Gene expression profiling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology

Gene expression profiling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology. Physiol Genomics 23: 182–191, 2005. First published July 26, 2005; doi:10.1152/physiolgenomics.00139.2005.—Large-scale gene expression studies were performed for one of the main European aquaculture species, the gilthead sea bream Sparus auratus L. For this purpose, a cDNA microarray containing 10,176 clones from a cDNA library of mixed embryonic and larval stages was constructed. In addition to its importance for aquaculture, the taxonomic position and the relatively small genome size of sea bream makes it a prospective model for evolutionary biology and comparative genomics. However, so far, no large-scale analysis of gene expression exists for this species. In the present study, gene expression was analyzed in gilthead sea bream during early development, a significant period in the determination of quantitative traits and therefore of considerable interest for aquaculture. Synexpression groups expressed primarily early and late in development were determined and were composed of both known and novel genes. Furthermore, it was possible to identify stress response genes induced by cortisol injections using the cDNA microarray generated. The creation of gene expression profiles for sea bream by microarray hybridization will accelerate identification of candidate genes involved in multifactorial traits and certain regulatory pathways and will also contribute to a better understanding of the genetic background of fish physiology, which may help to improve aquaculture practices.We thank Dr. M. Pankratz and lab for providing the microarray spotting facilities and Dr. C. Seiler for support in generating pictures of the developmental stages of sea bream. Sequences reported in this article have been submitted to the National Center for Biotechnology Information (NCBI) EST database under Accession Nos. CB184056–CB184594 and CV133223–CV133736. Microarray expression data have been submitted to ARRAYExpress under Accession Nos. E-MEXP-181 (experiment) and A-MEXP-110 (array) as well as to the NCBI Omnibus under Accession Nos. GSE 2064 and GSE 1887

Substantial Downregulation of Myogenic Transcripts in Skeletal Muscle of Atlantic Cod during the Spawning Period

Gonadal maturation is an extremely energy consuming process for batch spawners and it is associated with a significant decrease in growth and seasonal deterioration in flesh quality. Our knowledge about the molecular mechanisms linking sexual maturation and muscle growth is still limited. In the present study, we performed RNA-Seq using 454 GS-FLX pyrosequencing in fast skeletal muscle sampled from two-year-old Atlantic cod (Gadus morhua) at representative time points throughout the reproductive cycle (August, March and May). In total, 126,937 good quality reads were obtained, with 546 nucleotide length and 52% GC content on average. RNA-Seq analysis using the CLC Genomics Workbench with the Atlantic cod reference UniGene cDNA data revealed 59,581 (46.9%) uniquely annotated reads. Pairwise comparison for expression levels identified 153 differentially expressed UniGenes between time points. Notably, we found a significant suppression of myh13 and myofibrillar gene isoforms in fast skeletal muscle during the spawning season. This study uncovered a large number of differentially expressed genes that may be influenced by gonadal maturation, thus representing a significant contribution to our limited understanding of the molecular mechanisms regulating muscle wasting and regeneration in batch spawners during their reproductive cycle

Heat shock protein (HSP) expression and mitogen-activated protein kinase (MAPK) phosphorylation during early embryonic developmental stages of the Gilthead sea bream (Sparus aurata)

Both heat shock proteins (HSPs), which have key roles in vital cell functions, as well as members of the mitogen-activated protein kinases (MAPKs), which adjust gene expression by transducing cellular signals to the nucleus, are necessary for normal embryonic development in vertebrates. Therefore, protein expression levels of HSP70 and HSP90 and the activation of members of the MAPK protein family, such as p38 MAPK, ERKs, and JNKs were studied in the early developmental stages of the Gilthead sea bream, Sparus aurata Linnaeus, 1758. The protein expression of HSP70 and the phosphorylation ratio of JNKs remained at equal levels at all examined developmental stages, while the other examined proteins exhibited a differential profile. HSP90 levels were mostly increased at the 16-cell stage and towards the morula stages, and the lowest values were observed at the two- to four-cell and one-half epiboly stages. While p38 MAPK phosphorylation ratio exhibited increased values mostly in the early developmental stages, the opposite was observed concerning ERK phosphorylation ratio, where increased values were observed in the later embryonic stages (high blastula to one-half epiboly stages). These differential profiles of the examined protein expression levels highlight the importance of these proteins during embryogenesis and pave the way for further research to unveil their distinct role in early development

The Atlantic Bonito ( Sarda sarda,Bloch 1793) Transcriptome and Detection of Differential Expression during Larvae Development

Versión del editor

Species-specific secondary metabolites from Primula veris subsp. veris obtained In Vitro adventitious root cultures: an alternative for sustainable production

Primula veris subsp. veris L. is a perennial herbaceous and medicinal plant species the roots and flowers of which are a source of valuable pharmaceutical raw materials. The plant tissues are used to produce expectorant and diuretic drugs due to their high content of triterpene saponins and phenolic glycosides. Underground roots of P. veris can be obtained only through a destructive process during the plant’s harvesting. In the present study, an in vitro adventitious root production protocol was developed as an alternative way of production, focused on four species-specific secondary metabolites. Root explants were cultured in Murashing & Skoog liquid medium supplemented with 5.4 µM α-naphthaleneacetic acid, 0.5 µM kinetin, L-proline 100 mg/L, and 30 g/L sucrose, in the dark and under agitation. The effect of temperature (10, 15 and 22 ◦C) on biomass production was investigated. The content of two flavonoid compounds (primeverin and primulaverin), and two main triterpene saponins (primulic acid I and II) were determined after 60 days of culture and compared with 1.5-year-old soil-grown plants. The accumulated content (mg/g DW) of bioactive compounds of in vitro adventitious roots cultured under 22 ◦C was significantly higher than the other two temperatures of the study, being 9.71 mg/g DW in primulaverin, 0.09 mg/g DW in primeverin, 6.09 mg/g DW in primulic acid I, and 0.51 mg/g DW in primulic acid II. Compared to the soil-grown roots (10.23 mg/g DW primulaverin, 0.28 mg/g DW primeverin, 17.01 mg/g DW primulic acid I, 0.09 mg/g DW primulic acid II), the in vitro grown roots at 22 ◦C exhibited a 5.67-fold higher content in primulic acid II. However, primulic acid I and primeverin content were approximately three-fold higher in soil-grown roots, while primulaverin content were at similar levels for both in vitro at 22 ◦C and soil-grown roots. From our results, tissue culture of P. veris subsp. veris could serve not only for propagation but also for production of species-specific secondary metabolites such as primulic acid II through adventitious root cultures. This would therefore limit the uncontrolled collection of this plant from its natural environment and provide natural products free from pesticides in a sustainable wa

Genetic vs community diversity patterns of macrobenthic species: preliminary results from the lagoonal ecosystem

1 - The use of molecular data derived from multispecies assemblages in order to test ecological theory has only recently been introduced in the scientific literature.2 - As a first step, we compared patterns of abiotic environment, polychaeta distribution and their genetic diversity in five lagoon ecosystems in Greece. Our results confirm the hypothesis that higher genetic diversity is expected in the populations of the species occurring in the transitional waters rather than of those occurring in the marine environment.3 - Patterns derived from the polychaete community level and from the mitochondrial DNA (16S rRNA) obtained from Nephtys hombergii and Hediste diversicolor showed convergence, indicating the potential use of molecular matrices as surrogates in community analysis.4 - Finally, the high correlation between the genetic diversity pattern of H. diversicolor and the phosphorus concentration in the sediments may imply the broadening of the hierarchic-response-tostress hypothesis towards lower than species level

The sex-specific transcriptome of the hermaphrodite sparid sharpsnout seabream (Diplodus puntazzo)

Background: Teleosts are characterized by a remarkable breadth of sexual mechanisms including various forms of hermaphroditism. Sparidae is a fish family exhibiting gonochorism or hermaphroditism even in closely related species. The sparid Diplodus puntazzo (sharpsnout seabream), exhibits rudimentary hermaphroditism characterized by intersexual immature gonads but single-sex mature ones. Apart from the intriguing reproductive biology, it is economically important with a continuously growing aquaculture in the Mediterranean Sea, but limited available genetic resources. Our aim was to characterize the expressed transcriptome of gonads and brains through RNA-Sequencing and explore the properties of genes that exhibit sex-biased expression profiles. Results: Through RNA-Sequencing we obtained an assembled transcriptome of 82,331 loci. The expression analysis uncovered remarkable differences between male and female gonads, while male and female brains were almost identical. Focused search for known targets of sex determination and differentiation in vertebrates built the sex-specific expression profile of sharpsnout seabream. Finally, a thorough genetic marker discovery pipeline led to the retrieval of 85,189 SNPs and 29,076 microsatellites enriching the available genetic markers for this species. Conclusions: We obtained a nearly complete source of transcriptomic sequence as well as marker information for sharpsnout seabream, laying the ground for understanding the complex process of sex differentiation of this economically valuable species. The genes involved include known candidates from other vertebrate species, suggesting a conservation of the toolkit between gonochorists and hermaphrodites

A genetic linkage map of the hermaphrodite teleost fish Sparus aurata L.

The gilthead sea bream (Sparus aurata L.) is a marine fish of great importance for fisheries and aquaculture. It has also a peculiar sex-determination system, being a protandrous hermaphrodite. Here we report the construction of a first-generation genetic linkage map for S. aurata, based on 204 microsatellite markers. Twenty-six linkage groups (LG) were found. The total map length was 1241.9 cM. The ratio between sex-specific map lengths was 1:1.2 (male:female). Comparison with a preliminary radiation hybrid (RH) map reveals a good concordance, as all markers located in a single LG are located in a single RH group, except for Ad-25 and CId-31. Comparison with the Tetraodon nigroviridis genome revealed a considerable number of evolutionary conserved regions (ECRs) between the two species. The mean size of ECRs was 182 bp (sequence identity 60–90%). Forty-one ECRs have a known chromosomal location in the pufferfish genome. Despite the limited number of anchoring points, significant syntenic relationships were found. The linkage map presented here provides a robust comparative framework for QTL analysis in S. aurata and is a step toward the identification of genetic loci involved both in the determination of economically important traits and in the individual timing of sex reversal

Quantitative Trait Loci Involved in Sex Determination and Body Growth in the Gilthead Sea Bream (Sparus aurata L.) through Targeted Genome Scan

Among vertebrates, teleost fish exhibit a considerably wide range of sex determination patterns that may be influenced by extrinsic parameters. However even for model fish species like the zebrafish Danio rerio the precise mechanisms involved in primary sex determination have not been studied extensively. The zebrafish, a gonochoristic species, is lacking discernible sex chromosomes and the sex of juvenile fish is difficult to determine. Sequential protandrous hermaphrodite species provide distinct determination of the gender and allow studying the sex determination process by looking at the mechanism of sex reversal. This is the first attempt to understand the genetic basis of phenotypic variation for sex determination and body weight in a sequential protandrous hermaphrodite species, the gilthead sea bream (Sparus aurata). This work demonstrates a fast and efficient strategy for Quantitative Trait Loci (QTL) detection in the gilthead sea bream, a non-model but target hermaphrodite fish species. Therefore a comparative mapping approach was performed to query syntenies against two other Perciformes, the European sea bass (Dicentrarchus labrax), a gonochoristic species and the Asian sea bass (Lates calcarifer) a protandrous hermaphrodite. In this manner two significant QTLs, one QTL affecting both body weight and sex and one QTL affecting sex, were detected on the same linkage group. The co-segregation of the two QTLs provides a genomic base to the observed genetic correlation between these two traits in sea bream as well as in other teleosts. The identification of QTLs linked to sex reversal and growth, will contribute significantly to a better understanding of the complex nature of sex determination in S. aurata where most individuals reverse to the female sex at the age of two years through development and maturation of the ovarian portion of the gonad and regression of the testicular area. [Genomic sequences reported in this manuscript have been submitted to GenBank under accession numbers HQ021443–HQ021749.

Profiling of infection specific mRNA transcripts of the European seabass Dicentrarchus labrax

<p>Abstract</p> <p>Background</p> <p>The European seabass (<it>Dicentrarchus labrax</it>), one of the most extensively cultured species in European aquaculture productions, is, along with the gilthead sea bream (<it>Sparus aurata</it>), a prospective model species for the Perciformes which includes several other commercially important species. Massive mortalities may be caused by bacterial or viral infections in intensive aquaculture production. Revealing transcripts involved in immune response and studying their relative expression enhances the understanding of the immune response mechanism and consequently also the creation of vaccines. The analysis of expressed sequence tags (EST) is an efficient and easy approach for gene discovery, comparative genomics and for examining gene expression in specific tissues in a qualitative and quantitative way.</p> <p>Results</p> <p>Here we describe the construction, analysis and comparison of a total of ten cDNA libraries, six from different tissues infected with <it>V. anguillarum </it>(liver, spleen, head kidney, gill, peritoneal exudates and intestine) and four cDNA libraries from different tissues infected with Nodavirus (liver, spleen, head kidney and brain). In total 9605 sequences representing 3075 (32%) unique sequences (set of sequences obtained after clustering) were obtained and analysed. Among the sequences several immune-related proteins were identified for the first time in the order of Perciformes as well as in Teleostei.</p> <p>Conclusion</p> <p>The present study provides new information to the Gene Index of seabass. It gives a unigene set that will make a significant contribution to functional genomic studies and to studies of differential gene expression in relation to the immune system. In addition some of the potentially interesting genes identified by <it>in silico </it>analysis and confirmed by real-time PCR are putative biomarkers for bacterial and viral infections in fish.</p