalgae from ten different classes were analyzed. BL occurred in 14 species and were the less affected lipids by growth stage with diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-b-alanine (DGTA) showing the highest stability. PL were more influenced by growth stage with phosphatidylcholine (PC), phosphatidylglycerol (PG), and phosphatidyletanolamine (PE) declining towards older culture stages in some species. Glycolipids were the more common lipids, and no evident age-related variability pattern could be associated to taxonomic diversity. Selecting BL and PL as descriptor variables optimally distinguished microalgae taxonomic variability at all growth stages. Principal coordinate analysis arranged species through a main tendency from diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-b-alanine (DGCC) containing species (mainly dinoflagellates and haptophytes) to DGTA or PC containing species (mainly cryptophytes). Two diatom classes with similar fatty acid profiles could be distinguished from their respective content in DGTA (Bacillariophyceae) or DGCC (Mediophyceae). In green lineage classes (Trebouxiophyceae, Porphyridophyceae, and Chlorodendrophyceae), PC was a better descriptor than BL. BL and PL explained a higher proportion of microalgae taxonomic variation than did fatty acids and played a complementary role as lipid markers.

Interspecific variability in phosphorus-induced lipid remodelling among marine eukaryotic phytoplankton.

The response of marine microalgal lipids to phosphorus is of central importance in phytoplankton ecology but remains poorly understood. We determined how taxonomically diverse microalgal species remodelled their lipid class profile in response to phosphorus availability and whether these changes coincided with those already known to occur in land plants and in the limited number of phytoplankton species for which data are available. The complete lipid class profile and specific lipid ratios influenced by phosphorus availability were quantified in two green microalgae and seven Chromalveolates exposed to phosphorus repletion, deprivation and replenishment. Lipid class cell quota changes in the two green microalgae resembled the currently described pattern of betaine lipids substituting for phospholipids under phosphorus depletion, whereas only two of the studied Chromalveolates showed this pattern. Sulpholipids counterbalanced phosphatidylglycerol only in Picochlorum atomus. In all other species, both lipids decreased simultaneously under phosphorus deprivation, although sulpholipids declined more slowly. Phosphorus deprivation always induced a decrease in digalactosyl-diacylglycerol. However, the ratio of digalactosyl-diacylglycerol to total phospholipids increased in eight species and remained unchanged in Isochrysis galbana. Marine phytoplankton seems to have evolved a diversified mechanism for remodelling its lipid class profile under the influence of phosphorus, with cryptophytes and particularly haptophytes exhibiting previously unobserved lipid responses to phosphorus.

Exploring occurrence and molecular diversity of betaine lipids across taxonomy of marine microalgae.

Betaine lipids (BL) from ten microalgae species of the kingdoms Plantae and Chromista were identified and quantified by HPLC/ESI-TOF-MS. Diacylgyceryl-N-trimethylhomoserine (DGTS) was detected in Trebouxiophyceae and Eustigmatophyceae species, whereas Tetraselmis suecica was described as the first green algae containing diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-beta-alanine (DGTA). DGTA molecular species where also characterized in Cryptophyceae species as well as in the Bacillariophyceae diatom Phaeodactylum tricornutum. The Mediophyceae diatom Chaetoceros gracilis had no DGTA, but contained diacylglyceryl-carboxyhydroxymethylcholine (DGCC). A principal coordinate (PCO) analysis of microalgae species revealed the existence of three main clusters around each BL type. The first PCO axis (43.9% of total variation) grouped Chlorophyceae, Trebouxiophyceae and Eustigmatophyceae species and positively correlated with DGTS. The second PCO axis (27.8% of total variation) segregated DGTA from DGCC containing species. Cryptophyceae, Bacillariophyceae and Chlorodendrophyceae were the more closely associated species to DGTA. Mediophyceae and Dinophyceae species contained DGCC as the only BL. Molecular diversity varied from the simplest DGCC composition in Gyrodinium dorsum to the highest spectrum of ten different molecular species detected for DGTA (Rhodomonas baltica) and DGCC (C. gracilis). The fatty acid profile of DGTS was very dissimilar to that of the whole lipid cell content. DGTS from Nannochloropsis gaditana was highly unsaturated respecting to total lipids, whereas in Picochlorum atomus DGTS unsaturation was nearly one half to that of total lipids. Dissimilarity between DGTA and total lipid fatty acid profile was minimum among all BL and DGTA fatty acid unsaturation was the maximum observed in the study. New DGCC molecular species enriched in 20:5 were described in Mediophyceae diatoms. Multivariate microalgae ordination using BL as descriptors revealed a higher chemotaxonomic potential than that based on their respective BL fatty acid profile. Nevertheless, taxonomic resolution was improved when fatty acids from the whole cell lipid pool were used.

Proteomic analysis through larval development of Solea senegalensis flatfish.

The post-embryonic development of the Senegalese sole, Solea senegalensis, a flatfish of growing interest in fisheries and aquaculture, is associated with drastic morpho-physiological changes during metamorphosis. Although in the last two decades knowledge on sole culture has notably increased, especially in Southern Europe, its progress was restricted due to lack of methods to control reproduction, improve larval quality and increase juvenile disease resistance. A limited knowledge of the physiological, molecular and genetic mechanisms involved is at the base of such limitation. A proteomic study was carried out to explore the molecular events that occur during S. senegalensis ontogenesis. Protein expression changes were monitored in larvae from 5 to 21 dph by combining 2DE and protein identification with de novo MS/MS sequencing. An average of 6177 ± 282 spots was resolved in 2DE gels. Hierarchical cluster analysis of the 705 selected spots grouped them in eight patterns. Thirty-four proteins were identified and assigned biological functions including structure, metabolism highlighting energy metabolism, transport, protein folding, stress response, chromatin organization and regulation of gene expression. These changes provide a sequential description of the molecular events associated with the biochemical and biological transformations that occur during sole larval development.

Differences in betaine lipids and fatty acids between Pseudoisochrysis paradoxa VLP and Diacronema vlkianum VLP isolates (Haptophyta).

Two Haptophytes were isolated from extensive aquaculture ponds at Veta La Palma state (Guadalquivir estuary, SW Spain). They were identified as Pseudoisochrysis paradoxa VLP and Diacronema vlkianum VLP based on their SSU rDNA homology to other Haptophytes and positioned in the Isochrysidaceae and Pavlovaceae families, respectively. Both Haptophytes had phosphatidilglycerol (PG) as the only phospholipid (PL), representing a low proportion of the total lipid content (0.8% in P. paradoxa VLP and 3.3% in D. vlkianum VLP). Instead, they were found to have different types of betaine lipids (BL) that were identified and characterized by HPLC/ESI-TOF-MS operating in multiple reacting monitoring (MRM) modes. P. paradoxa VLP had 2.2% of total lipids as diacylgyceryl-N-trimethylhomoserine (DGTS): it is the first Haptophyte reported to have this BL. Its total lipid fraction also contained 12.0% of diacylglyceryl-carboxyhydroxymethylcholine (DGCC) as the main BL and no diacylglyceryl-hydroxymethyl-N,N,N-trimethyl-ß-alanine (DGTA) was detected. DGTA was only present (4.6% of total lipids) in D. vlkianum VLP: this was the main difference in BL content relative to P. paradoxa. D. vlkianum VLP also had DGTS (4.1%) and DGCC (7.6%): it is the first microalgae in which the simultaneous presence of these three BL has been demonstrated. The fatty acid profiles of P. paradoxa VLP and D. vlkianum VLP were close to those described for the major part of known members of the Isochrisidaceae and Pavlovaceae families, respectively, with the main differences due to the higher percentages of 18:1n9 (18.5%), 18:4n3 (12.6%) and 22:6n3 (9.3%) in the former. The corresponding fatty acid percentages for D. vlkianum VLP were 3.9%, 3.5% and 3.9%, respectively. D. vlkianum VLP showed higher 16:1n7 (16.1%) and 20:5n3 (9.4%) contents, whereas P. paradoxa VLP had significantly lower percentages of 16:1n7 (1.7%) and 20:5n3 (0.6%). Fatty acids of BL differed between both haptophytes. In DGTS from P. paradoxa VLP, 90.9% of total molecular species consisted of the 14:0-18:1 fatty acid combination, whereas DGTS from D. vlkianum showed a more diverse range of fatty acids. The unsaturation index (UI) of DGTS was lower (55.8) than that of total lipid UI (178.3) in P. paradoxa VLP. In D. vlkianum VLP the UI of DGTS was higher (146.9) and similar to that for total cell lipids (145.9). DGTA from D. vlkianum VLP had the highest UI (321.8) of all BL studied and it contained maximum levels (27.7%) of 22:6n3, representing 7.1 times the proportion of this fatty acid in the whole lipid extract. DGCC was enriched in 20:5n3 by a factor of around four in both microalgae. Due to different levels of this fatty acid in the two microalgae their respective 20:5n3 content in DGCC varied from 2.2% (P. paradoxa VLP) to 41.0% (D. vlkianum VLP) and these concentrations were also associated with UI values of 92.2 and 271.0, respectively. The specific differences in BL and fatty acids described in the present work for two phylogenetic distant Hatophytes is a contribution to a better understanding on the complex relationship between lipid composition and taxonomy of this important Division of microalgae. Present results can also be useful for a more accurate identification of primary producers in food web studies using fatty acids and intact polar lipids as trophic markers.

Effects of stocking density and feed ration on growth and gene expression in the Senegalese sole (Solea senegalensis): potential effects on the immune response.

Stocking density and ration size are two major factors influencing aquaculture production. To evaluate their effects on growth and immune system in Senegalese sole (Solea senegalensis) juveniles, a 2 x 2 experimental design using two rations (1.0% and 0.25% of the total fish biomass) and two different initial stocking densities (7 and 30 kg m(-2)) was performed throughout a 60 days culture period. Soles fed 1.0% showed a higher specific growth rate (SGR) than those fed 0.25% (3.3-fold). No differences in SGR at 60 days were found between densities in spite of reduced values were detected at high density after 20 days (soles fed 0.25%) and 40 days (soles fed 1%) suggesting a compensatory growth. Physiologically, plasma cortisol levels were elevated in soles at high density (45-fold higher than at 7 kg m(-2)) whereas no differences associated to the feeding ration were observed. To assess the effects at a molecular level, the mRNA levels of genes involved in cellular stress (heat shock proteins HSP70 and HSP90), growth (insulin-like growth factors IGF-I, the spliced variants IGF-Ia and IGFI-b, and IGF-II) and innate immune system (g-type lysozyme and hepcidin (HAMP1)) were quantified. No differences in HSP90 expression were detected between densities or rations. In contrast, IGF-I, IGF-Ia and IGF-II showed reduced transcript levels in liver and HSP70 in liver and kidney at high density. Finally, g-type lysozyme and HAMP1 expression was greatly affected by both factors exhibiting an important reduction in the transcript levels at high density and low ration. Overall, our results show that S. senegalensis juveniles might exhibit satisfactory SGR at high density although the high plasma cortisol levels indicate a crowding stress that could negatively affect the expression levels of some of the genes studied.

Molecular characterization, gene expression and transcriptional regulation of thyroid hormone receptors in Senegalese sole.

Thyroid hormones (THs) play a key role in larval development, growth and metamorphosis in flatfish. Their genomic effects are mediated by thyroid hormone receptors (TRs). In this study, cDNAs encoding for TRalphaA, TRalphaB, and TRbeta have been sequenced in Senegalese sole (Soleasenegalensis). Main domains and conserved motifs were identified. Also, a truncated TRalphaB isoform (referred to as TRalphaBtr) and a spliced TRbeta variant (referred to as TRbetav) were detected. A phylogenetic analysis grouped both TRalpha and TRbeta genes into two separate clusters with their fish and mammalian counterparts. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, TRalphaA, TRalphaB, TRbetav, and TRbeta showed distinct transcript levels in tissues. During metamorphosis, only TRbetav and TRbeta modified their mRNA levels in a similar way to the T4 contents. To evaluate the possible regulation of TRs by their cognate ligand T4 during sole metamorphosis, larvae were exposed to the goitrogen thiourea (TU). TRbeta transcripts decreased significantly at 11 and 15 days after treatment. Moreover, adding exogenous T4 hormone to TU-treated larvae restored the steady-state levels or even increased TRbeta and TRbetav mRNAs with respect to the untreated control. Overall, these results demonstrate that TRbeta transcription is up-regulated by THs playing a major role during metamorphosis in Senegalese sole.

Translational machinery of senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.): comparative sequence analysis of the complete set of 60s ribosomal proteins and their expression.

Ribosomal proteins (RPs) comprise a large set of highly evolutionarily conserved proteins that are often over-represented in complementary DNA libraries. They have become very useful markers in comparative genomics, genome evolution, and phylogenetic studies across taxa. In this study, we report the sequences of the complete set of 60S RPs in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species. Amino-acid sequence comparisons of the encoded proteins showed a high similarity both between these two flatfish species and with respect to other fish and human counterparts. Expressed sequence tag analysis revealed the existence of paralogous genes for RPL3, RPL7, RPL41, and RPLP2 in Atlantic halibut and RPL13a in Senegalese sole as well as RPL19 and RPL22 in both species. Phylogenetic analysis of paralogs revealed distinct evolutionary histories for each RP in agreement with three rounds of genome duplications and lineage-specific duplications during flatfish evolution. Steady-state transcript levels for RPL19 and RPL22 RPs were quantitated during larval development and in different tissues of sole and halibut using a real-time polymerase chain reaction approach. All paralogs were expressed ubiquitously although at different levels in different tissues. Most RP transcripts increased coordinately after larval first-feeding in both species but decreased progressively during the metamorphic process. In all cases, expression profiles and transcript levels of orthologous genes in Senegalese sole and Atlantic halibut were highly congruent. The genomic resources and knowledge developed in this survey will be useful for the study of Pleuronectiformes evolution.

Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup).

HSP90 proteins are chaperones that play a pivotal role in controlling multiple regulatory pathways such as stress defense, hormone signalling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, two cDNAs encoding for cytosolic HSP90, referred to as HSP90AA and HSP90AB, have been sequenced. Main features and sequence identities with other fish and mammals are described. Phylogenetic analysis grouped both genes into two separate clusters with their fish and mammalian counterparts. Expression profiles during larval development and in juvenile tissues were analyzed using a real-time PCR approach. In juvenile fish, HSP90AB was constitutively expressed with lower transcript levels in skeletal muscle. In contrast, HSP90AA was mainly expressed in heart, skeletal muscle and skin. During metamorphosis, HSP90AB mRNA levels did not change whereas HSP90AA transcripts decreased significantly at the beginning of metamorphosis with the lowest mRNA levels at the metamorphosis climax. Due to the role of thyroid hormones (THs) on sole metamorphosis, the transcriptional regulation of HSP90 genes by THs was evaluated. Larvae exposed to the goitrogen thiourea (TU) exhibited higher HSP90AA mRNA levels than untreated control. Moreover, adding exogenous T4 hormone to TU-treated larvae restored the steady-state levels with respect to the untreated control. Unlike HSP90AA, the transcript levels of HSP90AB did not vary under any treatments. The response of both HSP90 genes to thermal stress in post-metamorphic individuals was also studied. A heat shock treatment (+7.9 degrees C for 1 h) rapidly activated HSP90AA (but not HSP90AB) transcription, reaching a peak after 30 min and declining expression levels progressively in the following 24 h. No significant changes in HSP90AA or HSP90AB transcript levels after a cold shock (-10 degrees C for 1 h) were observed. Overall, these results demonstrate that HSP90AA transcription is down-regulated by THs and up-regulated after a heat shock in Senegalese sole.

Selection of housekeeping genes for gene expression studies in larvae from flatfish using real-time PCR.

BACKGROUND: Flatfish metamorphosis involves major physiological and morphological changes. Due to its importance in aquaculture and as a model for developmental studies, some gene expression studies have focused on the understanding of this process using quantitative real-time PCR (qRT-PCR) technique. Therefore, adequate reference genes for accurate normalization are required. RESULTS: The stability of 12 potential reference genes was examined during larval development in Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus) to determine the most suitable genes for qRT-PCR analysis. Transcription levels of genes encoding beta-Actin (ACTB), glyceraldehyde-3P-dehydrogenase (GAPDH), annexin A2 (ANXA2), glutathione S-transferase (GST), ornithine decarboxylase (ODC), hypoxanthine phosphoribosyltransferase (HPRT1), ubiquitin (UBQ), elongation factor 1 alpha (eEF1A1), 18S ribosomal RNA, and the ribosomal proteins S4 (RPS4) and L13a (RPL13a) were quantitated. Two paralogous genes for ACTB were analyzed in each of both flatfish species. In addition, two paralogous genes for GAPDH were studied in Senegalese sole. RPL13a represented non-orthologous genes between both flatfish species. GeNorm and NormFinder analyses for expression stability revealed RPS4, UBQ and eEF1A1 as the most stable genes in Senegalese sole, Atlantic halibut and in a combined analysis. In all cases, paralogous genes exhibited differences in expression stability. CONCLUSION: This work suggests RPS4, UBQ, and eEF1A1 genes as useful reference genes for accurate normalization in qRT-PCR studies in Senegalese sole and Atlantic halibut larvae. The congruent results between both species in spite of the drastic differences in larval development suggest that selected housekeeping genes (HKGs) could be useful in other flatfish species. However, the finding of paralogous gene copies differentially expressed during development in some HKGs underscores the necessity to identify orthologous genes.

Molecular characterization and expression analysis of five different elongation factor 1 alpha genes in the flatfish Senegalese sole (Solea senegalensis Kaup): differential gene expression and thyroid hormones dependence during metamorphosis.

BACKGROUND: Eukaryotic elongation factor 1 alpha (eEF1A) is one of the four subunits composing eukaryotic translation elongation factor 1. It catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome in a GTP-dependent manner during protein synthesis, although it also seems to play a role in other non-translational processes. Currently, little information is still available about its expression profile and regulation during flatfish metamorphosis. With regard to this, Senegalese sole (Solea senegalensis) is a commercially important flatfish in which eEF1A gene remains to be characterized. RESULTS: The development of large-scale genomics of Senegalese sole has facilitated the identification of five different eEF1A genes, referred to as SseEF1A1, SseEF1A2, SseEF1A3, SseEF1A4, and Sse42Sp50. Main characteristics and sequence identities with other fish and mammalian eEF1As are described. Phylogenetic and tissue expression analyses allowed for the identification of SseEF1A1 and SseEF1A2 as the Senegalese sole counterparts of mammalian eEF1A1 and eEF1A2, respectively, and of Sse42Sp50 as the ortholog of Xenopus laevis and teleost 42Sp50 gene. The other two elongation factors, SseEF1A3 and SseEF1A4, represent novel genes that are mainly expressed in gills and skin. The expression profile of the five genes was also studied during larval development, revealing different behaviours. To study the possible regulation of SseEF1A gene expressions by thyroid hormones (THs), larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae exhibited lower SseEF1A4 mRNA levels than untreated controls at both 11 and 15 days after treatment, whereas transcripts of the other four genes remained relatively unchanged. Moreover, addition of exogenous T4 hormone to TU-treated larvae increased significantly the steady-state levels of SseEF1A4 with respect to untreated controls, demonstrating that its expression is up-regulated by THs. CONCLUSION: We have identified five different eEF1A genes in the Senegalese sole, referred to as SseEF1A1, SseEF1A2, SseEF1A3, SseEF1A4, and Sse42Sp50. The five genes exhibit different expression patterns in tissues and during larval development. TU and T4 treatments demonstrate that SseEF1A4 is up-regulated by THs, suggesting a role in the translational regulation of the factors involved in the dramatic changes that occurs during Senegalese sole metamorphosis.

Molecular characterization and gene expression of six trypsinogens in the flatfish Senegalese sole (Solea senegalensis Kaup) during larval development and in tissues.

The application of large-scale genomics to Senegalese sole (Solea senegalensis) has allowed for the identification of six different trypsinogen genes. The catalytic triad (His-57, Asp-102, and Ser-195) and other residues required for trypsin functionality were conserved across all trypsinogens. Sequence identities, charges and phylogenetic analysis allowed them to be classified into three groups: group I or anionic trypsinogens (ssetryp1a, ssetryp1b and ssetryp1c), group II or cationic trypsinogen (ssetryp2) and group III or psychrophilic trypsinogens (ssetryp3 and ssetrypY). The expression profiles of these genes were studied in juvenile tissues and during larval development using a real-time PCR approach. In juvenile fish, trypsinogens were expressed mainly in the intestine. Transcripts of ssetryp1c were the highest in all tissues except in brain where those of ssetryp2 were the most abundant. During larval development, ssetryp1 variants and ssetryp2 transcript levels increased from 2 to 6 days after hatching, and decreased thereafter. In contrast, transcripts of group III trypsinogens increased slightly or not significantly in premetamorphosis and decreased at metamorphosis. The expression levels ssetryp3 and ssetrypY were the lowest in larvae (from 172- to 1391-fold lower than ssetryp1 and ssetryp2). In contrast, they were expressed at a similar level as ssetryp2, although lower than ssetryp1, in juvenile tissues.

Thyroid hormones down-regulate thyrotropin beta subunit and thyroglobulin during metamorphosis in the flatfish Senegalese sole (Solea senegalensis Kaup).

Thyroid hormones (TH) play a critical role in flatfish metamorphosis. Their levels are regulated by the pituitary-thyroid axis. The expression profile of thyroid stimulating hormone (TSH) beta subunit and thyroglobulin (Tg) was investigated using a real-time PCR approach. Both genes exhibited different expression patterns during larval development in Senegalese sole. TSH beta mRNAs reduced progressively at the commencement of metamorphosis. On the contrary, Tg transcripts increased sharply at the onset of metamorphosis and dropped after the metamorphosis climax. T4 levels, as determined by radioimmunoassay, clearly resembled the Tg expression profile with a peak at the metamorphosis climax. To investigate if such expression profiles were regulated by TH, premetamorphic larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae were not able to complete metamorphosis. However, the addition of exogenous T4 enabled to revert this effect. Expression analysis showed higher mRNA levels of both TSH beta and Tg in TU-treated larvae in comparison to control larvae. Moreover, the TU+T4 treated larvae exhibited similar or lower mRNA levels than in the control. Present results demonstrate that TH mediate metamorphosis and down-regulate TSH beta and Tg at transcriptional level in Senegalese sole.

Molecular characterization, gene expression and dependence on thyroid hormones of two type I keratin genes (sseKer1 and sseKer2) in the flatfish Senegalese sole (Solea senegalensis Kaup).

BACKGROUND: Keratins make up the largest subgroup of intermediate filaments, and, in chordates, represent the most abundant proteins in epithelial cells. They have been associated with a wide range of functions in the cell, but little information is still available about their expression profile and regulation during flatfish metamorphosis. Senegalese sole (Solea senegalensis) is a commercially important flatfish in which no keratin gene has been described yet. RESULTS: The development of large-scale genomics of Senegalese sole has facilitated the identification of two different type I keratin genes referred to as sseKer1 and sseKer2. Main characteristics and sequence identities with other fish and mammal keratins are described. Phylogenetic analyses grouped sseKer1 and sseKer2 in a significant clade with other teleost epidermal type I keratins, and have allowed for the identification of sseKer2 as a novel keratin. The expression profile of both genes was studied during larval development and in tissues using a real-time approach. sseKer1 and sseKer2 mRNA levels were significantly higher in skin than in other tissues examined. During metamorphosis, sseKer1 transcripts increased significantly at first stages, and reduced thereafter. In contrast, sseKer2 mRNA levels did not change during early metamorphosis although a significant drop at metamorphosis climax and late metamorphosis was also detected. To study the possible regulation of sseKer gene expressions by thyroid hormones (THs), larvae were exposed to the goitrogen thiourea (TU). TU-treated larvae exhibited higher sseKer1 and sseKer2 mRNA levels than untreated control at both 11 and 15 days after treatment. Moreover, addition of exogenous T4 hormone to TU-treated larvae restored or even reduced the steady-state levels with respect to the untreated control, demonstrating that expression of both genes is negatively regulated by THs. CONCLUSION: We have identified two keratin genes, referred to as sseKer1 and sseKer2, in Senegalese sole. Phylogenetic analyses revealed sseKer2 as a novel keratin. Although they exhibit different expression patterns during larval development, both of them are negatively regulated by THs. The co-regulation by THs could explain the reduction of both keratin transcripts after the metamorphosis climax, suggesting their role in the tissue remodelling processes that occur during metamorphosis.

Comparative sequence analysis of the complete set of 40S ribosomal proteins in the Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.) (Teleostei: Pleuronectiformes): phylogeny and tissue- and development-specific expression.

BACKGROUND: Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. Their levels can vary as a function of organism growth and development; however, some RPs have been associated with other cellular processes or extraribosomal functions. Their high representation in cDNA libraries has resulted in the increase of RP sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis. RESULTS: The development of large-scale genomics of Senegalese sole (Solea senegalensis) and Atlantic halibut (Hippoglossus hippoglossus), two commercially important flatfish species, has made possible the identification and systematic analysis of the complete set of RP sequences for the small (40S) ribosome subunit. Amino acid sequence comparisons showed a high similarity both between these two flatfish species and with respect to other fish and human. EST analysis revealed the existence of two and four RPS27 genes in Senegalese sole and Atlantic halibut, respectively. Phylogenetic analysis clustered RPS27 in two separate clades with their fish and mammalian counterparts. Steady-state transcript levels for eight RPs (RPS2, RPS3a, RPS15, RPS27-1, RPS27-2, RPS27a, RPS28, and RPS29) in sole were quantitated during larval development and in tissues, using a real-time PCR approach. All eight RPs exhibited different expression patterns in tissues with the lowest levels in brain. On the contrary, RP transcripts increased co-ordinately after first larval feeding reducing progressively during the metamorphic process. CONCLUSION: The genomic resources and knowledge developed in this survey will provide new insights into the evolution of Pleuronectiformes. Expression data will contribute to a better understanding of RP functions in fish, especially the mechanisms that govern growth and development in larvae, with implications in aquaculture.

Differential gene expression and dependence on thyroid hormones of two glyceraldehyde-3-phosphate dehydrogenases in the flatfish Senegalese sole (Solea senegalensis Kaup).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme of the glycolytic pathway. The application of large-scale genomics to Senegalese sole (Solea senegalensis) has facilitated the identification of two different genes referred to as GAPDH-1 and GAPDH-2. Main characteristics and sequence similarities with other fish and mammals are described. Phylogenetic analyses grouped both genes into two separate clusters with their mammalian counterparts as a sister clade. The expression profiles of both genes were studied during larval development and in juvenile tissues using a real-time PCR approach. In juvenile fish, GAPDH-1 was expressed mainly in muscle and GAPDH-2 in brain. During metamorphosis, GAPDH-2 mRNA levels did not change. In contrast, GAPDH-1 transcripts increased significantly between S1 and S2 metamorphic stages and reduced their levels thereafter. A treatment with the goitrogen thiourea indicated that GAPDH-1 expression was negatively regulated by thyroid hormones. The possible role of GAPDHs in metamorphosis is discussed.

Cytogenetic characterization of the sole Solea senegalensis (Teleostei: Pleuronectiformes: Soleidae): Ag-NOR, (GATA)n, (TTAGGG)n and ribosomal genes by one-color and two-color FISH.

A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for the nucleolus organizer region (Ag-NOR) identification, one-color FISH for chromosomal mapping of 45S and 5S ribosomal DNAs (rDNAs), (GATA)n, and (TTAGGG)n, and two-color FISH for co-localization of both rDNAs. The Ag-NORs and the 45S rDNA were mapped to a medium-sized submetacentric chromosomal pair. Hybridization with the 5S rDNA showed a major signal on the short arm of a medium-sized submetacentric chromosome pair and a minor signal on a centromeric site of a small acrocentric chromosome pair. Differences in the Ag-NOR and 45S and 5S rDNAs FISH signal sizes were observed between homologous chromosomes and among individuals. A two-color FISH co-localized 45S and 5S rDNAs to a medium-sized submetacentric chromosomal pair. The hybridization with the telomeric (TTAGGG)n repeat displayed small signals at all chromosomal telomeres. Finally, the (GATA)n probe produced dispersed and small hybridization signals on all chromosome spreads, showing its ubiquitous existence in the genome. These results were compared with those from other Pleuronectiformes and discussed in terms of karyotype evolution.