Early starvation in European seabass (Dicentrarchus labrax) larvae has no drastic effect on hepatic intermediary metabolism in juveniles.

The present study aims to investigate nutritional programming through early starvation in the European seabass (Dicentrarchus labrax). European seabass larvae were fasted at three different developmental periods for three durations from 60 to 65 dph (F1), 81 to 87 dph (F2), and 123 to 133 dph (F3). Immediate effects were investigated by studying gene expression of npy (neuropeptide Y) and avt (Arginine vasotocin) in the head, while potential long-term effects (i.e., programming) were evaluated on intermediary metabolism later in life (in juveniles). Our findings indicate a direct effect regarding gene expression in the head only for F1, with higher avt mRNA level in fasted larved compared to controls. The early starvation periods had no long-term effect on growth performance (body weight and body length). Regarding intermediary metabolism, we analyzed related key plasma metabolites which reflect the intermediary metabolism: no differences for glucose, triglycerides, and free fatty acids in the plasma were observed in juveniles irrespective of the three early starvation stimuli. As programming is mainly linked to molecular mechanisms, we then studied hepatic mRNA levels for 23 key actors of glucose, lipid, amino acid, and energy metabolism. For many of the metabolic genes, there was no impact of early starvation in juveniles, except for three genes involved in glucose metabolism (glut2-glucose transporter and pk-pyruvate kinase) and lipid metabolism (acly-ATP citrate lyase) which were higher in F2 compared to control. Together, these results highlight that starvation between 81 to 87 dph may have more long-term impact, suggesting the existence of a developmental window for programming by starvation. In conclusion, European seabass appeared to be resilient to early starvation during larvae stages without drastic impacts on intermediary metabolism later in life.

Plasma cortisol and production of miRNAs in red drum (Sciaenops ocellatus) exposed to three distinct challenges.

The red drum Sciaenops ocellatus is a marine fish species of high commercial interest. Despite improvements in current aquaculture practices, there are still concerns about the impact of daily manipulations regarding fish welfare. To investigate how does fish respond to various challenges, S. ocellatus juveniles were submitted to two acute challenges, namely a confinement stress and a cold-temperature shock, as well as a chronic stress challenge consisting of 18 days of repetitive challenge events. The level of cortisol produced by individuals was used as a measure of activation hypothalamic-pituitary-interrenal (HPI) axis. A significant increase in cortisol levels was detected only after the confinement stress. Interestingly, the fish exposed to a chronic stress for 18 days exhibited cortisol levels significantly lower than those of non-challenged fish. The small RNA-sequencing conducted for the chronic stress experiment only allowed us to identify two plasmatic microRNAs more abundant in non-challenged fish (miR-205-1-5p and let-7b-5p) compared to challenged fish. The miR-205-1-5p was of particular interest since it was already detected in previous studies on other fish species. In silico analysis allowed to predict potentially highly conserved mRNA targets of this specific miRNA, among which is tnfrsfa that plays a key role in the secondary stress response.

Gamma irradiation-induced offspring masculinization is associated with epigenetic changes in female zebrafish.

Sex ratio variation is a key topic in ecology, because of its direct effects on population dynamics and thus, on animal conservation strategies. Among factors affecting sex ratio, types of sex determination systems have a central role, since some species could have a sex determined by genetic factors, environmental factors or a mix of those two. Yet, most studies on the factors affecting sex determination have focused on temperature or endocrine-disrupting chemicals (EDCs), and much less is known regarding other factors. Exposure to gamma irradiation was found to trigger offspring masculinization in zebrafish. Here we aimed at deciphering the potential mechanisms involved, by focusing on stress (i.e. cortisol) and epigenetic regulation of key genes involved in sex differentiation in fish. Cortisol levels in exposed and control (F0) zebrafish females' gonads were similar. However, irradiation increased the DNA methylation level of foxl2a and cyp19a1a in females of the F0 and F1 generation, respectively, while no effects were detected in testis. Overall, our results suggest that parental exposure could alter offspring sex ratio, at least in part by inducing methylation changes in ovaries.

Altered ovarian transcriptome is linked to early mortality and abnormalities in zebrafish embryos after maternal exposure to gamma irradiation.

Recent laboratory studies focusing on multigenerational approach demonstrated drastic phenotypic effects after chronic fish irradiation exposure. No irradiation effect at phenotypic scale was observed for F0 (reproductive performances) while early mortality and malformations were observed in F1 offspring whether they were irradiated or not. The objective was to study molecular mechanisms likely to be involved in these phenotypic effects induced by parental irradiation. Thus, F0 adult zebrafish were irradiated for ten days until reproduction and maternal involvement in offspring development was assessed. Levels of maternal provided cortisol and vitellogenin, needed for embryo development, were not impacted by irradiation. However, maternal transcriptome highlighted irradiation effect on processes involved in oocyte development, as well as on essential maternal factors needed for offspring development. Therefore, this study highlighted the importance of parental exposure on offspring fate and of the importance of multigenerational exposure in risk assessment.

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.

Water temperature explains part of the variation in basal plasma cortisol level within and between fish species.

Within the thermal tolerance range of fish, metabolism is known to escalate with warming. Rapid thermic changes also trigger a series of physiological responses, including activation of the stress axis, producing cortisol. Fish have adapted to their environment by producing a low level of plasmatic cortisol when unstressed (basal), so that thriving in their natural temperature should not impact their basal cortisol levels. Yet, surprisingly, little is known on how temperature affects cortisol within and between fish species. Here, we conducted a phylogenetic meta-analysis to (1) test whether temperature can explain the differences in basal cortisol between species and (2) evaluate the role of temperature on differences in cortisol levels between individuals of a same species. To do this, we retrieved basal plasma cortisol data from 126 studies, investigating 33 marine and freshwater fish species, and correlated it to water temperature. Intra-species variability in basal plasma cortisol levels was further investigated in two species: the European sea bass Dicentrarchus labrax and the Nile tilapia Oreochromis niloticus. Factors such as life stage, sex and weight were also considered in the analyses. Overall, our phylogenetic analysis revealed a clear positive correlation between basal cortisol level and the temperature at which the fish live. The role of temperature has also been confirmed within D. labrax, while it failed to be significant in O. niloticus. In this paper, the influence of habitat, life stage, sex and weight on basal plasma cortisol levels is also discussed. Since some abiotic parameters were not included in the analysis, our study is a call to encourage scientists to systematically report other key factors such as dissolved oxygen or salinity to fully depict the temperature-cortisol relationship in fishes.

A simulated marine heatwave impacts European sea bass sperm quantity, but not quality.

Rapid environmental changes will be the major challenge that most biota will have to deal with in the near future. Extreme events, such as marine heatwaves, are becoming more frequent and could be spatially uniform at a regional scale for a relatively long period of time. To date, most research studies on heatwaves have focused on sessile organisms, but these extreme events can also impact mobile species. Here, a 3-week marine heatwave was simulated to investigate its effects on the male reproductive performance of a Mediterranean Sea emblematic species, the European sea bass Dicentrarchus labrax. Males from the control condition (c. 13°C) produced significantly more sperm than those exposed to a relatively warm thermal treatment (c. 16°C). Nonetheless, neither the percentage of motile spermatozoa nor most of the other sperm motility parameters were significantly affected by the rearing temperature over the whole period. Overall, the results of this study suggest only moderated effects of a potential winter heatwave on the reproductive performance of male European sea bass.

Adult sex ratios: causes of variation and implications for animal and human societies.

Converging lines of inquiry from across the social and biological sciences target the adult sex ratio (ASR; the proportion of males in the adult population) as a fundamental population-level determinant of behavior. The ASR, which indicates the relative number of potential mates to competitors in a population, frames the selective arena for competition, mate choice, and social interactions. Here we review a growing literature, focusing on methodological developments that sharpen knowledge of the demographic variables underlying ASR variation, experiments that enhance understanding of the consequences of ASR imbalance across societies, and phylogenetic analyses that provide novel insights into social evolution. We additionally highlight areas where research advances are expected to make accelerating contributions across the social sciences, evolutionary biology, and biodiversity conservation.

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.

Energy as the cornerstone of environmentally driven sex allocation.

In recent years, observations of distinct organisms have linked the quality of the environment experienced by a given individual and the sex it will develop. In most described cases, facing relatively harsh conditions resulted in masculinization, while thriving in favorable conditions promoted the development of an ovary. This was shown indistinctively in some species presenting a genetic sex determination (GSD), which were able to sex-reverse, and in species with an environmental sex determination (ESD) system. However, this pattern strongly depends on evolutionary constrains and is detected only when females need more energy for reproduction. Here, I describe the mechanisms involved in this environmentally driven sex allocation (EDSA), which involves two main energy pathways, lipid and carbohydrate metabolism. These pathways act through various enzymes and are not necessarily independent of the previously known transducers of environmental signals in species with ESD: calcium-redox, epigenetic, and stress regulation pathways. Overall, there is evidence of a link between energy level and the sexual fate of individuals of various species, including reptiles, fish, amphibians, insects, and nematodes. As energy pathways are evolutionarily conserved, this knowledge opens new avenues to advance our understanding of the mechanisms that allow animals to adapt their sex according to the local environment.

Transcriptomic profiles of consistent risk-taking behaviour across time and contexts in European sea bass.

Bolder individuals have greater access to food sources and reproductive partners but are also at increased risk of predation. Boldness is believed to be consistent across time and contexts, but few studies have investigated the stability of this trait across variable environments, such as varying stress loads or long periods of time. Moreover, the underlying molecular components of boldness are poorly studied. Here, we report that boldness of 1154 European sea bass, evaluated using group risk-taking tests, is consistent over seven months and for individuals subjected to multiple environments, including a chronically stressful environment. Differences in risk-taking behaviour were further supported by differences observed in the responses to a novel environment test: shy individuals displayed more group dispersion, more thigmotaxic behaviour and lower activity levels. Transcriptomic analyses performed on extreme phenotypes revealed that bold individuals display greater expression for genes involved in social and exploration behaviours, and memory in the pituitary, and genes involved in immunity and responses to stimuli in the head kidney. This study demonstrates that personality traits come with an underpinning molecular signature, especially in organs involved in the endocrine and immune systems. As such, our results help to depict state-behaviour feedback mechanisms, previously proposed as key in shaping animal personality.

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.

Group size, temperature and body size modulate the effects of social hierarchy on basal cortisol levels in fishes.

Social rank in a structured society has been linked to basal levels of glucocorticoids in various species, with dominant individuals generally presenting lower levels than subordinates. The biotic and abiotic factors influencing glucocorticoids levels across social ranks are still, however, unclear in fishes. We investigated the influences of group size, fish size, sex, age, and reproduction type, plus water salinity and temperature, on the basal levels of cortisol, the major stress hormone in fishes. A phylogenetically controlled meta-analysis was performed on data from 72 studies over 22 species of fishes. As expected, dominants generally exhibited lower levels of cortisol than subordinates. More importantly, the strength of the correlation between cortisol and rank was modulated by three main factors, group size, environmental temperature, and fish size. Differences in basal cortisol between dominants and subordinates were significantly greater in small groups (dyadic contexts) when compared to larger groups. Differences between dominants and subordinates were also greater in temperate regions when compared to the tropics, and in species with larger body size. These results provide valuable insights into the links among hierarchy, stress and metabolism in fishes.

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.

Human protection drives the emergence of a new coping style in animals.

Wild animals face novel environmental threats from human activities that may occur along a gradient of interactions with humans. Recent work has shown that merely living close to humans has major implications for a variety of antipredator traits and physiological responses. Here, we hypothesize that when human presence protects prey from their genuine predators (as sometimes seen in urban areas and at some tourist sites), this predator shield, followed by a process of habituation to humans, decouples commonly associated traits related to coping styles, which results in a new range of phenotypes. Such individuals are characterized by low aggressiveness and physiological stress responses, but have enhanced behavioral plasticity, boldness, and cognitive abilities. We refer to these individuals as "preactive," because their physiological and behavioral coping style falls outside the classical proactive/reactive coping styles. While there is some support for this new coping style, formal multivariate studies are required to investigate behavioral and physiological responses to anthropogenic activities.

Males can adjust offspring sex ratio in an adaptive fashion through different mechanisms.

Sex allocation research has primarily focused on offspring sex-ratio adjustment by mothers. Yet, fathers also benefit from producing more of the sex with greater fitness returns. Here, we review the state-of-the art in the study of male-driven sex allocation and, counter to the current paradigm, we propose that males can adaptively influence offspring sex ratio through a wide variety of mechanisms. This includes differential production and motility of X- versus Y-bearing sperms in mammals, variation in seminal fluid composition in haplo-diploid invertebrates, and epigenetic mechanisms in some fish and lizards exhibiting environmental sex determination. Conflicts of interest between mothers and fathers over offspring sex ratios can emerge, although many more studies are needed in this area. While many studies of sex allocation have focused on adaptive explanations with little attention to mechanisms, and vice versa, the integration of these two topics is essential for understanding male-driven sex allocation.

Evolutionary dynamics in the Anthropocene: Life history and intensity of human contact shape antipredator responses.

Humans profoundly impact landscapes, ecosystems, and animal behavior. In many cases, animals living near humans become tolerant of them and reduce antipredator responses. Yet, we still lack an understanding of the underlying evolutionary dynamics behind these shifts in traits that affect animal survival. Here, we used a phylogenetic meta-analysis to determine how the mean and variability in antipredator responses change as a function of the number of generations spent in contact with humans under 3 different contexts: urbanization, captivity, and domestication. We found that any contact with humans leads to a rapid reduction in mean antipredator responses as expected. Notably, the variance among individuals over time observed a short-term increase followed by a gradual decrease, significant for domesticated animals. This implies that intense human contact immediately releases animals from predation pressure and then imposes strong anthropogenic selection on traits. In addition, our results reveal that the loss of antipredator traits due to urbanization is similar to that of domestication but occurs 3 times more slowly. Furthermore, the rapid disappearance of antipredator traits was associated with 2 main life-history traits: foraging guild and whether the species was solitary or gregarious (i.e., group-living). For domesticated animals, this decrease in antipredator behavior was stronger for herbivores than for omnivores or carnivores and for solitary than for gregarious species. By contrast, the decrease in antipredator traits was stronger for gregarious, urbanized species, although this result is based mostly on birds. Our study offers 2 major insights on evolution in the Anthropocene: (1) changes in traits occur rapidly even under unintentional human "interventions" (i.e., urbanization) and (2) there are similarities between the selection pressures exerted by domestication and by urbanization. In all, such changes could affect animal survival in a predator-rich world, but through understanding evolutionary dynamics, we can better predict when and how exposure to humans modify these fitness-related traits.

Effects of global warming on sex ratios in fishes.

In fishes, sex is determined by genetics, the environment or an interaction of both. Temperature is among the most important environmental factors that can affect sex determination. As a consequence, changes in temperature at critical developmental stages can induce biases in primary sex ratios in some species. However, early sex ratios can also be biased by sex-specific tolerances to environmental stresses that may, in some cases, be amplified by changes in water temperature. Sex-specific reactions to environmental stress have been observed at early larval stages before gonad formation starts. It is therefore necessary to distinguish between temperature effects on sex determination, generally acting through the stress axis or epigenetic mechanisms, and temperature effects on sex-specific mortality. Both are likely to affect sex ratios and hence population dynamics. Moreover, in cases where temperature effects on sex determination lead to genotype-phenotype mismatches, long-term effects on population dynamics are possible, for example temperature-induced masculinization potentially leading to the loss of Y chromosomes or feminization to male-biased operational sex ratios in future generations. To date, most studies under controlled conditions conclude that if temperature affects sex ratios, elevated temperatures mostly lead to a male bias. The few studies that have been performed on wild populations seem to confirm this general trend. Recent findings suggest that transgenerational plasticity could mitigate the effects of warming on sex ratios in some populations.