Carlos Infante

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

BackgroundFlatfish 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.ResultsThe 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 β-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.ConclusionThis 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.

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 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.

Molecular characterization, phylogeny, and expression of c-type and g-type lysozymes in brill (Scophthalmus rhombus).

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 24h, respectively) and Photobacterium damselae subsp. piscicida (17-fold at 24h) 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.

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.

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.

De novo assembly, characterization and functional annotation of Senegalese sole (Solea senegalensis) and common sole (Solea solea) transcriptomes: integration in a database and design of a microarray

BackgroundSenegalese sole (Solea senegalensis) and common sole (S. solea) are two economically and evolutionary important flatfish species both in fisheries and aquaculture. Although some genomic resources and tools were recently described in these species, further sequencing efforts are required to establish a complete transcriptome, and to identify new molecular markers. Moreover, the comparative analysis of transcriptomes will be useful to understand flatfish evolution.ResultsA comprehensive characterization of the transcriptome for each species was carried out using a large set of Illumina data (more than 1,800 millions reads for each sole species) and 454 reads (more than 5 millions reads only in S. senegalensis), providing coverages ranging from 1,384x to 2,543x. After a de novo assembly, 45,063 and 38,402 different transcripts were obtained, comprising 18,738 and 22,683 full-length cDNAs in S. senegalensis and S. solea, respectively. A reference transcriptome with the longest unique transcripts and putative non-redundant new transcripts was established for each species. A subset of 11,953 reference transcripts was qualified as highly reliable orthologs (>97% identity) between both species. A small subset of putative species-specific, lineage-specific and flatfish-specific transcripts were also identified. Furthermore, transcriptome data permitted the identification of single nucleotide polymorphisms and simple-sequence repeats confirmed by FISH to be used in further genetic and expression studies. Moreover, evidences on the retention of crystallins crybb1, crybb1-like and crybb3 in the two species of soles are also presented. Transcriptome information was applied to the design of a microarray tool in S. senegalensis that was successfully tested and validated by qPCR. Finally, transcriptomic data were hosted and structured at SoleaDB.ConclusionsTranscriptomes and molecular markers identified in this study represent a valuable source for future genomic studies in these economically important species. Orthology analysis provided new clues regarding sole genome evolution indicating a divergent evolution of crystallins in flatfish. The design of a microarray and establishment of a reference transcriptome will be useful for large-scale gene expression studies. Moreover, the integration of transcriptomic data in the SoleaDB will facilitate the management of genomic information in these important species.

Poly I:C induces Mx transcription and promotes an antiviral state against sole aquabirnavirus in the flatfish Senegalese sole (Solea senegalensis Kaup)

Mx is an interferon-induced protein that protects against viral infections. In this study the absolute number of Mx transcripts after poly I:C injection (a synthetic dsRNA) or sole aquabirnavirus (solevirus) inoculation in Senegalese sole (Solea senegalensis Kaup) has been quantified. Mx expression profiles differed clearly in both experimental conditions; the induction response was faster and more intense after poly I:C injection than after solevirus inoculation. Moreover, pre-injection of soles with poly I:C prior to solevirus infection eliminated the induction of Mx expression associated with this virus. To evaluate the possible interference of poly I:C treatments on solevirus replication, the mRNA levels of the virus capsid protein (VP2) were determined by RT-PCR. VP2 transcripts were hardly detected in poly I:C pre-injected animals from 12 to 72 h after solevirus inoculation. All these data suggest that poly I:C is able to induce an antiviral state that interferes with solevirus replication, and support the suitability of Mx expression analysis as a marker to study the defensive response against solevirus.

Complete mitochondrial genome of the blackspot seabream, Pagellus bogaraveo (Perciformes: Sparidae), with high levels of length heteroplasmy in the WANCY region.

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.

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

BackgroundEukaryotic 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.ResultsThe 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.ConclusionWe 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.