Otolith morphogenesis during the early life stages of fish is temperature-dependent: Validation by experimental approach applied to European seabass (Dicentrarchus labrax).

Otolith shape is often used as a tool in fish stock identification. The goal of this study was to experimentally assess the influence of changing temperature and ontogenic evolution on the shape component of the European seabass (Dicentrarchus labrax) otolith during early-life stages. A total of 1079 individuals were reared in a water temperature of 16°C up to 232 days post hatch (dph). During this experiment, several specimens were transferred into tanks with a water temperature of 21°C to obtain at the end of this study four different temperature treatments, each with varying ratios between the number of days at 16 and 21°C. To evaluate the otolith morphogenesis, samples were examined at 43, 72, 86 and 100 dph. The evolution of normalized otolith shape from hatching up to 100 dph showed that there were two main successive changes. First, faster growth in the antero-posterior axis than in the dorso-ventral axis changed the circular-shaped otolith from that observed at hatching and, second, increasing the complexity relating to the area between the rostrum and the anti-rostrum. To test the effect of changing temperature, growing degree-day was used in three linear mixed-effect models. Otolith morphogenesis was positively correlated to growing degree-day, but was also dependent on temperature level. Otolith shape is influenced by environmental factors, particularly temperature, making it an efficient tool for fish stock identification.

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.

Low temperature has opposite effects on sex determination in a marine fish at the larval/postlarval and juvenile stages.

Temperature-dependent sex determination (TSD) can be observed in multiple reptile and fish species. It is adaptive when varying environmental conditions advantage either males or females. A good knowledge of the thermosensitive period is key to understand how environmental changes may lead to changes in population sex ratio. Here, by manipulating temperature during development, we confirm that cold temperature (16°C) increases the proportion of fish that develop as females in European sea bass (Dicentrarchus labrax) until 56 days posthatching, but show that it has an opposite effect at later stages, with the proportion of males reaching ~90% after 230 days at 16°C. This is the first observation of opposite effects of temperature at different time periods on the sex ratio of a vertebrate. Our results highlight the potential complexity of environmental effects on sex determination.