Temperature-Biased miRNA Expression Patterns during European Sea Bass (Dicentrarchus labrax) Development.

Environmental effects and, particularly, temperature changes have been demonstrated to influence the activity, function, and well-being of teleosts. Temperature may change seasonally in the wild, and in captivity under aquaculture operations. Moreover, climate change is expected to shift temperature profiles worldwide. MicroRNAs (miRNA) are important temperature-sensitive gene-expression regulators acting at the post-transcriptional level. They are known to be key regulators in development, reproduction, and immune responses. Therefore, early larval development of the European sea bass (Dicentrarchus labrax), one of the most extensively cultured species in Mediterranean aquaculture, was investigated at early rearing temperatures, i.e., 15, 17.5, and 20 °C, in regard to the impact of temperatures on miRNAs through sncRNA high-throughput sequencing but also at the phenotypic level in terms of growth, sex, vision, and skeletal deformities. Expression profiling revealed stage- and temperature-specific miRNA expression targeting genes with roles in reproduction and immune response mainly at the flexion and all-fins stages. Similar stage- and temperature-specific results were also observed concerning the number of rod cells and lower jaw elongation. The present work presents for the first time highly promising results on the influence of early rearing temperature at the post-transcriptional level during European sea bass development, with a putative impact on reproduction and immune response, as well as regarding teleost vision and larval development.

Sex-biased dynamics of three-spined stickleback (Gasterosteus aculeatus) gene expression patterns.

The study of the differences between sexes presents an excellent model to unravel how phenotypic variation is achieved from a similar genetic background. Sticklebacks are of particular interest since evidence of a heteromorphic chromosome pair has not always been detected. The present study investigated sex-biased mRNA and small non-coding RNA (sncRNA) expression patterns in the brain, adipose tissues, and gonads of the three-spined stickleback. The sncRNA analysis indicated that regulatory functions occurred mainly in the gonads. Alleged miRNA-mRNA interactions were established and a mapping bias of differential expressed transcripts towards chromosome 19 was observed. Key players previously shown to control sex determination and differentiation in other fish species but also genes like gapdh were among the transcripts identified. This is the first report in the three-spined stickleback demonstrating tissue-specific expression comprising both mRNA and sncRNA between sexes, emphasizing the importance of mRNA-miRNA interactions as well as new presumed genes not yet identified to have gender-specific roles.

Non-coding RNA Expression Patterns of Two Different Teleost Gonad Maturation Stages.

Non-coding RNAs (ncRNAs) are involved in several different regulatory pathways including reproduction. In teleost fish, efficacious reproduction is heavily dependent on the completion of the reproductive cycle. The presence of ncRNA, however, and their expression dynamics and putative regulatory role in mature and immature gonads have not yet been extensively explored. Therefore, the abundance of ncRNAs in mature and immature female sharpsnout seabream (Diplodus puntazzo) was investigated. The sharpsnout seabream is a rudimentary hermaphrodite which, in captivity, displays dysfunctions in the gonad maturation process. Our analyses revealed a gonad specific read length distribution with two main peaks representing miRNAs (21-26 nt) and PIWI RNA (27-34 nt). Besides, distinct expression patterns for several ncRNA biotypes including microRNAs (miRNAs), PIWI RNAs (piRNAs), and ribosomal RNAs (rRNAs) were detected. Identified miRNA accounted to 938, corresponding to ~ 13% of obtained transcripts. Among the differential expressed ncRNAs, 10 (~ 7%) were annotated as miRNA, out of which 2 were found in higher abundance in immature gonads (miR-125c and miR-24) and 8 (miR-451, miR-7a, miR-122-1, miR190a, miR129, ENSGACT00000029608, ENSGACT00000029489, and ENSGACT00000029667) were found to be higher expressed in mature gonads. Putative miRNA targets, including long non-coding RNAs (lncRNAs) and genes, are proposed. Target genes are involved in several processes of fish oocyte development, such as steroidogenesis, proteolysis, and apoptosis, and may explain hormone regulation. This study demonstrates a gonad maturation biased ncRNA profile which in turn may support the role of ncRNAs in ovarian physiology and reproductive performance of fish, stressing the specific function of each RNA biotype in oocyte development.

Unravelling paralogous gene expression dynamics during three-spined stickleback embryogenesis.

Development requires the implementation of a plethora of molecular mechanisms, involving a large set of genes to ensure proper cell differentiation, morphogenesis of tissues and organs as well as the growth of the organism. Genome duplication and resulting paralogs are considered to provide the raw genetic materials important for new adaptation opportunities and boosting evolutionary innovation. The present study investigated paralogous genes, involved in three-spined stickleback (Gasterosteus aculeatus) development. Therefore, the transcriptomes of five early stages comprising developmental leaps were explored. Obtained expression profiles reflected the embryo's needs at different stages. Early stages, such as the morula stage comprised transcripts mainly involved in energy requirements while later stages were mostly associated with GO terms relevant to organ development and morphogenesis. The generated transcriptome profiles were further explored for differential expression of known and new paralogous genes. Special attention was given to hox genes, with hoxa13a being of particular interest and to pigmentation genes where itgb1, involved in the melanophore development, displayed a complementary expression pattern throughout studied stages. Knowledge obtained by untangling specific paralogous gene functions during development might not only significantly contribute to the understanding of teleost ontogenesis but might also shed light on paralogous gene evolution.

Full genome survey and dynamics of gene expression in the greater amberjack Seriola dumerili.

Background: Teleosts of the genus Seriola, commonly known as amberjacks, are of high commercial value in international markets due to their flesh quality and worldwide distribution. The Seriola species of interest to Mediterranean aquaculture is the greater amberjack (Seriola dumerili). This species holds great potential for the aquaculture industry, but in captivity, reproduction has proved to be challenging, and observed growth dysfunction hinders their domestication. Insights into molecular mechanisms may contribute to a better understanding of traits like growth and sex, but investigations to unravel the molecular background of amberjacks have begun only recently. Findings: Illumina HiSeq sequencing generated a high-coverage greater amberjack genome sequence comprising 45 909 scaffolds. Comparative mapping to the Japanese yellowtail (Seriola quinqueriadiata) and to the model species medaka (Oryzias latipes) allowed the generation of in silico groups. Additional gonad transcriptome sequencing identified sex-biased transcripts, including known sex-determining and differentiation genes. Investigation of the muscle transcriptome of slow-growing individuals showed that transcripts involved in oxygen and gas transport were differentially expressed compared with fast/normal-growing individuals. On the other hand, transcripts involved in muscle functions were found to be enriched in fast/normal-growing individuals. Conclusion: The present study provides the first insights into the molecular background of male and female amberjacks and of fast- and slow-growing fish. Therefore, valuable molecular resources have been generated in the form of a first draft genome and a reference transcriptome. Sex-biased genes, which may also have roles in sex determination or differentiation, and genes that may be responsible for slow growth are suggested.

In Vivo Effects of Lipopolysaccharide on Peroxisome Proliferator-Activated Receptor Expression in Juvenile Gilthead Seabream (Sparus Aurata).

Fish are constantly exposed to microorganisms in the aquatic environment, many of which are bacterial pathogens. Bacterial pathogens activate the innate immune response in fish involving the production of pro-inflammatory molecules that, in addition to their immune-related role, can affect non-immune tissues. In the present study, we aimed at investigating how inflammatory responses can affect metabolic homeostasis in the gilthead seabream (Sparus aurata), a teleost of considerable economic importance in Southern European countries. Specifically, we mimicked a bacterial infection by in vivo administration of lipopolysaccharide (LPS, 6 mg/kg body weight) and measured metabolic parameters in the blood and, importantly, the mRNA expression levels of the three isotypes of peroxisome proliferator activated receptors (PPARα, ß, and γ) in metabolically-relevant tissues in seabream. PPARs are nuclear receptors that are important for lipid and carbohydrate metabolism in mammals and that act as biological sensors of altered lipid metabolism. We show here that LPS-induced inflammatory responses result in the modulation of triglyceride plasma levels that are accompanied most notably by a decrease in the hepatic mRNA expression levels of PPARα, ß, and γ and by the up-regulation of PPARγ expression only in adipose tissue and the anterior intestine. In addition, LPS-induced inflammation results in an increase in the hepatic mRNA expression and protein activity levels of members of the mitogen-activated protein kinase (MAPK) family, known in mammals to regulate the transcription and activity of PPARs. Our results provide evidence for the involvement of PPARs in the metabolic response to inflammatory stimuli in seabream and offer insights into the molecular mechanisms underlying the redirection of metabolic activities under inflammatory conditions in vertebrates.

Expression Patterns of Atlantic Sturgeon (Acipenser oxyrinchus) During Embryonic Development.

During teleost ontogeny the larval and embryonic stages are key stages, since failure during this period of tissue differentiation may cause malformations, developmental delays, poor growth, and massive mortalities. Despite the rapid advances in sequencing technologies, the molecular backgrounds of the development of economically important but endangered fish species like the Atlantic sturgeon (Acipenser oxyrinchus) have not yet been thoroughly investigated. The current study examines the differential expression of transcripts involved in embryonic development of the Atlantic sturgeon. Addressing this goal, a reference transcriptome comprising eight stages was generated using an Illumina HiSequation 2500 platform. The constructed de novo assembly counted to 441,092 unfiltered and 179,564 filtered transcripts. Subsequently, the expression profile of four developmental stages ranging from early (gastrula) to late stages of prelarval development [2 d posthatching (dph)] were investigated applying an Illumina MiSeq platform. Differential expression analysis revealed distinct expression patterns among stages, especially between the two early and the two later stages. Transcripts upregulated at the two early stages were mainly enriched in transcripts linked to developmental processes, while transcripts expressed at the last two stages were mainly enriched in transcripts important to muscle contraction. Furthermore, important stage-specific expression has been detected for the hatching stage with transcripts enriched in molecule transport, and for the 2 dph stage with transcripts enriched in visual perception and lipid digestion. Our investigation represents a significant contribution to the understanding of Atlantic sturgeon embryonic development, and transcript characterization along with the differential expression results will significantly contribute to sturgeon research and aquaculture.

Identification, Phylogeny, and Function of fabp2 Paralogs in Two Non-Model Teleost Fish Species.

Intestinal fatty-acid-binding protein (IFABP or FABP2) is a cytosolic transporter of long-chain fatty acids, which is mainly expressed in cells of intestinal tissue. Fatty acids in teleosts are an important source of energy for growth, reproduction, and swimming and a main ingredient in the yolk sac of embryos and larvae. The fabp2 paralogs, fabp2a and fabp2b, were identified for 26 teleost fish species including the paralogs for the two non-model teleost fish species, namely the gilthead sea bream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). Despite the high similarity of fabp2 paralogs, as well as the identical organization in four exons, paralogs were mapped to different chromosomes/linkage groups supporting the hypothesis that the identified transcripts are true paralogs originating from a single ancestor gene after genome duplication. This was also confirmed by phylogenetic analysis using fabp2 sequences of 26 teleosts and by synteny analysis carried out with ten teleosts. Differential expression analysis of the gilthead sea bream and European sea bass fabp2 paralogs in the intestine after fasting and refeeding experiment further revealed their altered implication in metabolism. Additional expression studies in seven developmental stages of the two species detected fabp2 paralogs relatively early in the embryonic development as well as possible complementary or separated roles of the paralogs. The identification and characterization of the two fabp2 paralogs will contribute significantly to the understanding of the fabp2 evolution as well as of the divergences in fatty acid metabolism.

Transcriptomic response of skeletal muscle to lipopolysaccharide in the gilthead seabream (Sparus aurata).

The physiological consequences of the activation of the immune system in fish are not well understood. In particular, skeletal muscle, due to its essential role in locomotion and whole-animal energy homeostasis, is a potentially important target of inflammation. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the white and red skeletal muscle transcriptome of the gilthead seabream (Sparus aurata) by microarray analysis at 24 and 72 h after injection. In white muscle, the transcriptomic response was characterized by an up-regulation of genes involved in carbohydrate catabolism and protein synthesis at 24 h and a complete reversal of this pattern at 72 h. In red muscle, an up-regulation of genes involved in carbohydrate catabolism and protein synthesis was observed only at 72 h after LPS administration. Interestingly, both white and red muscles showed a similar consistent down-regulation of immune genes at 72 h post-injection. However, genes involved in muscle contraction showed a general up-regulation in response to LPS in both types of muscle. In summary, LPS administration causes muscle type-specific responses regarding the expression of genes involved in carbohydrate and protein metabolism and a common decreased expression of immune genes in skeletal muscle, concomitant with increased expression of genes for contractile elements. Our results evidence a robust and tissue-specific transcriptomic response of the skeletal muscle to an acute inflammatory challenge.