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.

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.

Parental selection for growth and early-life low stocking density increase the female-to-male ratio in European sea bass.

In European sea bass (Dicentrarchus labrax), as in many other fish species, temperature is known to influence the sex of individuals, with more males produced at relatively high temperatures. It is however unclear to what extent growth or stress are involved in such a process, since temperature is known to influence both growth rate and cortisol production. Here, we designed an experiment aiming at reducing stress and affecting early growth rate. We exposed larvae and juveniles originating from both captive and wild parents to three different treatments: low stocking density, food supplemented with tryptophan and a control. Low stocking density and tryptophan treatment respectively increased and decreased early growth rate. Each treatment influenced the stress response depending on the developmental stage, although no clear pattern regarding the whole-body cortisol concentration was found. During sex differentiation, fish in the low-density treatment exhibited lower expression of gr1, gr2, mr, and crf in the hypothalamus when compared to the control group. Fish fed tryptophan displayed lower crf in the hypothalamus and higher level of serotonin in the telencephalon compared to controls. Overall, fish kept at low density produced significantly more females than both control and fish fed tryptophan. Parents that have been selected for growth for three generations also produced significantly more females than parents of wild origin. Our findings did not allow to detect a clear effect of stress at the group level and rather point out a key role of early sexually dimorphic growth rate in sex determination.

Sex dimorphism in European sea bass (Dicentrarchus labrax L.): New insights into sex-related growth patterns during very early life stages.

The European sea bass (Dicentrarchus labrax) exhibits female-biased sexual size dimorphism (SSD) early in development. New tagging techniques provide the opportunity to monitor individual sex-related growth during the post-larval and juvenile stages. We produced an experimental population through artificial fertilization and followed a rearing-temperature protocol (~16°C from hatching to 112 days post-hatching, dph; ~20°C from 117 to 358 dph) targeting a roughly balanced sex ratio. The fish were tagged with microchips between 61 and 96 dph in five tagging trials of 50 fish each; individual standard length (SL) was recorded through repeated biometric measurements performed between 83 to 110 dph via image analyses. Body weight (BW) was modelled using the traits measured on the digital pictures (i.e. area, perimeter and volume). At 117 dph, the fish were tagged with microtags and regularly measured for SL and BW until 335 dph. The experiment ended at 358 dph with the sexing of the fish. The sex-ratio at the end of the experiment was significantly in favor of the females (65.6% vs. 34.4%). The females were significantly longer and heavier than the males from 103 dph (~30 mm SL, ~0.44 g BW) to 165 dph, but the modeling of the growth curves suggests that differences in size already existed at 83 dph. A significant difference in the daily growth coefficient (DGC) was observed only between 96 and 103 dph, suggesting a physiological or biological change occurring during this period. The female-biased SSD pattern in European sea bass is thus strongly influenced by very early growth differences between sexes, as already shown in previous studies, and in any case long before gonadal sex differentiation has been started, and thus probably before sex has been determined. This leads to the hypothesis that early growth might be a cause rather than a consequence of sex differentiation in sea bass.