Circulating MicroRNAs Indicative of Sex and Stress in the European Seabass (Dicentrarchus labrax): Toward the Identification of New Biomarkers.

MicroRNAs (miRNAs) constitute a new category of biomarkers. Studies on miRNAs in non-mammalian species have drastically increased in the last few years. Here, we explored the use of miRNAs as potential, poorly invasive markers, to identify sex and characterize acute stress in fish. The European seabass (Dicentrarchus labrax) was chosen as a model because of its rapid response to stress and its specific sex determination system, devoid of sexual chromosomes. We performed a small RNA-sequencing analysis in the blood plasma of male and female European seabass (mature and immature) as well as in the blood plasma of juveniles submitted to an acute stress and sampled throughout the recovery period (at 0 h, 0.5 h, 1.5 h and 6 h). In immature individuals, both miR-1388-3p and miR-7132a-5p were up-regulated in females, while miR-499a-5p was more abundant in males. However, no miRNAs were found to be differentially expressed between sexes in the blood plasma of mature individuals. For the acute stress analysis, five miRNAs (miR-155-5p, miR-200a-3p, miR-205-1-5p, miR-143-3p, and miR-223-3p) followed cortisol production over time. All miRNAs identified were tested and validated by RT-qPCR on sequenced samples. A complementary analysis on the 3'UTR sequences of the European seabass allowed to predict potential mRNA targets, some of them being particularly relevant regarding stress regulation, e.g., the glucocorticoid receptor 1 and the mineralocorticoid receptor. The present study provides new avenues and recommendations on the use of miRNAs as biomarkers of sex or stress of the European seabass, with potential application on other fish species.

Natural cortisol production is not linked to the sexual fate of European sea bass.

In this study, we aimed to investigate the relationship between cortisol and the determination of sexual fate in the commercially important European sea bass (Dicentrarchus labrax). To test our hypothesis, we designed two temperature-based experiments (19 ℃, 21 ℃ and 23 ℃, experiment 1; 16 ℃ and 21 ℃, experiment 2) to assess the effects of these thermal treatments on European sea bass sex determination and differentiation. In the fish from the first experiment, we evaluated whether blood cortisol levels and expression of stress key regulatory genes were different between differentiating (149 to 183 dph) males and females. In the second experiment, we assessed whether cortisol accumulated in scales over time during the labile period for sex determination as well as the neuroanatomical localisation of brain cells expressing brain aromatase (cyp19a1b) and corticotropin-releasing factor (crf) differed between males and females undergoing molecular sex differentiation (117 to 124 dph). None of the gathered results allowed to detect differences between males and females regarding cortisol production and regulatory mechanisms. Altogether, our data provide strong physiological, molecular and histochemical evidence, indicating that in vivo cortisol regulation has no major effects on the sex of European sea bass.

Unraveling the genotype by environment interaction in a thermosensitive fish with a polygenic sex determination system.

In most animals, sex determination occurs at conception, when sex chromosomes are segregated following Mendelian laws. However, in multiple reptiles and fishes, this genetic sex can be overridden by external factors after fertilization or birth. In some species, the genetic sex may also be governed by multiple genes, further limiting our understanding of sex determination in such species. We used the European sea bass (Dicentrarchus labrax) as a model and combined genomic (using a single nucleotide polymorphism chip) and transcriptomic (RNA-Sequencing) approaches to thoroughly depict this polygenic sex determination system and its interaction with temperature. We estimated genetic sex tendency (eGST), defined as the estimated genetic liability to become a given sex under a liability threshold model for sex determination, which accurately predicts the future phenotypic sex. We found evidence that energetic pathways, concerning the regulation of lipids and glucose, are involved in sex determination and could explain why females tend to exhibit higher energy levels and improved growth compared to males. Besides, early exposure to high-temperature up-regulated sox3, followed by sox9a in individuals with intermediate eGST, but not in individuals showing highly female-biased eGST, providing the most parsimonious explanation for temperature-induced masculinization. This gonadal state was maintained likely by DNA methylation and the up-regulation of several genes involved in histone modifications, including jmjd1c Overall, we describe a sex determination system resulting from continuous genetic and environmental influences in an animal. Our results provide significant progress in our understanding of the mechanisms underlying temperature-induced masculinization in fish.