Characterization of the European Sea Bass (Dicentrarchus labrax) Gonadal Transcriptome During Sexual Development.

The European sea bass is one of the most important cultured fish in Europe and has a marked sexual growth dimorphism in favor of females. It is a gonochoristic species with polygenic sex determination, where a combination between still undifferentiated genetic factors and environmental temperature determines sex ratios. The molecular mechanisms responsible for gonadal sex differentiation are still unknown. Here, we sampled fish during the gonadal developmental period (110 to 350 days post fertilization, dpf), and performed a comprehensive transcriptomic study by using a species-specific microarray. This analysis uncovered sex-specific gonadal transcriptomic profiles at each stage of development, identifying larger number of differentially expressed genes in ovaries when compared to testis. The expression patterns of 54 reproduction-related genes were analyzed. We found that hsd17ß10 is a reliable marker of early ovarian differentiation. Further, three genes, pdgfb, snx1, and nfy, not previously related to fish sex differentiation, were tightly associated with testis development in the sea bass. Regarding signaling pathways, lysine degradation, bladder cancer, and NOD-like receptor signaling were enriched for ovarian development while eight pathways including basal transcription factors and steroid biosynthesis were enriched for testis development. Analysis of the transcription factor abundance showed an earlier increase in females than in males. Our results show that, although many players in the sex differentiation pathways are conserved among species, there are peculiarities in gene expression worth exploring. The genes identified in this study illustrate the diversity of players involved in fish sex differentiation and can become potential biomarkers for the management of sex ratios in the European sea bass and perhaps other cultured species.

Uncovering iron regulation with species-specific transcriptome patterns in Atlantic and coho salmon during a Caligus rogercresseyi infestation.

Salmon species cultured in Chile evidence different levels of susceptibility to the sea louse Caligus rogercresseyi. These differences have mainly been associated with specific immune responses. Moreover, iron regulation seems to be an important mechanism to confer immunity during the host infestation. This response called nutritional immunity has been described in bacterial infections, despite that no comprehensive studies involving in marine ectoparasites infestation have been reported. With this aim, we analysed the transcriptome profiles of Atlantic and coho salmon infected with C. rogercresseyi to evidence modulation of the iron metabolism as a proxy of nutritional immune responses. Whole transcriptome sequencing was performed in samples of skin and head kidney from Atlantic and coho salmon infected with sea lice. RNA-seq analyses revealed significant upregulation of transcripts in both salmon species at 7 and 14 dpi in skin and head kidney, respectively. However, iron regulation transcripts were differentially modulated, evidencing species-specific expression profiles. Genes related to heme degradation and iron transport such as hepcidin, transferrin and haptoglobin were primary upregulated in Atlantic salmon; meanwhile, in coho salmon, genes associated with heme biosynthesis were strongly transcribed. In summary, Atlantic salmon, which are more susceptible to infestation, presented molecular mechanisms to deplete cellular iron availability, suggesting putative mechanisms of nutritional immunity. In contrast, resistant coho salmon were less affected by sea lice, mainly activating pro-inflammatory mechanisms to cope with infestation.

Investigating the underlying mechanisms of temperature-related skin diseases in Atlantic salmon, Salmo salar L., as measured by quantitative histology, skin transcriptomics and composition.

Skin integrity is recognized as of vital consideration for both animal welfare and final product quality of farmed fish. This study examines the effects of three different rearing temperatures (4, 10 and 16 °C) on the skin of healthy Atlantic salmon post-smolts. Changes in skin condition were assessed by the means of skin composition analyses, quantitative histology assessments and transcriptome analysis. Level of protein, vitamin C and vitamin E was significantly higher at 16 °C compared with 4 °C. Quantitative histology measurements showed that the epidermal thickness decreased from low to high temperature, whereas the epidermal area comprising mucous cells increased. The difference was only significant between 4 and 16 °C. Both high and low temperature exhibited significant changes in the skin transcriptome. A number of immune-related transcripts responded at both temperatures. Contrary to well-described immunosuppressive effects of low water temperature on systemic immunity, a subtle increase in skin-mediated immunity was observed, suggesting a pre-activation of the mucosal system at 4 °C. Upregulation of a number of heat-shock proteins correlating with a decrease in epidermal thickness suggested a stress response in the skin at high temperature. The results demonstrate distinctive temperature-related effects on the skin of Atlantic salmon.

Lipopolysaccharides isolated from Aeromonas salmonicida and Vibrio anguillarum show quantitative but not qualitative differences in inflammatory outcome in Sparus aurata (Gilthead seabream).

In fish, the defence system recognises pathogenic microorganisms via pathogen recognition receptors (PRRs) that sense particular structures of the pathogens; the so-called pathogen associated molecular patterns (PAMPs) such as bacterial lipopolysaccharides (LPSs). The result of the PAMP-PRR interactions leads to complex and orchestrated immune responses. In this study, Sparus aurata (Gilthead seabream) were intraperitoneally injected with purified lipopolysaccharide (LPS) from Aeromonas salmonicida (As)- and Vibrio anguillarum (Va) (1 mg*Kgfish(-1)), both Gram negative bacteria responsible for vibriosis and furunculosis respectively, therefore causing an impact upon marine fish cultures. Head-kidney, intestine, spleen, liver and blood samples were collected at 3, 6, 12 and 24 h post-injection. Plasma levels of cortisol, prostaglandins and lactate were measured and were significantly increased after As-LPS and Va-LPS treatment. Furthermore, tissue-specific differences of the gene regulatory patterns were evident for each LPS. When monocyte/macrophage cell cultures were challenged with As-LPS and Va-LPS, the pro-inflammatory cytokine mRNA abundances present a similar pattern of response. However, As-LPS always triggered a stronger response concerning TNFα, IL1ß and cyclooxygenase-2 (COX2) mRNA abundance as well as PGE2 levels in the supernatant. Overall, the results indicate that specific LPSs do not activate different pro-inflammatory responses and that the observed gene expression pattern is tissue and concentration dependent.

Combining animal personalities with transcriptomics resolves individual variation within a wild-type zebrafish population and identifies underpinning molecular differences in brain function.

Resolving phenotype variation within a population in response to environmental perturbation is central to understanding biological adaptation. Relating meaningful adaptive changes at the level of the transcriptome requires the identification of processes that have a functional significance for the individual. This remains a major objective towards understanding the complex interactions between environmental demand and an individual's capacity to respond to such demands. The interpretation of such interactions and the significance of biological variation between individuals from the same or different populations remain a difficult and under-addressed question. Here, we provide evidence that variation in gene expression between individuals in a zebrafish population can be partially resolved by a priori screening for animal personality and accounts for >9% of observed variation in the brain transcriptome. Proactive and reactive individuals within a wild-type population exhibit consistent behavioural responses over time and context that relates to underlying differences in regulated gene networks and predicted protein-protein interactions. These differences can be mapped to distinct regions of the brain and provide a foundation towards understanding the coordination of underpinning adaptive molecular events within populations.

Gene expression and TNF-alpha secretion profile in rainbow trout macrophages following exposures to copper and bacterial lipopolysaccharide.

Fish macrophage function can be altered after exposure to pathogens as well as to xenobiotics. Considering that wild and farmed fish can be exposed in their habitats simultaneously to different types of stressors, including chemical contaminants (e.g. heavy metals) and pathogens (e.g. bacteria), it is fundamental to study their impact either isolated or in combination. Therefore, the present study aimed to evaluate the effects of copper and bacterial lipopolysaccharide (LPS), alone and in combination, on the transcription of target genes related with immune system, respiratory burst activity and cell death, using rainbow trout macrophages as in vitro model. A cell viability experiment was performed to determine the sub-lethal concentrations of copper for rainbow trout macrophages and the LC50-24 h was estimated at 60 µM. The expression of proinflammatory cytokines, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumour necrosis factor-α (TNFα) increased after copper and copper plus LPS exposure. Copper and LPS interact positively inducing an increase in cytokine expression, which may be indicative of an increased inflammatory response. However, the increase in TNFα mRNA expression induced by 50 µM copper was not accompanied by protein secretion indicating that mRNA abundance does not always reflect the level of protein and that the translation of the TNFα mRNA is somehow inhibited. Serum amyloid A (SAA) and trout C-polysaccharide binding protein (TCPBP) mRNA expression also increased after copper, LPS or LPS plus copper exposure, indicating a role of acute phase proteins in the local response to inflammation. NADPH oxidase and glutathione peroxidase gene expression increased in macrophages after 24 h exposure to copper, LPS or LPS plus copper. The results from the present study improve the understanding of mechanisms involved in copper toxicity, as well as the interaction with a simulated-inflammatory process.