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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.

Introduction: The sea bass, Dicentrarchus labrax, is an important species for European mariculture, mainly in Mediterranean countries. In this species, females grow faster and reach a higher weight than males. However, most farmed fish batches show a highly male-skewed sex-ratio. The sea bass has a complex sex determination system, combining both genetic (polygenic) and environmental (temperature) influences. Low initial temperatures (<17°C) favor female sex determination. In contrast, temperature in the late post-larval phase has the opposite effect, with cold temperature favoring male differentiation and high temperature (up to 23°C) favoring female differentiation (Clota et al., 2021). However, data on the combined effects of genotype and temperature are lacking. We need to know whether there is genotype-by-environment (GxE) interaction or not, to deploy a strategy towards producing monosex female individuals for production while keeping a balanced sex-ratio for hatchery and selective breeding. Results: Starting at 1 day and using incremental rearing periods at 16°C (31 to 244 days), followed by an increase to 21°C, we first showed that, while 31 days at 16°C led to 26% of females, increased cold exposure promoted the proportion of females, up to 46% after 74 days at 16°C. Conversely, exposure to 16°C for a period longer than 74 days progressively reduced the proportion of females, reaching a minimum of ≈ 10% after 230 days at 16°C. In a second experiment, we demonstrated that after an initial rearing of fish for 90 days at 16°C, a secondary exposure to four different temperatures (19, 21, 23, 25°C) resulted in different sex-ratios, with more females at higher temperatures (from 30% at 19°C to 49% at 25°C). Then, we examined the genotype-by-environment interaction in both periods, by genotyping fish with a 57K SNP chip, comparing 1) two groups reared at 16 or 21°C during the first 60 days and 2) four groups of fish reared at 19, 21, 23 or 25°C after 90 days at 16

Fish sex determination is often considered as governed by either genetic or environmental factors, but the European sea bass (Dicentrarchus labrax) defies this theory. In this species, a polygenic threshold sex determination system was demonstrated (Vandeputte et al. 2007), where the genetic sex tendency is influenced by larval rearing temperature to determine the phenotypic sex (Piferrer et al, 2005). In this study, we applied two thermal treatments during early larval stage, a low temperature protocol (16°C, LT), known to favour balanced sex-ratios and a high temperature masculinizing protocol (21°C, HT). We combined various “-omics” approaches to characterize this temperature-dependent polygenic sex determination of European sea bass. We produced 8 families by mating 8 males with the same female. The progenies were reared in common garden under two thermal treatments (LT, HT) in triplicate. Fish at four different key developmental stages encompassing the temperature sensitive period were sampled. We predicted the genetic sex tendency (eGST) of the animals using a genomic relationship matrix derived from 57K SNPs from the DLabCHIP array (Griot et al., 2021) with a threshold animal model. This was completed by a transcriptomic approach, whole-body energy measurements. The proportion of females was 53.4% at LT and 25.3% at HT, showing a marked masculinization at high temperature. We found that the eGST accurately predicted the future phenotypic sex. We provided 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. Overall, we describe for the first time a sex determination system resulting from continuous genetic and environmental influences in an animal, which provides significant progress in our understanding of the mechanisms underlying temperature-induced masculinization in fish

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.

Changes in ocean water temperature associated with global climate change are bound to enormously affect fish populations, with potential major economic consequences in the aquaculture and fisheries industries. A link between temperature fluctuations and changes in fish stress response is well established. In this study, we aimed to assess the effects of a short- (4 days) or a long-term (4 months) exposure to warm temperature in the stress physiology of European sea bass (Dicentrarchus labrax) larvae and juveniles. First, cortisol (i.e. the main stress hormone in fishes) analysis was used to confirm that a steady and short-term elevation of temperature acts as a physiological stressful event in these fish, and cortisol release is indeed above a metabolic increase linked to temperature. Moreover, our results verified that measurement of cortisol released into the water can be reliably employed as a non-invasive indicator of acute thermal stress in experimental conditions. Secondly, the different effects on the genetic cascade underlying the stress response between long-term low or high thermal treatments were evaluated at two larval development stages via candidate-gene and whole-transcriptome approaches. Interestingly, opposite expression for some key stress genes (nr3c1, nr3c2 and hsd11b2) were observed between developmental stages, highlighting the distinct adaptive mechanisms controlling the primary and secondary responses to a stressor. Surprising expression patterns for some understudied genes involved in the stress axis were also revealed, including crhr1, mc2r, mc5r, trh or trhr, which should be further explored. Finally, evaluation of cortisol content in scales was successfully used as a biomarker of chronic thermal stress, with 10x more cortisol in fish kept at 21 °C vs 16 °C after 4 months, supporting the gene expression results observed. The use of such a method as a proxy of long-term stress, unprecedented in the literature, holds a vast array of applicat

European sea bass (Dicentrarchus labrax) is an important European aquaculture species. Females, performing better than males with later maturation and larger size, are preferred. Unfortunately, in aquaculture conditions, the sex-ratio is typically biased towards males. Thus, deciphering the sex determinism system and finding routes for controlling the sexratio of this species has been a long-lasting challenge to support sea bass production and selective breeding programmes. A polygenic threshold sex determination system was demonstrated (Vandeputte et al. 2007), where the genetic sex tendency is influenced by larval rearing temperature to determine the phenotypic sex (Piferrer et al, 2005). In this study, we applied two thermal treatments during early larval stage, a low temperature protocol (16°C, LT), known to favour more balanced sex-ratios, and a high temperature masculinizing protocol (21°C, HT). We used genome-wide SNP genotypes to estimate the genetic parameters and genotype-by-temperature interaction of sex tendency in European sea bass

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

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.