Background: Keratins make up the largest subgroup of intermediate filaments, and, in chordates, r... more 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.
The entire sequence of the mitochondrial cytochrome b gene and 2 partial sequences of the ribosom... more The entire sequence of the mitochondrial cytochrome b gene and 2 partial sequences of the ribosomal RNA12S and 16S genes have been used to study the molecular phylogeny in 10 species of soles belonging to the genera Solea, Monochirus, Microchirus, Dicologlossa, and Synaptura from the Atlantic waters of the Gulf of Cádiz (Spain). The results obtained by means of different phylogenetic analyses (maximum likelihood, maximum parsimony, and neighbor-joining) were quite similar, supporting the monophyly of the Solea species. Nevertheless, they favor the differentiation of Dicologlossa cuneata and Dicologlossa hexophthalma in 2 distinct genera, since the most closely related species to the last one is Microchirus azevia. The fact that M. azevia is also more closely linked to Monochirus hispidus than to its congeneric Microchirus boscanion argues in favor of a taxonomic reorganization of these genera.
Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role as modulators of d... more Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role as modulators of development, growth, and reproduction. This study aimed to isolate the IGF-I and IGF-II cDNAs and determine their temporal expression pattern in diVerent organs and throughout larval development in Senegal sole. The rapid ampliWcation of cDNA ends (RACE) was used to obtain both full-length IGFs sequences. A high sequence similarity with other teleosts sequences was observed. Domains B and A revealed as the most evolutionary conserved. Steady-state copy numbers of IGF-I and IGF-II were also quantiWed in diVerent Senegal sole tissues by real-time PCR. IGF-I and IGF-II expressed ubiquitously with the highest mRNA levels in liver (88 £ 10 6 molecules/ g total RNA) and gills (14.0 £ 10 6 molecules/ g total RNA) respectively. IGF-II mRNA levels were higher than IGF-I in prehatching embryos and premetamorphic larvae with a signiWcant drop before the commencement of eye migration in metamorphosis. The abundance of IGF-II transcripts correlated positively with the growth rate during larval development. The putative role of IGF-II on metamorphosis and larval growth is discussed.
Background: Flatfish metamorphosis involves major physiological and morphological changes. Due to... more 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.
Lysozymes are key proteins of the innate immune system against bacterial infections. In this stud... more Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25-and 23-fold at 12 and 24 h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24 h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion. ª
Background: Keratins make up the largest subgroup of intermediate filaments, and, in chordates, r... more 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.
In the classical taxonomy, three Scomber species are distinguished: S. scombrus, S. australasicus... more In the classical taxonomy, three Scomber species are distinguished: S. scombrus, S. australasicus, and S. japonicus. Yet, some fish taxonomists have recently recognized Scomber colias, inhabiting the Atlantic Ocean, as a separate species from S. japonicus, distributed in the Pacific Ocean. Such proposal was based on significant mitochondrial DNA divergence as well as great phenotypic variation among individuals from these two ocean basins. However, in the absence of nuclear DNA data this issue remains still controversial. In this study, a phylogenetic analysis of nuclear 5S rDNA sequences was performed. A total of 30 individuals of S. colias collected in the Atlantic and 34␣specimens of S. japonicus from the Pacific were characterized. Moreover, nine individuals of Pacific S.␣australasicus and eight of Atlantic S. scombrus were included. Maximum likelihood, maximum parsimony, and neighbor-joining analyses revealed the presence of␣two well-supported distinct clades corresponding to S. colias and S. japonicus, respectively. Altogether, morphologic and genetic data are in agreement with the recognition of two different species, S. colias in the Atlantic, and S. japonicus in the Pacific.
Stocking density and ration size are two major factors influencing aquaculture production. To eva... more 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 Â 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 À2 ) 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 À2 ) 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 (insulinlike 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.
The complete nucleotide sequence of the mitochondrial genome for the Atlantic bluefin tuna Thunnu... more The complete nucleotide sequence of the mitochondrial genome for the Atlantic bluefin tuna Thunnus thynnus was determined. The entire genome was purified by gene amplification using the long polymerase chain reaction (PCR), and length was determined to be 16 526 base pairs (bp). The overall gene content, order and nucleotide composition correspond to those of other teleosts. Sequence features for protein coding genes, ribosomal RNAs and transfer RNAs are discussed. Origin of L-strand replication (O L ) forms a stable stem-loop structure. Moreover, typical conservative blocks in the non-coding region D-loop have been observed.
A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for t... more 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.
A multiplex-PCR assay for the authentication of the Atlantic mackerel Scomber scombrus in commerc... more A multiplex-PCR assay for the authentication of the Atlantic mackerel Scomber scombrus in commercial canned products has been developed. This novel method consists of a S. scombrus-specific fragment (123 bp) corresponding to the mitochondrial NADH dehydrogenase subunit 5, and a positive amplification control corresponding to the small rRNA 12S subunit (188 bp). The system was assayed using six different canned products labeled as S. scombrus. We observed a positive identification in all samples, revealing this methodology as a potential molecular tool for direct application in the authentication of S. scombrus canned products.
Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main P... more Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main PCR products (227, 441, and 2166 bp) were identified. The 227-and 441-bp fragments were characterized by highly divergent nontranscribed spacer sequences (referred to as NTS-I and NTS-II) that were 109 and 324 bp long, respectively, yet their coding sequences were nearly identical. The 2166-bp 5S rDNA unit was composed of two 5S rRNA genes separated by NTS-I and followed by a 1721-bp spacer containing the U2, U5, and U1 small nuclear RNA genes (snRNAs). They were inverted and arranged in the transcriptional direction opposite that of the 5S rRNA gene. This simultaneous linkage of 3 different snRNAs had never been observed before. The PCR products were used as probes in fluorescence in situ hybridization experiments to locate the corresponding loci on the chromosomes of S. senegalensis. A major 5S rDNA chromosomal site was located along most of the short arm of a submetacentric pair, while a minor site was detected near the centromeric region of an acrocentric pair.
Mackerels of the genus Scomber are commercially important species, but their taxonomic status is ... more Mackerels of the genus Scomber are commercially important species, but their taxonomic status is still controversial. Although previous phylogenetic data support the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as separate species, it is only based on the analysis of partial mitochondrial and nuclear DNA sequences. In an attempt to shed light on this relevant issue, we have determined the complete mitochondrial DNA sequence of S. colias, S. japonicus, and Scomber australasicus. The total length of the mitogenomes was 16,568 bp for S. colias and 16,570 bp for both S. japonicus and S. australasicus. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs, and 22 tRNAs) and organization similar to that observed in Scomber scombrus and most other vertebrates. The major noncoding region (control region) ranged between 865 and 866 bp in length and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Scomber species with regard to other scombrid fish. The major finding of this study is that S. colias and S. japonicus were significantly grouped in distinct lineages within Scomber cluster, which phylogenetically constitutes evidence that they may be considered as separate species. Additionally, molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.
The complete mitochondrial genome sequence of the blackspot seabream, Pagellus bogaraveo, was obt... more The complete mitochondrial genome sequence of the blackspot seabream, Pagellus bogaraveo, was obtained using the long PCR/cloning method. The total length of the mitogenome was 16,941 bp, and had a gene content (13 protein-coding, two ribosomal RNAs, and 22 transfer RNAs) and organization similar to those observed in most other vertebrates. Nevertheless, two main features in the WANCY region revealed as unique in P. bogaraveo mitogenome. First, the O L was disrupted by the insertion of a 66 bp long element that was determined to be a tRNA Cys pseudogene; second, the existence of high levels of length heteroplasmy, both intra-and inter-individuals, as a result of sequence duplications and deletions. The tandem duplication and random loss (TDRL) model as well as recombination are proposed to account for the length heteroplasmy and gene rearrangements in the P. bogaraveo WANCY region.
Thyroid hormones (TH) play a critical role in flatfish metamorphosis. Their levels are regulated ... more 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) b 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 b 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 b 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 b and Tg at transcriptional level in Senegalese sole.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme of the glycolytic pathway... more 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.
A novel procedure for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in com... more A novel procedure for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercially canned products has been developed. Three mitochondrial regions were simultaneously amplified by multiplex-Polymerase Chain Reaction, one corresponding to the small rRNA 12S subunit as a positive amplification control and two species-specific fragments corresponding to cytochrome b for A. rochei and ATPase 6 for A. thazard, respectively. Testing of two different detection systems revealed the fluorescence-based approach as the most sensitive. The results demonstrate that this rapid, low-cost methodology is a reliable molecular tool for direct application in the authentication of canned products.
Senegal sole ( Solea senegalensis Kaup) is a high-value marine flatfish exploited in both fisheri... more Senegal sole ( Solea senegalensis Kaup) is a high-value marine flatfish exploited in both fisheries and aquaculture. Here we describe the isolation of seven tetranucleotide and three dinucleotide polymorphic microsatellite loci. Characteristics of a captive broodstock and a wild population are described. Three loci displayed a significant departure from Hardy-Weinberg expectations in both populations, suggesting a substantial frequency of null alleles. The remaining seven loci were found to be in equilibrium in the wild population, whereas only four of them were in the cultured population. Cross-species amplification was successful for three loci in other five flatfishes.
Background: Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle resp... more 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.
Background: Keratins make up the largest subgroup of intermediate filaments, and, in chordates, r... more 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.
The entire sequence of the mitochondrial cytochrome b gene and 2 partial sequences of the ribosom... more The entire sequence of the mitochondrial cytochrome b gene and 2 partial sequences of the ribosomal RNA12S and 16S genes have been used to study the molecular phylogeny in 10 species of soles belonging to the genera Solea, Monochirus, Microchirus, Dicologlossa, and Synaptura from the Atlantic waters of the Gulf of Cádiz (Spain). The results obtained by means of different phylogenetic analyses (maximum likelihood, maximum parsimony, and neighbor-joining) were quite similar, supporting the monophyly of the Solea species. Nevertheless, they favor the differentiation of Dicologlossa cuneata and Dicologlossa hexophthalma in 2 distinct genera, since the most closely related species to the last one is Microchirus azevia. The fact that M. azevia is also more closely linked to Monochirus hispidus than to its congeneric Microchirus boscanion argues in favor of a taxonomic reorganization of these genera.
Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role as modulators of d... more Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role as modulators of development, growth, and reproduction. This study aimed to isolate the IGF-I and IGF-II cDNAs and determine their temporal expression pattern in diVerent organs and throughout larval development in Senegal sole. The rapid ampliWcation of cDNA ends (RACE) was used to obtain both full-length IGFs sequences. A high sequence similarity with other teleosts sequences was observed. Domains B and A revealed as the most evolutionary conserved. Steady-state copy numbers of IGF-I and IGF-II were also quantiWed in diVerent Senegal sole tissues by real-time PCR. IGF-I and IGF-II expressed ubiquitously with the highest mRNA levels in liver (88 £ 10 6 molecules/ g total RNA) and gills (14.0 £ 10 6 molecules/ g total RNA) respectively. IGF-II mRNA levels were higher than IGF-I in prehatching embryos and premetamorphic larvae with a signiWcant drop before the commencement of eye migration in metamorphosis. The abundance of IGF-II transcripts correlated positively with the growth rate during larval development. The putative role of IGF-II on metamorphosis and larval growth is discussed.
Background: Flatfish metamorphosis involves major physiological and morphological changes. Due to... more 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.
Lysozymes are key proteins of the innate immune system against bacterial infections. In this stud... more Lysozymes are key proteins of the innate immune system against bacterial infections. In this study we report the molecular cloning and characterization of the c-type and g-type lysozymes in brill (Scophthalmus rhombus). Catalytic and other conserved residues required for functionality were identified. Phylogenetic analysis revealed distinct evolutionary histories for each lysozyme type. Expression profiles of both lysozyme genes were studied in juvenile tissues using a real-time PCR approach. c-Type lysozyme was expressed mainly in stomach and liver, whereas the g-type was detected in all tissues with highest mRNA levels observed in the spleen. Induction experiments revealed that g-type transcripts increased significantly in head kidney after lipopolysaccharide (25-and 23-fold at 12 and 24 h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24 h) treatments. In contrast, no induction was observed for c-type lysozyme. All these data suggest that g-type lysozyme is involved in the response against bacterial infections, whereas c-type lysozyme may also play a role in digestion. ª
Background: Keratins make up the largest subgroup of intermediate filaments, and, in chordates, r... more 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.
In the classical taxonomy, three Scomber species are distinguished: S. scombrus, S. australasicus... more In the classical taxonomy, three Scomber species are distinguished: S. scombrus, S. australasicus, and S. japonicus. Yet, some fish taxonomists have recently recognized Scomber colias, inhabiting the Atlantic Ocean, as a separate species from S. japonicus, distributed in the Pacific Ocean. Such proposal was based on significant mitochondrial DNA divergence as well as great phenotypic variation among individuals from these two ocean basins. However, in the absence of nuclear DNA data this issue remains still controversial. In this study, a phylogenetic analysis of nuclear 5S rDNA sequences was performed. A total of 30 individuals of S. colias collected in the Atlantic and 34␣specimens of S. japonicus from the Pacific were characterized. Moreover, nine individuals of Pacific S.␣australasicus and eight of Atlantic S. scombrus were included. Maximum likelihood, maximum parsimony, and neighbor-joining analyses revealed the presence of␣two well-supported distinct clades corresponding to S. colias and S. japonicus, respectively. Altogether, morphologic and genetic data are in agreement with the recognition of two different species, S. colias in the Atlantic, and S. japonicus in the Pacific.
Stocking density and ration size are two major factors influencing aquaculture production. To eva... more 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 Â 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 À2 ) 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 À2 ) 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 (insulinlike 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.
The complete nucleotide sequence of the mitochondrial genome for the Atlantic bluefin tuna Thunnu... more The complete nucleotide sequence of the mitochondrial genome for the Atlantic bluefin tuna Thunnus thynnus was determined. The entire genome was purified by gene amplification using the long polymerase chain reaction (PCR), and length was determined to be 16 526 base pairs (bp). The overall gene content, order and nucleotide composition correspond to those of other teleosts. Sequence features for protein coding genes, ribosomal RNAs and transfer RNAs are discussed. Origin of L-strand replication (O L ) forms a stable stem-loop structure. Moreover, typical conservative blocks in the non-coding region D-loop have been observed.
A cytogenetic analysis of the sole Solea senegalensis was carried out using silver staining for t... more 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.
A multiplex-PCR assay for the authentication of the Atlantic mackerel Scomber scombrus in commerc... more A multiplex-PCR assay for the authentication of the Atlantic mackerel Scomber scombrus in commercial canned products has been developed. This novel method consists of a S. scombrus-specific fragment (123 bp) corresponding to the mitochondrial NADH dehydrogenase subunit 5, and a positive amplification control corresponding to the small rRNA 12S subunit (188 bp). The system was assayed using six different canned products labeled as S. scombrus. We observed a positive identification in all samples, revealing this methodology as a potential molecular tool for direct application in the authentication of S. scombrus canned products.
Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main P... more Some units of the 5S rDNA of Solea senegalensis were amplified by PCR and sequenced. Three main PCR products (227, 441, and 2166 bp) were identified. The 227-and 441-bp fragments were characterized by highly divergent nontranscribed spacer sequences (referred to as NTS-I and NTS-II) that were 109 and 324 bp long, respectively, yet their coding sequences were nearly identical. The 2166-bp 5S rDNA unit was composed of two 5S rRNA genes separated by NTS-I and followed by a 1721-bp spacer containing the U2, U5, and U1 small nuclear RNA genes (snRNAs). They were inverted and arranged in the transcriptional direction opposite that of the 5S rRNA gene. This simultaneous linkage of 3 different snRNAs had never been observed before. The PCR products were used as probes in fluorescence in situ hybridization experiments to locate the corresponding loci on the chromosomes of S. senegalensis. A major 5S rDNA chromosomal site was located along most of the short arm of a submetacentric pair, while a minor site was detected near the centromeric region of an acrocentric pair.
Mackerels of the genus Scomber are commercially important species, but their taxonomic status is ... more Mackerels of the genus Scomber are commercially important species, but their taxonomic status is still controversial. Although previous phylogenetic data support the recognition of Atlantic Scomber colias and Pacific Scomber japonicus as separate species, it is only based on the analysis of partial mitochondrial and nuclear DNA sequences. In an attempt to shed light on this relevant issue, we have determined the complete mitochondrial DNA sequence of S. colias, S. japonicus, and Scomber australasicus. The total length of the mitogenomes was 16,568 bp for S. colias and 16,570 bp for both S. japonicus and S. australasicus. All mitogenomes had a gene content (13 protein-coding, 2 rRNAs, and 22 tRNAs) and organization similar to that observed in Scomber scombrus and most other vertebrates. The major noncoding region (control region) ranged between 865 and 866 bp in length and showed the typical conserved blocks. Phylogenetic analyses revealed a monophyletic origin of Scomber species with regard to other scombrid fish. The major finding of this study is that S. colias and S. japonicus were significantly grouped in distinct lineages within Scomber cluster, which phylogenetically constitutes evidence that they may be considered as separate species. Additionally, molecular data here presented provide a useful tool for evolutionary as well as population genetic studies.
The complete mitochondrial genome sequence of the blackspot seabream, Pagellus bogaraveo, was obt... more The complete mitochondrial genome sequence of the blackspot seabream, Pagellus bogaraveo, was obtained using the long PCR/cloning method. The total length of the mitogenome was 16,941 bp, and had a gene content (13 protein-coding, two ribosomal RNAs, and 22 transfer RNAs) and organization similar to those observed in most other vertebrates. Nevertheless, two main features in the WANCY region revealed as unique in P. bogaraveo mitogenome. First, the O L was disrupted by the insertion of a 66 bp long element that was determined to be a tRNA Cys pseudogene; second, the existence of high levels of length heteroplasmy, both intra-and inter-individuals, as a result of sequence duplications and deletions. The tandem duplication and random loss (TDRL) model as well as recombination are proposed to account for the length heteroplasmy and gene rearrangements in the P. bogaraveo WANCY region.
Thyroid hormones (TH) play a critical role in flatfish metamorphosis. Their levels are regulated ... more 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) b 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 b 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 b 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 b and Tg at transcriptional level in Senegalese sole.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an essential enzyme of the glycolytic pathway... more 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.
A novel procedure for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in com... more A novel procedure for the authentication of frigate tunas (Auxis thazard and Auxis rochei) in commercially canned products has been developed. Three mitochondrial regions were simultaneously amplified by multiplex-Polymerase Chain Reaction, one corresponding to the small rRNA 12S subunit as a positive amplification control and two species-specific fragments corresponding to cytochrome b for A. rochei and ATPase 6 for A. thazard, respectively. Testing of two different detection systems revealed the fluorescence-based approach as the most sensitive. The results demonstrate that this rapid, low-cost methodology is a reliable molecular tool for direct application in the authentication of canned products.
Senegal sole ( Solea senegalensis Kaup) is a high-value marine flatfish exploited in both fisheri... more Senegal sole ( Solea senegalensis Kaup) is a high-value marine flatfish exploited in both fisheries and aquaculture. Here we describe the isolation of seven tetranucleotide and three dinucleotide polymorphic microsatellite loci. Characteristics of a captive broodstock and a wild population are described. Three loci displayed a significant departure from Hardy-Weinberg expectations in both populations, suggesting a substantial frequency of null alleles. The remaining seven loci were found to be in equilibrium in the wild population, whereas only four of them were in the cultured population. Cross-species amplification was successful for three loci in other five flatfishes.
Background: Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle resp... more 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.
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Papers by Carlos Infante