Coping styles in European sea bass: The link between boldness, stress response and neurogenesis
Introduction
Coping styles have been defined as “a coherent set of individual physiological and behavioral differences in stress responses consistent across time and context” by Koolhaas et al. [1]. They have been described in various animal species, including fish, as a continuum between two extreme phenotypes, called proactive and reactive [[2], [3], [4], [5]]. In terms of behavior, proactive fish are bolder, more aggressive, explore their environment faster, and display less flexible behavior than reactive fish [[4], [5], [6], [7], [8]]. Moreover, correlations between these behavioral responses across time and context have been established in several fish species [2,5,9], as well as between behavior and physiology [4,[10], [11], [12], [13], [14], [15]], generally used for defining coping styles since.
Stress can be defined as the cascade of biological events that occur when an organism faces a challenge out of the normal range and attempts to reestablish physiological equilibrium [16]. Typical stress responses of fish towards adverse events involve two main neuroendocrine axes, the brain-chromaffin axis and the hypothalamo-pituitary-interrenal (HPI) axis [[17], [18], [19]]. Both axes are important in the primary response to stress [[20], [21], [22]], which is activated in various stressful contexts [21,[23], [24], [25], [26], [27]] and leads to the release of stress hormones, catecholamines (mostly epinephrine and norepinephrine), and corticosteroids (cortisol in teleost fish) into the systemic circulation. At the central level, several monoaminergic neurotransmitters, such as serotonin (5-HT), dopamine (DA), and norepinephrine (NE), are also known to be involved in the organization of stress responses in vertebrates [27]. Stress induces changes in brain monoaminergic systems, both acutely and chronically, which is especially true for the serotonergic system. For example, serotonergic activity is consistently activated upon both chronic or acute stress exposure in fish [17,26,28]. Acute net chasing was for example demonstrated to induce a rapid increase in forebrain serotonergic activity in rainbow trout [17]. Similarly, chronic confinement stress (three times a day during 4 weeks) also induced elevated concentration of 5HIAA and serotonergic activity in the telencephalon of Artic Charr [28]. The concentration of the brain monoamines DA, NE, and 5HT and the concentration of their associated main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4-hydroxyphenylglycol (MHPG), and hydroxyindoleacetic acid (5HIAA), respectively, are generally used to quantify monoaminergic system activity using the turnover ratio between the monoamine and associated metabolite [26,27,29]. This activity has been suggested to vary between individuals with different behavioral responses [26,27,[30], [31], [32]]. Both the stress axis (HPI) and monoaminergic systems (brain-chromaffin) have been shown to be reliable physiological markers correlated with differences in coping styles [7,26,[33], [34], [35]] even if, in some species, physiological markers were not directly linked to behavioral traits related to coping styles [36].
The HPI axis, which produces cortisol, its major end-product hormone, acts as an activator of physiological and behavioral responses [18,37,38]. Glucocorticoid receptors (Grs) and mineralocorticoid receptor (Mr) bind cortisol [39] and are detected in various fish tissues. They are involved in different physiological functions linked for instance to growth, reproduction, immunity and they participate in the regulation of control cortisol release [[40], [41], [42], [43]]. The production and release of cortisol occurs in interrenal cells, located in the teleost head kidney, and is mainly controlled by adrenocortidotropic hormone (Acth), which is released by the pituitary gland into the blood [18]. In turn, the release of Acth is mainly controlled by corticotropin releasing-factor (Crf), a neuropeptide produced in the hypothalamus [20,44]. In addition, recent studies suggest that the regulation of stress axis and neurogenesis are closely related [[45], [46], [47]] and we previously proposed that fish displaying high sensitivity to stress also exhibit high levels of neurogenesis in a non-stressful context [45]. Proliferating cell nuclear antigen (Pcna), neurogenic differentiation factors 1 and 2 (Neurod1 and Neurod2), and the early growth response protein (Egr1) are important markers of neurogenesis and neuronal development [34,48]. While the regulation of stress axes might be an important feature to characterize coping styles in fish, there is still a paucity of information regarding the differences in neurogenesis between coping styles. Additional data are therefore needed in order to better understand the mechanisms associated to the behavioral responses.
Differences in behavioral and physiological responses to stress may have direct implications on growth, foraging activities, and the resulting fitness of wild fish species [37,49,50]. In the context of aquaculture, better knowledge of individual diversity can help to improve the welfare and management of the fish [5], as proactive fish sometimes show better feeding motivation and feed efficiency than reactive fish [2,5,51,52].
The European sea bass Dicentrarchus labrax is a predatory species, widely distributed around Europe and West Africa [53], that has a key ecological role in the marine food chain [54]. It is also one of the most highly farmed fish species in Europe [55]. Therefore, additional knowledge of coping styles in European sea bass is required to improve both its management and welfare under conditions of aquaculture [4,5,56] and to better understand and predict population dynamics [57,58].
Here, we had two main objectives. First, we monitored the boldness of the fish, by assessing the behavior of European sea bass over time and context testing for behavioral consistency between shy and bold fish. A group risk taking test (GRT) and open field test (OFT) were performed at 227 and 749 days post fertilization (dpf), respectively. Second, we aimed to describe the underlying physiological mechanisms associated with boldness. The blood and brains of the fish were sampled after the OFT to measure cortisol concentration in the plasma, the transcription of genes involved in stress regulation (mr, gr1, gr2, crf), neurogenesis (pcna, neurod1, neurod2), and neuronal development (egr1) and, finally, the concentration of monoamines (DA, 5HT, NE) and associated metabolites (DOPAC, 5HIAA) in whole brain.
Section snippets
Material and methods
Experiments were authorized by ethics committee agreement APAFIS#7098 and all procedures involving animals were in accordance with the ethical standards of the institution and followed the recommendations of Directive 2010/63/EU.
Behavioral consistency across context and time between the GRT and OFT
During the GRT, 63% of the fish exited the sheltered area within the 24 h of the test and were designated as bold. Consequently, 37% of the fish were designated as shy in the GRT. In the OFT, 58% of the fish exhibited bold behavior and exited the sheltered compartment vs. 42% which exhibited shy behavior and stayed in the sheltered compartment during the 20 min of the test. The proportion of fish with bold/shy behavior was similar between the GRT and OFT (χ2 = 0.51; p = .48, Fig. 2A). However,
Discussion
Here, we evaluated the boldness of European sea bass using two different challenges, which were group risk taking test (GRT) and open field test (OFT) at 227 and 749 dpf, respectively. The proportion of bold/shy fish did not differ between the two tests (approximately 60% of the fish were categorized as bold). The proportion of bold/shy fish however differed from a previous study in European sea bass in which ~80% were identified as shy using a group risk-taking test [56]. The high proportion
Acknowledgements
We are grateful to Marie-Odile Vidal, François Ruelle, and Alain Vergnet for hatching and rearing the fish. We would also like to thank Mako Pegart for valuable help with the gene transcription analyses. SA received a PhD grant from ERANET ANIHWA WINFISH (ANR-14-ANWA-0008) and Ifremer and BS received a postdoctoral grant from ERANET COFASP SUSHIFISH (ANR-15-COFA-0002). We also thank the anonymous reviewers for helpful comments on the previous version of the manuscript.
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