Bruno Guinand
email: bruno.guinand@umontpellier.fr

Athanasios Samaras

THE EPIGENETICS OF STRESS IN AQUACULTURE FISH -AN APPRAISAL

Keywords: allostasis, homeostasis, stress response, neuroepigenetics, brain, welfare

As the rearing environment and hatchery practices are stressful, there is an increased interest on how epigenetics could impact or monitor health and welfare of aquaculture species, and further participate to improve production. However, while epigenetic modifications inducing neurobehavioral changes after exposure to acute or chronic stress have been shown to be integral parts of the observed stress response in mammals, the present-day literature on the socalled 'epigenetics of stress' in fish is scarce. As factors that cause stress affect the central nervous system are mediated to the whole body by the hypothalamic-pituitary-adrenal (interrenal) (HPA/HPI) system, we argue that concentrating on the brain is of prime importance to progress in the epigenetics of stress in aquaculture. This knowledge should promote new opportunities for perhaps more integrative and comparative epigenetic monitoring of the impact of early-life stress in cultured fish species.

Most of the time, stress refers to negative outcomes experienced at any time during life that affect the health and the well-being of individuals. It may translate into neurobehavioral and neuropsychiatric disorders impacting the individual itself, but also its sociality and links with its environment. Stress can however be divided into "good", "tolerable" and "toxic" stress [START_REF] Selye | Stress and the general adaptation syndrome[END_REF][START_REF] Shonkoff | Neuroscience, molecular biology, and the childhood roots of health disparities[END_REF].

It might be experienced positively to biologically embed an appropriate response that may promote robustness and/or performance of the individuals through short-or long-lasting mechanisms. Managing the stress response of organisms is generally achieved by modifying the hatchery practices and rearing conditions organisms are submitted to. While certainly unachieved [START_REF] Saraiva | Domestication and welfare in farmed fish[END_REF], stress monitoring is increasingly devoted to improve animal welfare and sustainability of production [START_REF] Ellis | Cortisol and finfish welfare[END_REF][START_REF] Conte | Stress and the welfare of cultured fish[END_REF][START_REF] Sneddon | Stress management and welfare[END_REF][START_REF] Castanheira | Coping styles in farmed fish: consequences for aquaculture[END_REF][START_REF] Martos-Sitcha | Welfare and stressors in fish: Challenges facing aquaculture[END_REF][START_REF] Chandararathna | Animal Welfare Issues in Capture-Based Aquaculture[END_REF]. However, aquaculture systems invariably impose some level of stress on organisms [START_REF] Afonso | Identifying and managing maladaptive physiological responses to aquaculture stressors[END_REF], and be thoroughly monitored to keep welfare in the "good" to "tolerable" range. Thus, the effects of chronic exposure to common hatchery stressors on fish growth, development, immune system, reproduction, and survival, has to be evaluated [START_REF] Afonso | Identifying and managing maladaptive physiological responses to aquaculture stressors[END_REF].

Over the years, a wide range of indicators of fish stress has been proposed, ranging from the cellular to the organismal level [START_REF] Afonso | Identifying and managing maladaptive physiological responses to aquaculture stressors[END_REF][START_REF] Barreto | Emerging indicators of fish welfare in aquaculture[END_REF]. This includes classical monitoring of blood parameters [START_REF] Rebl | Blood will tell: what hematological analyses can reveal about fish welfare[END_REF][START_REF] Burgos-Aceves | Multidisciplinary haematology as prognostic device in environmental and xenobiotic stress-induced response in fish[END_REF], especially cortisol [START_REF] Sadoul | Measuring cortisol, the major stress hormone in fishes[END_REF]. Monitoring of gene expression using transcriptomics [START_REF] Eissa | Transcriptional stress responses to environmental and husbandry stressors in aquaculture species[END_REF][START_REF] Chandhini | Transcriptomics in aquaculture: current status and applications[END_REF], protein production using proteomics (e.g., [START_REF] De Magalhães | Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare research[END_REF]), behavior and/or physiological states using bio-sensing technologies [START_REF] Barreto | Emerging indicators of fish welfare in aquaculture[END_REF][START_REF] Endo | Biosensors for the assessment of fish health: a review[END_REF][START_REF] Brijs | Bio-sensing technologies in aquaculture: how remote monitoring can bring us closer to our farm animals[END_REF] are increasingly used. Granada et al. [START_REF] Granada | Epigenetics in aquaculture-the last frontier[END_REF] emphasized epigeneticsdefined as changes in the molecular machinery that would induce phenotypic variation without change in the DNA sequence as a 'last frontier' in aquaculture, and more generally livestock [START_REF] Ibeagha-Awemu | Epigenetic marks: regulators of livestock phenotypes and conceivable sources of missing variation in livestock improvement programs[END_REF][START_REF] Triantaphyllopoulos | Epigenetics and inheritance of phenotype variation in livestock[END_REF][START_REF] Wang | Impacts of epigenetic processes on the health and productivity of livestock[END_REF]. They put stimulii.e. positively defined stressors -at the forefront of induced changes in disease resistance, nutrition and growth, sex ratio management, and stress resilience to build more robust aquaculture species [START_REF] Granada | Epigenetics in aquaculture-the last frontier[END_REF].

Epigenetics is thought to shape the stress response and individual performances, with some hopes that epigenetic inputs may fuel aquaculture breeding programs [START_REF] Pittman | Fantastically plastic: fish larvae equipped for a new world[END_REF][START_REF] Gavery | Epigenetic considerations in aquaculture[END_REF][START_REF] Moghadam | Epigenetics-potential for programming fish for aquaculture[END_REF][START_REF] Houston | Harnessing genomics to fast-track genetic improvement in aquaculture[END_REF]. Indeed, the relationship between rate and timing of stress, epigenetics and performance classically refers to "early-life stress" (ELS, review in [START_REF] Fogelman | Early Life Stress, Physiology, and Genetics: A Review[END_REF]) [ Box 1]. Thus, stress-induced epigenetic processes could boost the effectiveness of the stress response and impact productivity. However, mostly loose, discursive relationships between hatchery-related stress and epigenetics have been provided so far [START_REF] Li | The implications for aquaculture practice of epigenomic programming of components of the endocrine system of teleostean embryos: lessons learned from mammalian studies[END_REF]. Furthermore, while ELS is recognized to alter the neural architecture to respond to stressors (e.g., [START_REF] Shonkoff | Neuroscience, molecular biology, and the childhood roots of health disparities[END_REF][START_REF] Mcewen | Mechanisms of stress in the brain[END_REF][START_REF] Kundakovic | Early-life experience, epigenetics, and the developing brain[END_REF] and many others), brain epigenetics or neuroepigenetics that might contribute to shape patterns of gene expression and organismal performance, including production traits is actually missing in farmed fish.

After an introduction to stress and the stress response, this chapter will focus on brain epigenetics to illustrate and promote further work in aquaculture. It further extends on how epigenetics might be used for the biomonitoring of the stress response for issues relevant to «epigenetic selection», production, and domestication.

Stress and stress response

Stress

Stress is a concept that is widely used in our everyday lives, although no universally accepted definition of stress exists. Stress was first described by Selye [START_REF] Selye | The general adaptation syndrome and the diseases of adaptation[END_REF] as "the nonspecific response of the body on any demand on it". More complex definitions have been suggested, including "stress is a state in which homeostasis is actually threatened or perceived to be so; homeostasis is re-established by a complex repertoire of behavioral and physiological adaptive responses of the organism" [START_REF] Chrousos | Stress and disorders of the stress system[END_REF]. This definition incorporates the stressor itself, the perception of the stressor by the organism, as well as a set of adaptive responses in order to re-establish homeostasis. It is, therefore, obvious that stress is a multidimensional concept that includes both the actors (stressors) and the responses (stress response) [ Box 2].

Regarding the stressors, these can be divided into many dichotomies, such as internal and environmental, acute and chronic, adaptive and maladaptive or causing eustress or distress, to name a few. Of particular interest is the (i) acute and chronic stress division that sets different demands on animals, yet the distinction between them can be fuzzy, and the (ii) eustress and distress dichotomy, which was described by Selye [START_REF] Selye | Stress and the general adaptation syndrome[END_REF] [ Box 2]. Incorporating Selye's idea of "not all stress is bad" to advances in neurobiology, a recent theory, called allostasis, has been proposed. Allostasis, in contrast to homeostasis, suggests that an organism "achieves constancy through change" [START_REF] Sterling | Handbook of life stress, cognition and health, in Allostasis: a new paradigm to explain arousal pathology[END_REF][START_REF] Mcewen | The concept of allostasis in biology and biomedicine[END_REF]. This is performed by adjusting set points of the regulatory mechanism and involves synthesis of prior knowledge and predicted current needs. When the cumulative cost of allostasis to the body exceeds certain thresholds, allostatic overload occurs, and pathological conditions start to arise. The latter condition is usually observed under chronic stress [START_REF] Korte | A new animal welfare concept based on allostasis[END_REF].

HPI/HPA axis and the stress hormones

The mechanisms regulating the response to stress are complex, involving the actions of many biological systems of the organism. First, the stressful stimuli are perceived by the central nervous system, which subsequently regulates the response. The first level of response is referred to as the primary response and describes the hormonal (mainly corticosteroids and catecholamines) responses. These responses are observed from early larval stages in most teleost fish species [START_REF] Pavlidis | Onset of the primary stress in European sea bass Dicentrarchus labrax, as indicated by whole body cortisol in relation to glucocorticoid receptor during early development[END_REF], depicting therefore the importance of early development in the physiological and epigenetic regulation of subsequent developmental stages.

Upon perception of a stressful stimulus the hypothalamicsympathetic axis is activated, followed by the activation of the hypothalamuspituitaryinterrenal (HPI) axis, which is homologous to the hypothalamuspituitaryadrenal (HPA) axis in mammals. The hypothalamus-sympathetic axis results in the release of epinephrine and norepinephrine to induce hyperglycemia and fuel the fight or flight response [START_REF] Gorissen | The endocrinology of the stress response in fish-an adaptation-physiological view[END_REF]. The release of catecholamines after stress is rapid because these monoamines are stored in vesicles in chromafin cells [START_REF] Barton | Stress in Fishes : A diversity of responses with particular reference to changes in circulating corticosteroids[END_REF],

called chromaffin cells located in the head kidney -or interrenal -of fish, while the recovery to baseline levels is also fast [START_REF] Milligan | Metabolic recovery from exhaustive exercise in rainbow trout[END_REF]. The activation of the HPI axis starts with the secretion of corticotrophin-releasing hormone (CRH) from the hypothalamic preoptic area, which stimulates the secretion of ACTH from the pituitary gland [START_REF] Flik | CRF and stress in fish[END_REF]. ACTH stimulates the interrenal cells to synthesize and secrete cortisol into the bloodstream [START_REF] Flik | CRF and stress in fish[END_REF][START_REF] Wendelaar Bonga | The stress response in fish[END_REF]. Cortisol actions are regulated by corticosteroid receptors, which in most teleost fish species are one mineralocorticoid (MR) and two glucocorticoid receptors (GRs). Once cortisol is bound, these receptors act as transcription factors and bind to specific DNA sequences (GR-and MRresponsive elements) in target-gene promoters controlling glucocorticoid and mineralocorticoid activities [START_REF] Wendelaar Bonga | The stress response in fish[END_REF][START_REF] Mommsen | Cortisol in teleosts:dynamics, mechanisms of action, and metabolic regulation[END_REF]. GR is a major site for epigenetic control of the stress response (e.g. [START_REF] Gray | Genomic and epigenomic mechanisms of glucocorticoids in the brain[END_REF]) (see below).

-Figure 1 -Apart from the feedforward mechanisms described above, cortisol synthesis is also regulated by negative feedback mechanisms of the HPI axis. Specifically, cortisol exerts a negative feedback regulation in the production of CRH and ACTH [START_REF] Mommsen | Cortisol in teleosts:dynamics, mechanisms of action, and metabolic regulation[END_REF], and an ultra-short-loop negative feedback on the site of cortisol production, the head kidney [START_REF] Bradford | Evidence for ultra-short-loop feedback in ACTH-induced interrenal steroidogenesis in coho salmon: Acute self-suppression of cortisol secretion in vitro[END_REF]. Finally, ACTH can also regulate its secretion by inhibition of hypothalamic CRH release [START_REF] Suda | A short negative feedback mechanism regulating corticotropin-releasing hormone release[END_REF].

There are also various other signals that can stimulate cortisol production, such as the hormones arginine vasotocin, AVT [START_REF] Balment | Arginine vasotocin a key hormone in fish physiology and behaviour: A review with insights from mammalian models[END_REF] (homologue of the arginine vasopressin [AVP] in mammals), -melanocyte stimulating hormone (-MSH [START_REF] Gorissen | The endocrinology of the stress response in fish-an adaptation-physiological view[END_REF]), and catecholamines [START_REF] Rotllant | Intra-adrenal interactions in fish: Catecholamine stimulated cortisol release in sea bass (Dicentrarchus labrax L.)[END_REF]. On the other hand, other hormones (e.g., somatostatin, leptin) also inhibit the production of ACTH, and therefore cortisol [START_REF] Eigler | Somatostatin system: Molecular mechanisms regulating anterior pituitary hormones[END_REF][START_REF] Gorissen | Recombinant human leptin attenuates stress axis activity in common carp (Cyprinus carpio L.)[END_REF].

Stress responses

Stress response comprised of secondary and tertiary responses. Secondary responses refer to the primary response-derived alterations that take place in the animal's physiology, including metabolic, biochemical, haematological, immunological, and osmoregulatory responses [START_REF] Barton | Stress in Fishes : A diversity of responses with particular reference to changes in circulating corticosteroids[END_REF].

These responses mainly act towards the production of energy in order to cope with the stressful event. Initially, catecholamines stimulate the production of glucose mainly via hepatic glycogenolysis, while cortisol participates at a later stage of the stress response by stimulating hepatic gluconeogenesis [START_REF] Wendelaar Bonga | The stress response in fish[END_REF]. When the energy demands set by the stressor cannot be met by the aerobic metabolism, fish are forced to rely on the utilization of anaerobic metabolism and produce lactate [START_REF] Milligan | Metabolic recovery from exhaustive exercise in rainbow trout[END_REF][START_REF] Samaras | Stress and metabolic indicators in a relatively high (European sea bass, Dicentrarchus labrax) and a low (meagre, Argyrosomus regius) cortisol responsive species, in different water temperatures[END_REF].

Apart from affecting carbohydrate metabolism, cortisol exerts proteolytic and lipolytic actions, resulting in increased amino and free fatty acids concentrations in circulation [START_REF] Wendelaar Bonga | The stress response in fish[END_REF][START_REF] Mommsen | Cortisol in teleosts:dynamics, mechanisms of action, and metabolic regulation[END_REF] to fuel glucose production via gluconeogenesis. Additionally, through binding to the mineralocorticoid receptor (MR) cortisol plays a crucial role in fish osmoregulation and ionic balance [START_REF] Wendelaar Bonga | The stress response in fish[END_REF][START_REF] Mommsen | Cortisol in teleosts:dynamics, mechanisms of action, and metabolic regulation[END_REF].

Stress, acting mainly through cortisol, is believed to increase the susceptibility of vertebrates to pathogenic diseases [START_REF] Tort | Stress and immune modulation in fish[END_REF]. Both activating and suppressive responses of the immune system to different stressors have been described, with the outcome depending on the timing as well as the nature of the stressor [START_REF] Tort | Stress and immune modulation in fish[END_REF][START_REF] Yada | Stress and disease Resistance: immune system and immunoendocrine interactions[END_REF]. In this context, acute stress in the form of short-term challenges can potentially enhance the function of the innate immune system by acting as eustress. Long-lasting, repeatable and severe stressors, on the other hand, may lead to suppression of the immune system and therefore act as distress [START_REF] Tort | Stress and immune modulation in fish[END_REF][START_REF] Yada | Stress and disease Resistance: immune system and immunoendocrine interactions[END_REF] [ Box 2].

Finally, tertiary responses involve changes at the whole animal level, like swimming behavior [START_REF] Juell | Use of artificial light to control swimming depth and fish density of Atlantic salmon (Salmo salar) in production cages[END_REF], reproduction [START_REF] Schreck | Stress and fish reproduction: the roles of allostasis and hormesis[END_REF], feeding behavior and growth [START_REF] Leal | Stress-induced effects on feeding behavior and growth performance of the sea bass (Dicentrarchus labrax): A self-feeding approach[END_REF]. Such behaviors are adversely affected by stress, as for instance the reduced appetite and cessation of feeding the follows a stressful event even for days [START_REF] Rubio | Compensatory feeding in the sea bass after fasting and physical stress[END_REF].

Individual differences in stress response and coping styles

It is well documented that basal circulating cortisol levels and the stress response pattern can differ between fish species [START_REF] Fanouraki | Species specificity in the magnitude and duration of the acute stress response in Mediterranean marine fish in culture[END_REF]. Apart from that, variation in cortisol concentrations can exist between individuals within the same species as well, as a result of different environmental conditions (temperature [START_REF] Madaro | Effects of acclimation temperature on cortisol and oxygen consumption in Atlantic salmon (Salmo salar) post-smolt exposed to acute stress[END_REF][START_REF] Samaras | Water temperature modifies the acute stress response of European sea bass, Dicentrarchus labrax L[END_REF], photoperiod [START_REF] Pascoli | Seasonal effects on hematological and innate immune parameters in sea bass Dicentrarchus labrax[END_REF], salinity [START_REF] Tsui | The effects of a sudden salinity change on cortisol, glucose, lactate, and osmolality levels in grouper Epinephelus malabaricus[END_REF]), but also husbandry [START_REF] Samaras | Scale matters: performance of European sea bass, Dicentrarchus labrax, L. (1758), reared in cages of different volumes[END_REF][START_REF] Santos | Chronic stress impairs performance, energy metabolism and welfare indicators in European seabass (Dicentrarchus labrax): The combined effects of fish crowding and water quality deterioration[END_REF].

Moreover, genetic origin can affect the stress response within the same species [START_REF] Pottinger | Consistency in the stress response of two strains of rainbow trout, Oncorhynchus mykiss[END_REF][START_REF] Samaras | Repeatability of cortisol stress response in the European sea bass (Dicentrarchus labrax) and transcription differences between individuals with divergent responses[END_REF].

In this context, divergence in cortisol response has been described in several fish species [START_REF] Pottinger | Consistency in the stress response of two strains of rainbow trout, Oncorhynchus mykiss[END_REF][START_REF] Samaras | Repeatability of cortisol stress response in the European sea bass (Dicentrarchus labrax) and transcription differences between individuals with divergent responses[END_REF][START_REF] Øverli | Evolutionary background for stresscoping styles: Relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates[END_REF], and consistent differences among individuals of the same species have been described (i.e.

identification of low and high responding fish). Cortisol responsiveness has also been associated with behavioral differences and coping styles [START_REF] Øverli | Behavioral and neuroendocrine correlates of selection for stress responsiveness in rainbow trout-a review[END_REF][START_REF] Ruiz-Gomez | Response to environmental change in rainbow trout selected for divergent stress coping styles[END_REF], as well as animal performance, such as differential growth [START_REF] Pottinger | Context dependent differences in growth of two rainbow trout (Oncorhynchus mykiss) lines selected for divergent stress responsiveness[END_REF], and metabolic functions [START_REF] Samaras | Repeatability of cortisol stress response in the European sea bass (Dicentrarchus labrax) and transcription differences between individuals with divergent responses[END_REF][START_REF] Tort | Consistency of stress response to repeated handling in the gilthead sea bream Sparus aurata Linnaeus, 1758[END_REF]. The main driving forces behind these differences are the biosynthetic capacity of the head kidney [START_REF] Hori | The mRNA expression of cortisol axis related genes differs in Atlantic cod (Gadus morhua) categorized as high or low responders[END_REF][START_REF] Samaras | Regulation of divergent cortisol responsiveness in European sea bass, Dicentrarchus labrax L[END_REF] and its sensitivity to adrenocorticotropin hormone (ACTH) [START_REF] Hori | The mRNA expression of cortisol axis related genes differs in Atlantic cod (Gadus morhua) categorized as high or low responders[END_REF][START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF]. Moreover, cortisol response seems to be affected by a strong genetic [START_REF] Chatziplis | QTL for stress and disease resistance in European sea bass, Dicentrarchus labrax[END_REF][START_REF] Fevolden | Poststress levels of lysozyme and cortisol in adult rainbow trout: heritabilities and genetic correlations[END_REF] and epigenetic [START_REF] Moghadam | Impacts of early life stress on the methylome and transcriptome of Atlantic salmon[END_REF][START_REF] Krick | Family-effects in the epigenomic response of red blood cells to a challenge test in the European sea bass (Dicentrarchus labrax, L.)[END_REF] component.

Common measurements of the stress response

Since hormones regulate the physiological response to stress, measuring their concentration is one of the most common and informative techniques about the stress response. Among the major stress hormones, circulating cortisol is the most commonly used acute stress indicator [START_REF] Sadoul | Measuring cortisol, the major stress hormone in fishes[END_REF]. However, care should be taken when measuring circulating cortisol, since (i) cortisol response is variable among species [START_REF] Barton | Stress in Fishes : A diversity of responses with particular reference to changes in circulating corticosteroids[END_REF][START_REF] Samaras | Stress and metabolic indicators in a relatively high (European sea bass, Dicentrarchus labrax) and a low (meagre, Argyrosomus regius) cortisol responsive species, in different water temperatures[END_REF][START_REF] Fanouraki | Species specificity in the magnitude and duration of the acute stress response in Mediterranean marine fish in culture[END_REF], and (ii) cortisol can be influenced by environmental factors such as temperature, dissolved oxygen and water quality in general [START_REF] Madaro | Effects of acclimation temperature on cortisol and oxygen consumption in Atlantic salmon (Salmo salar) post-smolt exposed to acute stress[END_REF][START_REF] Samaras | Water temperature modifies the acute stress response of European sea bass, Dicentrarchus labrax L[END_REF][START_REF] Santos | Chronic stress impairs performance, energy metabolism and welfare indicators in European seabass (Dicentrarchus labrax): The combined effects of fish crowding and water quality deterioration[END_REF]. Catecholamines, on the other hand, display a very fast release and recovery rendering their measurement unreliable due to the fact that, unless fish are catheterized, it is almost impossible to achieve unstressed levels of both epinephrine and norepinephrine [START_REF] Milligan | Metabolic recovery from exhaustive exercise in rainbow trout[END_REF][START_REF] Wendelaar Bonga | The stress response in fish[END_REF].

Other components of the HPI axis, such as ACTH and CRH cannot be easily and reliably measured in fish, either as direct measurement of their concentration or through molecular techniques [START_REF] Gorissen | The endocrinology of the stress response in fish-an adaptation-physiological view[END_REF][START_REF] Flik | CRF and stress in fish[END_REF].

Common measurements of the stress response also target the secondary responses, and specifically parameters of carbohydrates metabolism, such as glucose and lactate [START_REF] Samaras | Stress and metabolic indicators in a relatively high (European sea bass, Dicentrarchus labrax) and a low (meagre, Argyrosomus regius) cortisol responsive species, in different water temperatures[END_REF][START_REF] Fanouraki | Species specificity in the magnitude and duration of the acute stress response in Mediterranean marine fish in culture[END_REF][START_REF] Madaro | Effects of acclimation temperature on cortisol and oxygen consumption in Atlantic salmon (Salmo salar) post-smolt exposed to acute stress[END_REF],

and less commonly lipids, such as non-esterified fatty acids, cholesterol and triglycerides, and proteins [START_REF] Gorissen | Recombinant human leptin attenuates stress axis activity in common carp (Cyprinus carpio L.)[END_REF]. Stress effects on hematology are commonly assessed through hematocrit and hemoglobulin [START_REF] Samaras | Repeatability of cortisol stress response in the European sea bass (Dicentrarchus labrax) and transcription differences between individuals with divergent responses[END_REF], while the effects on osmoregulation are assessed through osmolality and ion concentration measurements [START_REF] Samaras | Stress and metabolic indicators in a relatively high (European sea bass, Dicentrarchus labrax) and a low (meagre, Argyrosomus regius) cortisol responsive species, in different water temperatures[END_REF][START_REF] Samaras | Water temperature modifies the acute stress response of European sea bass, Dicentrarchus labrax L[END_REF][START_REF] Tort | Consistency of stress response to repeated handling in the gilthead sea bream Sparus aurata Linnaeus, 1758[END_REF]. It is also common to measure innate immune system bioindicators (lysozyme, complement C) [START_REF] Tort | Stress and immune modulation in fish[END_REF][START_REF] Tort | Serum haemolytic and agglutinating activity as indicators of fish immunocompetence: Their suitability in stress and dietary studies[END_REF]. Finally, tertiary response indicators include aspects of feeding, appetite and growth [START_REF] Leal | Stress-induced effects on feeding behavior and growth performance of the sea bass (Dicentrarchus labrax): A self-feeding approach[END_REF][START_REF] Rubio | Compensatory feeding in the sea bass after fasting and physical stress[END_REF][START_REF] Samaras | Allostatic load and stress physiology in European seabass (Dicentrarchus labrax L.) and gilthead seabream (Sparus aurata L.) Front[END_REF], swimming behavior, and respiration rate. Specifically, increase in the respiration rate (opercular beat rate), and in the time spent at the bottom of tanks (depending on species), as well as induction of "freezing behaviors", preference for dark areas and loss of schooling consistency are all cited as characteristic examples of behavioral responses to stress [START_REF] Millot | Behavioural stress responses predict environmental perception in European sea bass (Dicentrarchus labrax)[END_REF][START_REF] Kleinhappel | Stress-induced changes in group behaviour[END_REF].

Overall, "stress is in the brain" as the stress response primarily depends on encoding of stressful events in the central nervous system (CNS). However, this response is better monitored by targeting downstream manifestation of stressors on the organisms. Focus on upstream epigenetically-driven brain processes on, e.g., neurogenesis and neuronal regulation that may modulate the HPI axis, the stress response and phenotypic or behavioural trait plasticity should certainly deserve more attention.

12.3. Is there an epigenetics of stress in cultured fish?

A brief state of the art

Fish in aquaculture are exposed to various stimuli perceived as stressors. The effects of stress in fish performance depend on the (i) developmental stage of the fish, (ii) condition of the fish, in terms of nutritional, physiological and health status, and (iii) type of the stressor, including whether it is acute or chronic, its severity and duration, the time of application and whether it is repeatable, predictable, controllable or not. In larval stages, factors that evoke stress include husbandry practices, water quality such as temperature, dissolved oxygen concentration and salinity, tank size and water velocity [START_REF] Lika | Do experimental units of different scale affect the biological performance of European sea bass Dicentrarchus labrax larvae[END_REF], as well as sudden changes of these parameters. This may induce early life stress (ELS) during sensitive periods of development can affect physiology later in life, depending on stress intensity (e.g., [START_REF] Moghadam | Impacts of early life stress on the methylome and transcriptome of Atlantic salmon[END_REF][START_REF] Vindas | Early life stress induces long-term changes in limbic areas of a teleost fish: The role of catecholamine systems in stress coping[END_REF][START_REF] Varsamos | Husbandry stress during early life stages affects the stress response and health status of juvenile sea bass, Dicentrarchus labrax[END_REF]) [ Box 1]. In later developmental stages, fry, juveniles and adults are subjected to stressful husbandry practices (crowding, capture and air-exposure, transportation, sorting, administration of veterinary treatments), which can be added to the stressors described above. Finally, broodstocks are usually maintained in separate rearing systems and/or are exposed to different husbandry practices than fish grown to be harvested. It is common that broodstock are tagged, anaesthesized for sexual maturation stage analysis, involving stripping, ultrasounds or biopsies, as well as hormonal therapies and alterations in environmental parameters for out-of-season egg production. For example, stressful practices in females during oocyte maturation and spawning can affect the cortisol egg content [START_REF] Faught | Maternal stress-associated cortisol stimulation may protect embryos from cortisol excess in zebrafish[END_REF][START_REF] Sopinka | Glucocorticoids in fish eggs: variation, interactions with the environment, and the potential to shape offspring fitness[END_REF]. Finally, it is common that stressful factors are presented for long periods or in a repeated form, and/or in combination between them and/or in manners that render them uncontrollable and unpredictable. They become chronic stressors with detrimental effects on fish because they may arrest feeding and growth, dysregulate the HPI axis [START_REF] Millot | Behavioural stress responses predict environmental perception in European sea bass (Dicentrarchus labrax)[END_REF][START_REF] Samaras | Cortisol concentration in scales is a valid indicator for the assessment of chronic stress in European sea bass, Dicentrarchus labrax[END_REF], suppress the immune functioning [START_REF] Tort | Stress and immune modulation in fish[END_REF][START_REF] Yada | Stress and disease Resistance: immune system and immunoendocrine interactions[END_REF][START_REF] Balasch | Netting the stress responses in fish[END_REF] and disrupt reproductive processes [START_REF] Eissa | Transcriptional stress responses to environmental and husbandry stressors in aquaculture species[END_REF].

The impact of rearing and/or manipulation of the rearing environment on the epigenome has been repeatedly reported in fish [77,90-97; see also 98 for an introduction to mechanisms],

and intragenerational epigenetic programming associated to ELS [Box 1] is emphasized in order to improve production trait in a near future (e.g. [START_REF] Gavery | Epigenetic considerations in aquaculture[END_REF][START_REF] Houston | Harnessing genomics to fast-track genetic improvement in aquaculture[END_REF][START_REF] Li | The implications for aquaculture practice of epigenomic programming of components of the endocrine system of teleostean embryos: lessons learned from mammalian studies[END_REF]). However, a relationship between ELS, epigenetic changes and improved fish performance has been demonstrated only once in Atlantic salmon (Salmo salar) in a hatchery context [START_REF] Moghadam | Impacts of early life stress on the methylome and transcriptome of Atlantic salmon[END_REF]. Using whole fry, authors showed (i) consistent differential epigenetic signatures between stressed (submitted to cold-shock and airexposure) and non-stressed (control) individuals, (ii) a correlation between methylation (i.e., a methyl group added to some cytosines in the DNA sequence) and transcriptional differences for a set of genes, and (iii) superior growth performance observed in stressed fish compared to controls after one year. A companion study showed that relationships between gene expression variation and cytosine methylation differences at candidate genes including genes linked to the HPI axis such as the proopiomelanocortin (pomc), crh and nr3c1 genesthe last one encoding for GR -could be complex and dependent on the life-stage stressor acted upon fish (egg, posthatch, or at both stages) [START_REF] Robinson | Early developmental stress affects subsequent gene expression response to an acute stress in Atlantic salmon: an approach for creating robust fish for aquaculture?[END_REF].

The role of stress-related genes is however reinforced by other studies. For example, using whole genome bisulfite sequencing, it was shown that captivity of adult Atlantic salmon induced differential methylation in F1 offspring that was associated to several biological functions, but primarily genes involved in the nervous system (glutamate receptors), together with genes associated to the immune system [START_REF] Wellband | Environment-driven reprogramming of gamete DNA methylation occurs during maturation and is transmitted intergenerationally in Atlantic Salmon[END_REF]. Results are in line with studies reporting epigenetic modifications at stress-susceptibility genes involved in the glutamatergic system in fish [100] and mammals (e.g., [101]), but also illustrate a potential intergenerational inheritance of stress. Putative multigenerational inheritance (i.e., parental influence to the F1) of epigenetic changes have been reported at numerous occasions in fish (e.g. [START_REF] Berbel-Filho | Environmental enrichment induces intergenerational behavioural and epigenetic effects on fish[END_REF][START_REF] Venney | Rearing environment affects the genetic architecture and plasticity of DNA methylation in Chinook salmon[END_REF][102][103][104][105][106][107]), but brain tissues were not targeted. To the best of our knowledge, only Reiser et al. [108] studied brain epigenetic profiles of individuals submitted to a barren vs an enriched environment (i.e. a stress gradient) across life-stages in trout (Oncorhynchus mykiss). The enriched environment was shown to modulate epigenetic patterns, but phenotypic counterparts were few and nonsignificant in this study, furthermore the method used did not allow for the identification of differentially imprinted genes. Concurrently, when investigating some aspects of epigenetic inheritance of eighteen genes in European sea bass (Dicentrarchus labrax), Anastasiadi et al.

[107] showed that some marks were related to stress responsive genes and conserved from parents to offspring, but this was shown for sperm and not brain. This included nr3c1 which is of primarily importance in the regulation of the HPI axis and regularly screened for gene expression variation in fish (e.g., [109][110][111][112][113]).

Furthermore, emphasizing ELS might obscure other important epigenetic contributions to the stress response [Box 1], notably in studies aiming to better apprehend the impact of intergenerational inheritance. For example, in the brook charr (Salvelinus alpinus), it was shown that thermal regime during parental sexual maturation, but not during rearing of offspring modulates DNA methylation [114]. This study is not directly related to the stress response, but it does ask about stressful practices during broodstock management.

.

Unbalances and misperceptions

Apart from their number which is still low, actual limitations in the study of the epigenetic of stress in cultured fish can be summarized as follows:

1-Studies have poor relationships with the stress axis. ELS is an acronym indicating that individuals were submitted to a stressor that may influence epigenetic outputs (e.g. lists of differentially methylated genes), but brain epigenetics which represents a fundamental driver of the process is ignored.

2-Studies were interested in changes in epigenetic profiles, but only few of them considered the impact of stressor(s) on traits relevant to the hatchery environment (e.g., growth, weight at age, immune competence).

3-Stress-responsive genes from the HPI axis (e.g. nr3c1, crh, pomc) and others are not routinely screened for epigenetic modifications although they have been the subject of an impressive number of studies in mammals. At best, these genes or the pathway they belong to are detected by chance [START_REF] Wellband | Environment-driven reprogramming of gamete DNA methylation occurs during maturation and is transmitted intergenerationally in Atlantic Salmon[END_REF]107], or proposed as candidates [START_REF] Best | Epigenetics in teleost fish: From molecular mechanisms to physiological phenotypes[END_REF]115], but not systematically studied yet.

In the next section, we focus on results obtained in mammals at epigenetically imprinted genes within brain tissues.

12.4. The neuroepigenetics of stress: Fishing with mammalian models Rationales and limitations -Since data have rapidly accumulated in mammalian models, we propose below a short review of epigenetic variation observed at some key genes of the HPA/HPI axis, also including the main players of the vasopressin and oxytocin systems (Fig. 2). A review is provided in [122] for the epigenetics of the catecholaminergic system. Within the HPI, the choice of genes is subjective, as complex molecular processes including epigenetic modifications cannot be summarized to few genes. However, stress-induced variation in the expression of genes presented in Fig. 2 has been studied in fish, notably in the brain (e.g., [START_REF] Vindas | Early life stress induces long-term changes in limbic areas of a teleost fish: The role of catecholamine systems in stress coping[END_REF]109,123]). Their study could then represent an affordable first step for a better understanding on the (epi)genetic impact of stress in aquaculture. Furthermore, variation in gene expression and/or observed polymorphisms have been shown to be associated to the stress response, neuropsychiatric disorders, neurodegenerative pathologies and/or aberrant behavioral phenotypes in mammals [124,125], and this set of genes include the hormonal/endocrine (e.g., crh, crhr, pomc), and the neuronal/synaptic (e.g., c-fos, egr-1, slca4c) activation of the stress axis to emphasize a potential stressor-specific and dual activation of epigenetic programming (Fig. 2). A stimulus-induced neural activation could primarily initiate modifications in the underlying epigenetic regulatory systems, while parallel induction of the HPA axis activity could lead to hormonal priming of the epigenetic machinery for long-term effects via the release of glucocorticoids.

-Figure 2 -

Even if the mammalian HPA and fish HPI axes are homologous, increased knowledge on the neuroepigenetics of stress has been mostly gained from placental mammalian species with high social behavior. The transfer of knowledge to fish species that are oviparous and generally show more limited social behavior or parental care than mammals remains presumptive. While zebrafish has been proposed as a translational model for the study of stressrelated disorders, it is not fully established that comparisons with mammals are always effective. Some evidence is reported for anxiety-like behaviors [126], and for post-traumatic stress disorder symptoms [127]. However, authors noted that micro-RNA networks (i.e. base pair long, non-coding RNA molecules) that are important dynamic players of epigenetic tuning at the post-transcriptional level have undergone extensive rewiring in teleost fish, compared to mammalian models [128]. This induces limitations for functional analysis [START_REF] Best | Epigenetics in teleost fish: From molecular mechanisms to physiological phenotypes[END_REF],

while micro-RNAs are major regulators of the brain response to stress [129,130] and of glucocorticoid actions [131]. Furthermore, the teleost-specific genome duplication is a common issue in both genetics and epigenetics of fish. It implies the testing of more genes than in mammalian models and assessing how the functions of these additional genes evolved or not [START_REF] Best | Epigenetics in teleost fish: From molecular mechanisms to physiological phenotypes[END_REF]. For example, Campos et al. [132] showed that the three DNA methyl transferase 3 (DNMT3) genes present in mammals correspond to six paralogs in zebrafish that showed differential sensitivity to external stimuli. Duplications of the main HPA axis genes are however relatively well-known [133], but the functions of some duplicated receptors still remain unanswered. Furthermore, because the stress response is variable among fish species (e.g. cortisol peak, duration) and reflects physiological variation that has a root in the regulation of molecular interactions, detailed comparison with the zebrafish model should be cautiously considered. Species-specific epigenetic knowledge should be developed.

Immediate early genes in the hippocampus -The hippocampus is particularly sensitive to the effects of glucocorticoids secreted during the diurnal rhythm and chronic stress that may suppress neurogenesis [134]. C-fos (FBJ osteosarcoma oncogene, a proto-oncogene) and Egr-1 (Early growth receptor-1) are transcription factors also defined as stress-induced immediate early genes that are transiently induced in the brain, and notably in the dentate gyrus of the mammalian hippocampus when submitted to an acute stressor. They are used as functional markers of activity in neurons and neuronal circuitries after a variety of stimuli/stressors (e.g., [135][136][137][138][139]). C-fos can be used as a potent indicator of the neuronal activity of memory retrieval after conditioning to aversive stimuli, and a marker of the stress response [140], including in the aquaculture environment [141,142]. Early-life stress and acute restrain stress were found to change the expression of c-fos in brain areas of seabream (Sparus aurata) [START_REF] Vindas | Early life stress induces long-term changes in limbic areas of a teleost fish: The role of catecholamine systems in stress coping[END_REF] and zebrafish

[143], respectively. Gene expression variation at egr-1 was found to predict glucocorticoid receptor expression in the brain of a cichlid (Neolamprologus pulcher) [144]. However, related epigenetic signaling was not studied so far in fish.

While c-fos and egr-1 are not the only genes affected by stress-induced epigenetic modifications in the hippocampus, they are widely studied in fish. Transcriptional control of cfos and egr-1 is traditionally thought to occur predominantly by the activation of the extracellular signal-regulated kinase mitogen-activated protein kinase (Erk-Mapk) signaling cascade that interacts with GRs. This control involves the activation of signaling molecules, which results in specific phospho-acetylation of histone H3, epigenetic chromatin modelling and modification of gene expression. It includes the upregulation of c-fos and egr-1, and combines with changes in DNA methylation patterns of both genes. In rats, methylationdependent interactions between GR and the EGR-1 protein were also observed [145,146]. This suggests that the methylation status observed at the NR3C1 promoter and downstream expression of at least one of the genes could be functionally related. How the epigenetic status of NR3C1 and ERG-1 precisely interact to shape distinct stress-sensitive phenotypes with different stress-induced memories remains unknown. In the European sea bass, Krick et al. [78] also mentioned Bdnf, but methylation variation between pre-and post-stress challenge fish pointed to genes that regulate Bdnf synthesis, but not Bdnf itself. This however suggested that the epigenetic machinery surrounding bdnf gene itself is fairly complex and misunderstood.

Brain-Derived Neurotrophic Factor (BDNF) -

Finally, strain-dependent BDNF methylation inducing complex modulation of RNA transcription and expression has been reported in mice [168]. This relationship was dependent on the early social environment, and might be of some interest for aquaculture. Furthermore, mice reared in enriched social environment had increased acetylation in the BDNF promoter, translating to increases in BDNF expression when responding to a stress challenge later in life [169].

Serotonin signaling -The brain serotoninergic system including serotonin (5-HT), its transporter (5-HTT ; SLC6A4 gene [Solute Carrier Family 6 Member A4], aka SERT) and numerous associated receptors (e.g., 5-HT1A or 5-HT2A ; HTR1A and HTR2A genes [5-Hydroxytryptamine Receptor 1A/2A], respectively) is widely conserved across taxa [170,171].

Serotonin is considered to be involved in more behavioral, physiological and pathological mechanisms than any other neurotransmitter [172]. Serotonin is an important regulator of neural plasticity, behavior, and stress coping in fish [173][174][175]. It interacts with corticosteroid metabolism and the HPI axis to regulate stress coping style [173,176]. Accordingly, studies monitoring the concentration and/or expression of serotonin and its allies to assess stress and stress coping are widespread in fish (e.g., [START_REF] Vindas | Early life stress induces long-term changes in limbic areas of a teleost fish: The role of catecholamine systems in stress coping[END_REF]147,148]), but epigenetics studies are lacking.

However, increased methylation in the proximal promoter region of SLC6A4 was shown positively correlated to threat [177] and aberrant compulsive-disorders [178]. Methylation landscapes of the HTR1A and HTR2A receptor genes have also been shown to respond to stress exposure and be related to neurobehavioral impairments (e.g., schizophrenia, panic disorders) [179,180]. The expression of SLC6A4, HTR1A and OXTR (oxytocin receptor, see below) have been shown to correlate in stressed zebrafish [181,182], but epigenetic changes have not been investigated yet. Interestingly, correlated methylation diffrences at these genes have been suggested to 'imprint' the wolf-to-dog domestication process [183].

It should be briefly mentioned that Several studies have demonstrated the role of serotonin and BDNF on emotion and memory-related networks (e.g., [184][185][186]. For example, it has been shown that Atlantic salmon submitted to uncontrollable chronic stress presented higher growth rates than control fish, with the expression of BDNF negatively or positively interacting with serotonergic activity in the controls and the stressed fish, respectively [148].

The epigenetics of this system has to be documented. CRH release, hence promoting the return of cortisol levels to normal after stress is experienced [188]. AVT (AVP) and IT (OXT) have also been shown to be implied in the stress axis, with further implication in resulting group behavior and social interactions [189][190][191][192].

In spite of differences in the nature of some hormones or neuropeptides, stress-induced epigenetic changes have been observed for most of these genes. This includes epigenetic modulation of OXT and its receptor OXTR [193][194][195], and AVP [196,197]. The epigenetic regulation of AVP through interaction with MeCP2 has been reviewed [198,199]. Stressinduced epigenetic modulation of AVPR1A has been investigated in mice [200], and in prairie voles (Microtus ochrogaster), and shown to be associated to changes in social behavior [201,202].

Nevertheless, because of their interaction and the impact of stressors on HPA axis dysregulation, methylation changes at NR3C1 encoding for GR [145,[203][204][205], and its cochaperone FKBP5 [206][207][208] are the most thoroughly studied regarding stress vulnerability and resilience. FKBP5 was particularly studied in relation to stress sensitivity and induced pain, of particular interest for welfare [204]. Methylation at exon 17 (rat) or 1F (human) of NR3C1 has been the target of many studies (review in [209]), but other methylated sites in the promoter region or the gene body of NR3C1 have also been investigated resulting in a complex regulation of the phenotypic outputs [204,210]. For FKBP5, methylation at intron 7 of the gene was exclusively investigated so far. It has been shown that intronic demethylation increased FKBP5 expression and induced symptoms of GR resistance [211]. The precise interplay between the two genes and their methylation status in regulating the HPA axis remains complex, and necessitates that further genes involved in the epigenetic regulation of the stress and coping styles be considered and studied.

Furthermore, no signifcant relationship between FKBP5 methylation status and cortisol output was found [212,213]. Conversely, a relationship to cortisol production under various stress exposure and NR3C1 methylation was observed [214][215][216]. Intergenerational programming of NR3C1 methylation profiles in offspring has been reported in humans [217][218][219], also including methylation at the alternative receptor (NR3C2, encoding for MR) [220].

However, epigenetic data related to NR3C2 are far less developed, perhaps neglected.

Apart from methylation, histone modifications are also important in the stress-induced regulation of the HPA axis and glucocorticoid receptors [145,221]. For example, breeddependent epigenetic modulation at the promoter of NR3C1 by histone deacetylases was found in the liver of piglets (brain not investigated), and associated to differences in weigth among individuals [222].

Other hypothalamic and pituitary candidates -Data regarding the epigenetic regulation of the main hypothalamic hormone CRH and its receptor CRHR1 when submitted to various stressors have also accumulated. They concern methylation, histone modifications, MeCP2 levels or the impact of miRNAs [223][224][225]. Different methylation status of the CRH/CRHR1 duo has also been recorded in the hippocampus of stressed vs control rats and may translate over at least two generations [226].

POMC is produced in the brain stem and in the arcuate nucleus of hypothalamus, but it is also the main hormone produced by the pituitary and cleaved to ACTH (Fig. 1). Methylation and other epigenetic modifications of POMC induced by ELS have been observed (e.g., [215,227]). For example, methylation of POMC has been shown to be related to the methylation of upstream genes -notably FKBP5and to impact ACTH and cortisol production depending on the stressor type, duration and frequency [215]. Impairments in the regulation of food intake and nutritional programming related to the epigenetic modifications observed at POMC were also evaluated in human [228,229], and should certainly deserve more attention in aquaculture.

Epigenetic modifications occurring at or close to these and other genes appear essential for the regulation of many stress-induced mood or anxiety-like disorders that will affect fish welfare and regulation of the full stress response later in life. How this can be managed to improve traits of interests in a productive environment is not understood.

By targeting the main genes of the HPI axis and few others for which information have been gained over years, we hereby favor a candidate gene approach over epigenome-wide, highthroughput approaches. High-throughput studies are now achievable in many important aquaculture species that have their genome sequenced. Such studies could provide important functional information on the impact of stress at a (epi)genome-wide level and to propose new candidates that may complement patterns investigated at commonly studied genes. On the contrary, although the candidate gene approach targets only a part of the genome, it may participate to translate observations made in humans and rodents to fish, including species with different stress responses.

12.5. Epigenetic biomonitoring of stress

Tracking changes

Epigenetic profiles can be analyzed to estimate a cumulative impact of stress over the organism and be compared to other estimators (e.g. cortisol, glucose, lysozyme). As biomonitoring could be broadly defined as a method for assessing exposure to stressors, it could be important to address this issue for a set of epigenetic markers acting as biomarkers. Epigenetic profiles of genes related to the HPA/HPI axis are interesting target to address this issue by tracking dynamic epigenetic modifications from early to important developmental stages (including sexual differentiation), and during adulthood under different conditions to estimate the impact of different rearing conditions and/or practices. This longitudinal tracking is essential to capture the full complexity of the epigenotype-to-phenotype relationship, but a difficult task.

Actually, progress has been made mainly for sex determination-related issues [230], and a general framework has certainly to be established for longitudinal stress monitoring of fish cohorts within rearing tanks at different life-stages. However, this biomonitoring is already made in fish through blood parameters, including cortisol that establishes a causal link with the stress axis [START_REF] Rebl | Blood will tell: what hematological analyses can reveal about fish welfare[END_REF][START_REF] Sadoul | Measuring cortisol, the major stress hormone in fishes[END_REF]. As blood cells are nucleated in fish, a genome-wide epigenetic information is accessible without sacrificing fish, also limiting invasiveness compared to tissue biopsies.

Blood cells may provide complimentary data to other blood metabolite parameters such as cortisol, glucose, lactate and other stress indicators. Monitoring of blood epigenetics has already been performed in fish and it is thus technically amenable [START_REF] Krick | Family-effects in the epigenomic response of red blood cells to a challenge test in the European sea bass (Dicentrarchus labrax, L.)[END_REF][START_REF] Gavery | Temporal dynamics of DNA methylation patterns in response to rearing juvenile steelhead (Oncorhynchus mykiss) in a hatchery versus simulated stream environment[END_REF]231], but it was never correlated to common proxy of stress such as cortisol. In hens, methylation of red blood cells correlated to rearing conditions (open aviaries vs cages), authors envisioned methylation as a proxy of long term stress [232]. They initiated the search for epigenetic stress biomarkers in hens [233], and reported differentially methylated candidate genes in the blood that could reflect methylation at stress-responsive brain genes [234]. Similar observations were made for the European sea bass [START_REF] Moghadam | Impacts of early life stress on the methylome and transcriptome of Atlantic salmon[END_REF]. Results remain presumptive as comparisons with methylation of target genes directly in brain tissues were not performed yet (see below).

Besides blood that might be very difficult to sample from small fish, any other cells could potentially integrate a portion of the epigenetic stress response in fish. This includes scales that are also a source of cortisol monitoring [START_REF] Sadoul | Measuring cortisol, the major stress hormone in fishes[END_REF][START_REF] Samaras | Cortisol concentration in scales is a valid indicator for the assessment of chronic stress in European sea bass, Dicentrarchus labrax[END_REF], but it could be also possible to extend this approach to water by translating environmental DNA (eDNA) protocols on the epigenetic side (epi-eDNA). Epi-eDNA has been proposed for wild populations of aquatic organisms [235,236], but could be fuitful to monitor an « epigenetic stress index » in the enclosed hatchery environment for welfare assessment at the tank level. This remains a challenging task to obtain well-preserved samples useful to epigenetic analyses that depend on many factors such as water chemistry (basically fresh-vs seawater) or temperature [236]. However, targeting epigenetic profiles of a tissue or of a few candidate genes, as presented in the previous section, or genes traditionnaly used to monitor differences in the stress response and/or coping styles might be achievable. A final advantage of blood -or even water -is the capacity to store samples in biobanks for future assessment [237].

Tissue-dependency

Although blood offers many advantages, drawbacks are present. Perhaps the main one derives from the fact that epigenetic profiles are tissue-dependent. It is thus necessary to establish if the epigenetic profiles of a peripheral tissue such as blood are a reasonable proxy of stress, and also if they are valid across species and conditions. Most of the literature in human is based on the use of blood (lymphocytes not red blood cells), buccal cells and saliva samples and impressive progress has been made in the field. Studies provided support for the blood-brain comparison or provided tools to evaluate this concordance [238][239][240]. It is however clear that evidence at some of the most studied genes is still partly contentious as recently shown for BDNF [241]. In humans, it was reported the impossibility to translate a recognized signature between kit-ligand methylation, adversity and cortisol level in monocytes compared to previous analysis that used a variety of tissues [242].

12.5. Methylation values and rate of accumulation might be modified under stress. Therefore, a discrepancy between epigenetic and chronological ages could be predictive of cumulative stress periods over the organism, impaired future events, and may be a pre-requisite for phenotypic prediction [246,247]. Many epigenetic clocks use blood samples as starting tissue and, in human, it was shown that 'epigenetic aging' measured at methylation sites occurred in glucocorticoid response elements, suggesting that the history of stress exposure can be strongly related to glucorticoid signaling [248]. Epigenetic clocks have to be established for brain and blood tissues in aquaculture fishes and could be indices of fish welfare and well being in hatchery. Such data are lacking and certainly challenging asfor exampledistrinct brain areas may put different imprinting of stress exposures shaping their own epigenetic clocks.

Critical period

Time-dependency also refer to critical periods at which organisms are sensitive to ELS. This is however a very general definition that triggers neural plasticity in early life, but does not stipulate what the extent and the main boundaries of a critical period are. For example, in humans, most of the ELS literature focuses on pre-and early post-natal periods as critical periods that would translate in phenotypic effects later in life. However, the activity of specific processses peaked after birth, could last until adolescence, and large epigenetic alterations are known to occur until five years of age before decreasing [249,250]. Therefore, more than one or at least one extended critical period with different sensitivity to stressors may occur This suggests that some information on 'windows of opportunity' for stress to imprint the epigenetic machinery and induce allostatic load limiting performance and affecting wellbeing later in life is lacking in fish. It is clear that early developmental stages (e.g., hatching, mouth-opening) should deserve attention, but the dynamics of epigenetic changes over the first months of life have to be investigated in greater detail in order to concentrate improvements of hatchery practices over critical stressful periods that may extend beyond larval rearing. From the epigenetics side, if unstressed individuals are sufficient to investigate how experimental manipulations of stressors affect the epigenome in individual studies, a standardized protocol would improve further comparisons across laboratories, strains and species. This will facilitate the design of collaborative studies to prime eustress and adequately modify a phenotype.

Conclusions

While epigenetic modifications have been shown to be linked to numerous neurobehavioral disorders in human and rodents, the epigenetics of stress in fish and aquaculture is still in its infancy. This research area has to be improved by targeting more specifically systems or genes that mediate the stress response. The HPI axis represents a starting point as the epigenetic regulation of stress-responsive genes has received considerable attention in mammals, but also in gene expression studies in fish. Other systems (e.g., the sympathetic nervous system) may deserve parallel attention. At the scale of a single generation, it is necessary adopt a more longitudinal approach, to go beyond ELS and to define more precisely the 'critical period' at which a stressor may program an epigenome and translate in a significant impact on health, welfare, and production. Such knowledge should open new avenues to an epigenetic biomonitoring of stress using peripheral tissues (i.e., blood) to complement more traditional cortisol evaluation of well-being and create another level of evaluation of coping styles. This necessitates costly programmes and the development of new resources targeting the analyses of epigenowide variation. Increased accessibility to (epi)genomic resources for most aquaculture species as well as the development of models and tools are promising to make the epignetics of stress an exciting new research area.

Box 1: Early life stress, epigenetic programming and the epigenetics of stress -Early-life stress (ELS) or "early life adversity" refers to critical period(s) during early developmental stages (aka 'critical window' or 'sensitive period') at which an organism is especially sensitive to priming by environmental stressors. According to Waddington's definition of epigenetics, ELS induces the complex and dynamic interactions between the developmental environment and the genome that led to the production of an individual phenotype without considering change at the DNA level. ELS thus refers to early intra-generational reprogamming of some organismal functions, with stressors leaving a signature in the whole epigenetic machinery ('epigenetic imprinting' or 'memory') that would further drive and regulate genome-wide expression of the organism, including neuronal function and future behaviors. In that sense, stressors experimented during early-life may favour phenotypic plasticity and adaptability to the surrounding developmental environment, while maintaining canalization to ensure organismal functions. In production settings, ELS have to be monitored to ensure organisms that, e.g., protective immunity, social behaviours, physical and cognition performances are maintained in a resting zone favouring health and welfare, and should should thus promote eustress rather than distress.

Sensitivity to ELS is classically considered to extend from the fertilized egg stage to hatching, plus a post-hatch period that is certainly species-specific. It could possibly be extended over a larger developmental period, and the late-developmental boundary of the critical period at which ELS could remain active is generally misunderstood, or neglected. Furthermore, how stressors that differ in strength and/or duration interplay during the critical period to shape a phenotype and promote eustress condition is also misunderstood, and certainly dependent on the individual genotypes that represent a landscape for epigenetic programming.

ELS is an important term in the field of epigenetics, and certainly the best entry to deal with the epigenetics of stress. However, ELS could be not considered as a substitute as it only captures the action of a particular stressor or a set of stressors in early-life, while perhaps neglecting those that may arise at other important developmental periods (e.g., sexual differentiation, sexual and gamete maturation) remain poorly assessed. Furthermore, the focus is made on stressor(s) and a phenotype of interest (e.g., growth rate) that could be enough for production, rather than on the stress response which is a complex set of responses that cannot be equated to a phenotype of interest, but rather to the processes mediated by organs, hormones and behaviors that participate to build this phenotype while preserving welfare, and perhaps promoting the onset of domestication for cultured species like fish. It is important not to turn ELS as the alpha and omega of the epigenetics of stress that has also to be fueled by multidisciplinary inputs from, e.g., physiology, neurobiology and behavioural sciences. Chronic stress is less well-defined than acute stress. Stressors can be severe or mild, predictable or unpredictable, and controllable or uncontrollable. Chronic stress can be comprised of various combinations of the above-mentioned parameters. The responses of animals to chronic stress vary between adaptation through habituation and the development of pathological conditions like hypersensitivity, hyposensitivity or prolonged activation of the stress response mechanism.

Eustress refers to the responses of an organism that lead to enhanced survival, performance, and adaptation.

Distress refers to the physiological alterations caused by a stressor that can induce detrimental effects to the organism.

  BDNF is a neurotrophic factor widely expressed in the brain, for which stress-related expression is commonly reported[147][148][149]. BDNF has been shown to directly regulate the HPA axis[150][151][152], and has particular functions over dendritic remodeling, neuron survival and synaptic plasticity in the hippocampus and the basal amygdala at early life-stages[153][154][155]. One of the BDNF receptors, TrkB (Tropomyosinrelated kinase receptor B, aka NTRK2: Neurotrophic Receptor Kinase 2), and GR are coexpressed in hippocampal neurons, suggesting a strong interplay between the hormonal microenvironment and the neurotrophin regulation for the control of behavioral tasks[156].BDNF-TrkB signalling pathway has been reviewed[157], and its role for the development and maintenance of aminergic neuronal populations reported for zebrafish[158].Under stress, methylation or histone acetylation of BDNF varies in humans[159,160] and rodents[161][162][163]. Epigenetic modifications at BDNF and especially the methylation status of the BDNF promoter was shown to impact binding of MeCP2 (Methyl CpG Binding-Protein) therefore repressing transcription. The MeCP2 protein has been suggested to regulate critical periods[164], and has a central role in activity-dependent neuronal plasticity and transcription during brain development. Early life experiences are thought to imprint BDNF methylation and induce changes in the HPA axis regulation and behavior later in life[165,166]. Epigeneticallydriven regulation within the BDNF-TrkB signaling pathway also impacts long-term fear memory[167].

  Hypothalamic connections -CRH and AVT (in fish) or AVP (in mammals) released from the hypothalamus are the essential regulators of the HPA/HPI axis in mammals and fish (Figs1 &2). Together with BDNF and other upstream regulators such as the pituitary-adenylate cyclase protein, CRH and vasotocin interactions with specific receptor subtypes have been shown to induce responses to anxiogenic stimuli[187]. In mammals, in addition to CRH and AVT, oxytocin (OXT, a mammalian homologue of isotocin, IT in fish) moderates the release of ACTH and cortisol by either altering sensitivity to the feedback mechanism or by decreasing

Box 2 .

 2 Definitions of stressors and stress responseStressor is a factor that triggers a stress response. Stressors can vary in nature: environmental, biological, physical, psychological, and others.Stress response is the organism's set of responses to the stressor. Common examples of stress responses are the "fight-flight" response and the release of cortisol into circulation.Hypothalamus -Pituitary -Interrenal (HPI) axis is a collection of structures that provide feedforward and feedback signals for the regulation of glucorticoid release in fish. It is a system homologous to the hypothalamuspituitaryadrenal (HPA) axis in mammals.Acute stress is the effect of stressors usually of short duration and severe intensity. The animal's responses are mainly the fight-flight response, through the activation of the sympathetic nervous system and the stimulation of the HPI axis, which result in the release of catecholamines and cortisol into the circulation, respectively.

Fig. 1 :

 1 Fig. 1: NOR REPORTED for COPYRIGHT REASONS

.4. Individual differences in stress response and coping styles

	12.1. Introduction	p. 5
	12.2. Stress and stress response	p. 6
	12.2.1. Stress	p. 6
	12.2.2. HPI/HPA axis and the stress hormones	p. 7
	12.2.3. Stress responses	p. 8
	12.2

p. 9

12.2.5. Common measurements of the stress response

p. 9

12.3.

Is there an epigenetics of stress in cultured fish? p. 10 12.3.1. State of the art p. 10 12.3.2. Unbalances and misperceptions p. 12 12.4. The neuroepigenetics of stress : Fishing with mammalian models p. 13 12.5. Epigenetic biomonitoring of stress p. 19

	12.1. Introduction
	12.5.1. Tracking changes	p. 20
	12.5.2. Tissue-dependency	p. 21
	12.5.3. Time-dependency	p. 21
	12.5.4. Critical period	p. 22
	12.6. Conclusions	p. 22

  Mechanisms producing epigenetic alterations in the CNS participate to brain functional plasticity and encode stressful life events and experiencesnotably in early life, but also adulthood[116,117] following stress exposure. These events are encoded in discrete neuronal populations that, once activated, would propagate or promote specific responses to stress across the whole lifespan and influence health and well-being of organisms. In human, it was shown that glucocorticoid-induced DNA methylation altered certain gene expression patterns throughout lifetime by priming the GR response of neighboring loci, rendering them more

responsive (i.e., enhanced transcriptional expression) to a subsequent acute glucocorticoid exposure

[118]

. Various authors

[116,[119][120][121]

produced reviews documenting how stressors in early life and sometimes later in adulthood induced neuroepigenetic changes at the major central stress axis genes and could mediate HPA dysfunctions, but also behavioral changes in human and mice.

3. Time-dependency

  Some epigenetics profiles can also be age-dependent and allow building epigenetic clocks for age estimation. An epigenetic clock is a mathematical procedure that estimates the age of one individual through determining and combining the methylation values of selected loci across the genome. Age-predictive models of epigenetic clocks are tissue dependent and necessitate rigorous calibration[243,244]. One has been proposed in European sea bass however[245].

Acknowledgements p. 23

Acknowledgements:

We greatly thank colleagues that provided support during this work, and especially E. Desmarais and S. Ben Chehida. The kindness and patience of editors have been appreciated. Funding by the EU ERA-NET FP7 programme for the Cooperation in Fisheries, Aquaculture and Seafood Processing (COFASP) and the Agence Nationale pour la Recherche (ANR; grant number: ANR-16-0001-COFA) has also to be acknowledged.

Captions of the figures

A focus is made onto the HPI axis, the hippocampus and other systems for genes in which stress-induced epigenetic modifications -mainly methylation. These modifications can rewire brain/neuronal molecular pathways and translate into neurobehavioral disorders in human and rodents, sometimes resulting in differences in cortisol production. Gene names are reported as in mammals. Solid lines refer to epigenetic interactions among genes or action of genes on specific organs (serotonergic system). (-): negative feedback loop induced by the cortisol on the hypothalamus. Abbreviations: ACTH: adrenocorticotropin hormone; AVP: arginine vasopressin (AVT: arginine-vasotocin in fish); AVPR1A: arginine vasopressin receptor 1A; BDNF: brain-derived neurotrophic factor; cfos: FBJ osteosarcoma oncogene; CRH: corticotropin-releasing hormone; CRHR1: corticotropin-releasing hormone receptor 1; EGR-1: early growth receptor-1; FKBP5: FK506 Binding Protein 5; HTR1A: 5-hydroxytryptamine recptor 1A; HTR2A: 5--hydroxytryptamine recptor 2A; POMC: proopiomelanocortin; OXT: oxytocin (IT: isotocin in fish); OXTR: oxytocin receptor.


B Sperelakis-Beedham
email: brian.sperelakis-beedham@aphp.fr

M Lopez

E Bourrat

N Gaitch

F Houriez

B Martinez

I Fajac

PR Burgel

G Hickman

M P Audrézet

D Gonde

F Cabet

M Gerfaud- Valentin
email: mathieu.gerfaud-valentin@chu-lyon.fr

R Nove-Josserand
email: raphaele.nove-josserand@chu-lyon.fr

C Raynal

A Pagin

MP Reboul

A De Becdelièvre
email: alix.debecdelievre@aphp.fr

T Bienvenu
email: thierry.bienvenu@aphp.fr

I Callebaut
email: isabelle.callebaut@sorbonne-universite.fr

E Girodon

AQP5, a second gene at play with CFTR in aquagenic palmoplantar keratoderma

Keywords: Aquagenic palmoplantar keratoderma, Aquagenic wrinkling, CFTR, Aquaporin 5, AQP5

AQP5 , a second gene at play with CFTR in aquagenic

palmoplantar keratoderma

Aquagenic palmoplantar keratoderma (APPK), also known as Aquagenic Wrinkling, is a rare skin disease characterized by transient skin wrinkling with oedema and whitish papules mostly on the palms, rarely on the soles, after water immersion 1 . It is frequently observed in patients with cystic fibrosis (CF) 2 but also occurs sporadically, often in young women. An excess of carriers of variants in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene has been observed in patients and a mild phenotype such as a CFTR-related disorder (CFTR-RD) could be unmasked [START_REF] Raynal | CFTR gene variants: a predisposition factor to aquagenic palmoplantar keratoderma[END_REF] . Other genes might be at play, such as the Aquaporin 5 (AQP5) gene, involved in Non-Epidermolytic Bothnian-type palmoplantar keratoderma (NEPPK), which is a permanent condition worsened by water immersion 4 .

We investigated the involvement of AQP5 in 198 patients with an isolated APPK (86.9% of women, 92% of Caucasian ethnicity), including those from our previously published cohort [START_REF] Raynal | CFTR gene variants: a predisposition factor to aquagenic palmoplantar keratoderma[END_REF] .

Diagnosis was based on clinical examination by a dermatologist and familial data were collected, along with sweat test results when available (Table 1). All patients gave their written informed consent to the genetic studies and had an in-depth CFTR gene study, leading to unmask CF in one and a CFTR-RD in 13. Massive parallel sequencing of the AQP5 coding regions led to identify an AQP5 variant in six patients (3.0%), of whom two also carried a heterozygous CFTR variant (Table 1). Five AQP5 variants are clustered in the cytoplasmic Cterminal tail, where variants implicated in NEPPK are absent from. These C-terminal variants could correspond to a mild form of NEPPK, mimicking APPK. Indeed, NEPPK-causing variants are located in the extracellular end of the water channel or in extracellular loops, thus the mechanism for NEPPK seems to be water conductance dysregulation [START_REF] Blaydon | Mutations in AQP5, Encoding a Water-Channel Protein, Cause Autosomal-Dominant Diffuse Nonepidermolytic Palmoplantar Keratoderma[END_REF] . On the contrary, the C-terminal AQP5 variants found in this study are expected to moderately impact the 3D structure of AQP5 but they could significantly impair the ability of AQP5 to interact with partners, as well as its subcellular trafficking or stability at the membrane (Fig. 1). Recent studies revealed that AQP5's C-terminal tail is compulsory for AQP5 to pass protein quality controls [START_REF] Muroi | C-Terminal Domain of Aquaporin-5 Is Required to Pass Its Protein Quality Control and Ensure Its Trafficking to Plasma Membrane[END_REF] . Specific interactions between this region and multiple junctional proteins also suggest an influence on cell-to-cell adhesion regulation [START_REF] Login | Aquaporin-5 regulation of cellcell adhesion proteins: an elusive "tail" story[END_REF] . Hence, differences in variant location may cause different pathological processes and explain differences in the PPK phenotype. Epidemiological data also support the role of C-terminal AQP5 variants, which are significantly more frequent in patients with APPK than in a control population taken from the GnomAD database 7 (OR:10.0, 95% CI 4.1 to 24.4, p<0.0001).

The sixth AQP5 variant found in this study, p.Asn125del, located in the membrane-spanning domain, could lead to disturb the overall conformation of the extracellular C-loop, which is important for optimizing water transport [START_REF] Dingwell | Structure of the Functionally Important Extracellular Loop C of Human Aquaporin 1 Obtained by Solid-State NMR under Nearly Physiological Conditions[END_REF] . An effect on the stability of the tetramer could also be expected, as the extremities of the C-loop interact with each other in the neighboring monomers.

This study eventually raises the hypothesis of a multifactorial aetiology of APPK, given the

  role of CFTR and AQP5 in fluid homeodynamics. Heterozygous CFTR and AQP5 variants may therefore be risk factors predisposing to APPK. An oligogenic model could account for the absence of family history of APPK in patients carrying inherited AQP5 variants. It compels us to consider AQP5 variants, even frequent ones, as potential risk factors, especially when combined with CFTR variants. Our results prompt searching for permanent PPK, even if discrete, as well as other cases of PPK in the patients' families. Ultimately, our study underlines the interest of genetic exploration of APPK, including AQP5 alongside CFTR, for medical care, genetic counselling of CF and a better understanding of the mechanisms underlying APPK.

Figure legend Fig 1 .

 legend1 Figure legend Fig 1. AQP5 variants found in patients referred for aquagenic palmoplantar keratoderma. 3D structure coordinates were taken from the Protein Data Bank (http: https://www.rcsb.org/) and from the AlphaFold database (https://alphafold.ebi.ac.uk/) and were visualized using Chimera (https://www.cgl.ucsf.edu/chimera/). (a) : Variants (green balls) are mapped on the AlphaFold2 model of the AQP5 3D structure (AF-P55064-F1-model_v1 9 ), colored according to the prediction confidence score (pLDDT>90: blue, 90≥pLDDT>70: cyan, 70≥pLDDT>50: yellow). The cytoplasmic Cterminal tail is predicted as an alpha-helix (yellow). The position of the membrane bilayer was predicted using the PPM server 10 . (b) : The monomeric model of the full length protein was superimposed on the experimental 3D structure of human AQP5, organized as a tetramer (in grey, pdb 3D9S 11 , RMSD 0.67 Å). The position of the C-terminal, cytoplasmic helix (shown in yellow on the AlphaFold2 model and absent from the experimental 3D structures (3 other subunits in grey) is predicted to be variable, according to the position of its homologous sequence on the experimental 3D structure of human AQP2 (pdb 4OJ2, not shown).

  Figure 1

Table 1 :

 1 Genotype and clinical data of patients found to carry an AQP5 variant African/African American, Oth : Other (population not assigned), ASJ : Ashkenazi Jewish, SA : South Asian, NFE : Non-Finnish European *** CFTR variants were classified according to the CFTR-France database (https://cftr.iurc.montp.inserm.fr/cgi-bin/home.cgi?) +/-: heterozygous; CF: CF-causing; CFTR-RD: CFTR-RD causing

	Patient	Genotype

Acknowledgements:

The authors are indebted to all clinicians who referred the patients for genetic analysis.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability statement: AQP5 variants identified in this study have been deposited in the Leiden Open Variation Databases (http://www.lovd.nl/3.0/home).

Conflict of interest: none to declare

Each author has provided a disclosure statement.

Ethics statement: the study was in accordance with the ethical standards in France and all patients gave their written informed consent to the genetic study.