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The hypothalamic–pituitary–inter‐renal (HPI) axis activity is related to the stress coping styles of olive flounder, Paralichthys olivaceus. But the relationship between hypothalamic–pituitary–gonadal (HPG) axis activity and stress coping styles remains unknown. Here, we combine physiological measurements with ovarian development to examine the reproduction differences between proactive and reactive flounders. The GSI was significantly higher in the proactive fish than the reactive type during the breeding season. In April, all the proactive fish under investigation had ovaries at mid‐vitellogenic stage while reactive fish had ovaries at pre‐vitellogenic stage, but both the proactive fish and the reactive ones had ovaries at post‐vitellogenic stage in May. In HPG axis, the plasma E2 levels and the mRNA levels of fshβ, fshr and lhcgr was higher in the proactive flounder than the reactive ones in April yet there was no difference between them in May. In HPI axis, the concentration of cortisol was lower in the proactive fish than the reactive ones in April, yet the results indicate no difference in May. Surprisingly, the transcript gr in ovary displays no differences between the proactive and the reactive fish and mr shows significant up‐regulation for the proactive fish in April and the results indicated no difference again in May. Overall, these data suggested that the proactive individuals matured earlier than the reactive ones, the activity of HPI axis was lower, and the activity of HPG axis was higher in the proactive fish than the reactive ones during the breeding season. [ABSTRACT FROM AUTHOR]

Individual differences in behaviour and reactions to various environmental stress stimulus could reflect substantial differences in coping patterns among animals, including immune–endocrine responses. However, few studies have been conducted in this domain. In this study, two types of flounder (Paralichthys olivaceus) (bold individuals (BI) and shy individuals (SI)) were screened out and the expression of genes related to the immune–endocrine was in their head kidneys under temperature stress. BI and SI had the long‐term and stable differential expression of IRAF6 (tumour necrosis factor receptor‐associated factor 6), IL10 (Interleukin 10) and UII (urotensin Ⅱ) genes in the head kidney at optimum temperature (18℃). During acute hypothermal stress, the SI inflammatory gene TNFα (tumour necrosis factor‐α) was significantly downregulated, and IL6 (interleukin 6) and IL10 (interleukin10) were significantly upregulated. In acute hyperthermal stress, the SI inflammatory genes TNFα and IL6 were significantly downregulated, while IL10 continued to be significantly upregulated. Immune genes in BI showed no significant changes under acute temperature stress. We found that endocrine genes CRH (corticotropin‐releasing hormone), UI (Urotensin I) and UII showed opposite expression patterns in the head kidney of BI and SI after temperature stress. Meanwhile, gene expression of Hsp70 (heat shock protein 70) responded to hypothermal stress only in SI but not in BI, indicating that Hsp70 is involved in resistance to hypothermal stress only in SI. Through the correlation analysis of the genes discovered in this study, there is an opposite correlation between the part of endocrine–immune genes of BI and SI. It indicates differences in the regulatory mechanisms of the immune and endocrine systems in the head kidney of BI and SI under temperature stress. [ABSTRACT FROM AUTHOR]

Ectotherm animals, such as fish, are vulnerable when facing an extreme temperature fluctuation as a result of their inability to maintain body temperature. The caudal neurosecretory system (CNSS) is unique to fish and has been shown to maintain homeostasis in response to seasonal changes. However, its temperature sensitivity is unknown. Here, we used in vitro electrophysiological and anatomical approaches to investigate a thermosensory pathway in the CNSS. We showed that the CNSS responds directly to local hypothermal challenge via the TRP channel, and transmits this signal using the neurotransmitter, GABA, to the neurosecretory Dahlgren cells of the CNSS. These findings are the first demonstration of the thermal perception of the CNSS and add to our understanding of the physiological role of the CNSS in thermoregulation and seasonal adaptation. [ABSTRACT FROM AUTHOR]

Temperature is a critical environmental factor that affect most biological and physiological processes in fish. The caudal neurosecretory system (CNSS) is unique to fish and is proved to maintain homeostasis during seasonal alterations. However, the dynamic expression and secretion pattern of its major hormones, corticotrophin-releasing hormone (CRH), urotensin I (UI), and urotensin II (UII), and their response to thermal stress has not been studied. CRH, UII and cortisol in plasma, gene expression levels of CRH, UI, and UII in the CNSS of olive flounder (Paralichthys olivaceus) were therefore characterized. UI- and UII-positive Dahlgren cells, as well as cell proliferation in the CNSS, were also quantified. The results showed that plasma cortisol and CRH were increased in both low temperature (LT) and high temperature (HT) groups. However, there was no difference in plasma UI and UII during thermal stress. In CNSS, CRH, UI, and UII mRNA levels were all significantly elevated in response to acute hypothermal stress and recovered back to the control (normal) level after 8 days of adaptation. During hyperthermal challenge, gene expression of CRH and UI only significantly increased after 8-days of transfer but no change in UII was observed. We also demonstrated an increasing percent of UI-positive Dahlgren cells in the CNSS of 8-days hyperthermal stressed fish. However, no BrdU-labeled Dahlgren cells were found among the three treatment groups. Collectively, our results demonstrate that the CNSS is subjected to dynamic responses under thermal stress and expands upon the role of the CNSS in thermoregulation. The dynamic responses of hormone levels and the gene expression of CRH, UI and UII in CNSS are all involved in the process of hyper- or hypo-thermal stress and adaptation. [ABSTRACT FROM AUTHOR]

To examine how different fish coping strategies respond to salinity challenge, olive flounder (Paralichthys olivaceus) with active coping style (AC) and passive coping style (PC) were transferred from seawater (SW) to freshwater (FW) and their behavior and physiology were analyzed. Different behavioral coping strategies, in terms of escape and feeding tendencies, were confirmed in AC and PC fish without FW exposure. Differences in swimming distance between AC and PC flounder were then assessed after 1 and 2 days of FW transfer. Plasma parameters and branchial gene expression were also determined 2, 5, 8, and 14 days after transfer, with comparisons between AC and PC fish and against a SW-acclimated control group. The results showed that: (1) PC flounder exhibited a significant reduction in swimming activity, while AC flounder significantly increased locomotion 2 days after transfer. (2) The plasma osmolality and plasma ionic (Na+ and Cl−) concentration of FW-acclimated PC flounder declined in a continuous fashion over time but this contrasted against the plasma parameters of AC flounder which fluctuated below the baseline level of a SW-acclimated control group. (3) The expression of NKA-α1 and NHE-3-like mRNA in PC flounder gill increased significantly from 5 days, but the expression of these two genes in AC flounder only increased after 8 days of transfer. (4) There were no remarkable differences observed in Rhcg expressions between AC and PC flounder. This study indicates for the first time that PC flounder adopt a "freeze-passive tolerance" strategy while AC flounder adopt a "flight-active resistance" defense strategy in response to salinity challenge. [ABSTRACT FROM AUTHOR]

Urotensin I (UI), a fish corticotropin-releasing hormone (CRH) like peptide, has been found throughout vertebrate species that has great effects on adaptive physiology comprising stress-related responses, and osmotic regulation by binding with CRH type I receptor (CRHR1) and CRH type II receptor (CRHR2) in fish. Dynamic expression and regulation of UI and CRH receptors in the olive flounder ovarian follicle were studied so as to make further efforts to understand the role of UI in the development of teleost ovary. The results showed that stage-specific change in UI mRNA levels in ovarian follicles of olive flounder. UI and CRHR1 mRNA levels were higher in stage III follicles (300∼500 μm diameter) compared to stage II (90∼300 μm diameter) and IV (500∼800 μm diameter) follicles, however, the levels of CRHR2 mRNA were decreased in line with the ovarian development from stage II to stage IV. A strong signal of UI protein was observed in the follicular cells and oocyte in stage III and IV follicles by immunohistochemistry. In vitro treatment of olive flounder ovarian follicles with human chorionic gonadotropin (hCG) showed that the mRNA expression of UI increased significantly at low concentration and decreased at high concentration at 6 h, but the CRHR1 and CRHR2 mRNA did not change obviously. In addition, the results of incubation with 17α, 20β-dihydroxy-4-oregnen-3-one (DHP) show that UI and CRHR1 mRNA expression were elevated with increasing concentrations at 9 h. All above results indicated that UI and CRH receptors may have a vital effect on olive flounder ovarian development. [ABSTRACT FROM AUTHOR]

In the present study the influence of long photoperiod (LP, 16L:8D) and short photoperiod (SP, 8L:16D) on hepatic energy metabolism in the olive flounder ( Paralichthys olivaceus) was investigated. Flounders were maintained under LP or SP conditions for 2 weeks then plasmatic and hepatic parameters were assessed. At the plasmatic level, the concentration of cortisol was enhanced in flounder maintained under LP compared to SP. Alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities in plasma also increased in LP flounder. There was no significant difference in plasma glucose levels between the two experimental groups. Plasma osmotic pressure, Na and Cl levels were significantly higher in LP compared to the SP group. In liver, a significant decrease of triglycerides together with an increase in glycogen was observed in the LP group. Hepatic hsl and pepck and muscle hsl mRNA expression in LP was significantly higher in the SP group. Overall the results indicate that the LP treatment caused a mild stress response and increased hepatic energy metabolism in the flounder, which in turn could affect osmoregulation. In conclusion, it would appear that LP treatment can adversely influence hepatic energy metabolism in adult olive flounder under fasting condition. [ABSTRACT FROM AUTHOR]

The immunity of bony fish depends on the conserved components and functional complexity of immune cytokines. Herein, we used the pleiotropic cytokine IL-6 receptor as a target gene to study the defense response of PoIL-6R resist pathogens in different immune tissues of olive flounder, we found that PoIL-6R is widely expressed in these tissues studied and shows a significant immune response after stimulation with bacteria in vivo. In addition, we found a broad-spectrum immune response of PoIL-6R to bacterial and pathogen-associated molecular patterns through in vitro stimulation of olive flounder embryonic cell lines (FEC), and further revealed the function of PoIL-6R in positively regulating the natural immunity of olive flounder through activation of the NF-κB signaling pathway by overexpression of PoIL-6R in FEC. Collectively, these results demonstrate for the involvement of interleukin-6 receptors in the innate immune response of olive flounder to resist pathogens, and provides new perspectives and theoretical foundations for assessing the evolutionary mechanisms of adaptive immunity in vertebrates. [Display omitted] • PoIL-6R can participate in the novel innate immune response of flounder against pathogens. • The expression of PoIL-6R mRNA was positively correlated with the expression of IL-1β, IL-6, IFN-α and IFN-γ mRNA. • PoIL-6R can significantly activate the NF-kB signaling pathway. [ABSTRACT FROM AUTHOR]

In previous studies we employed multiple behavior assays, including propensity to feed, simulated trawl capture and escape response, to prove the presence of bold and shy personality (BP,SP) in olive flounder (Paralichthys olivaceus). However, the molecular mechanism of the different personality has not been elucidated. In this study, firstly, we found that the SP flounder had lower red blood cell count (RBC) and haemoglobin concentration (HBG) than BP flounder. Secondly, the transcriptomic profiles of the hindbrain in flounder with distinct personality were compared. A total of 144 differently expressed genes (DEGs) were identified, including 70 up-regulated and 74 down-regulated genes in SP flounder compared with BP flounder. Genes involved in hypoxia stress were detected in SP flounder, accompanied with down-regulation of ribosomal RNA synthesis. In addition, genes related with calcium signaling pathway, including endothelin, b-Fos, c-Fos and c-Jun were up-regulated in SP flounder. Furthermore, personality-related genes including UI, CCK, c-Fos showed significantly higher level in SP flounder than in BP flounder. GO enrichment analysis indicated that the GO categories "the tight junction pathway" and "lipid transport or localization pathway" were enriched in SP flounder, suggesting that the central nervous system homeostasis would be compromised. Thirdly, using a simple and scalable DNA methylation profiling method (MethylRAD), which allows for methylation analysis for DEGs in RNA-seq, we found that only part of gene expression was negatively associated with promoter methylation. Altogether, our study will not only lay a foundation for further studies on animal personality but also facilitate the selective breeding of olive flounder in aquaculture. • Flounder showed different behavioral individuality and oxygen metabolism. • Genes involved in hypoxia stressor were up-regulated in SP flounder. • SP flounder showed down-regulation pathway in ribosomal RNA synthesis. • Genes related with personality showed significantly higher level in SP flounder. [ABSTRACT FROM AUTHOR]

The pleiotropic cytokine IL -1 is involved in important immune responses such as thymocyte proliferation and B cell growth and differentiation. Activation of the IL -1 pathway requires its functional receptor IL -1RI, making IL -1RI the critical point of the IL -1 pathway. In-depth study of IL -1RI will help to understand the immune mechanism involved in IL -1. In this study, we identified the cDNA of the IL -1RI gene of olive flounder (PoIL-1RI). The total length of the PoIL-1RI cDNA is 2490 bp, the open reading frame is 1689 bp long and encodes a protein of 562 amino acids. The protein has three Ig domains and a typical TIR domain, as in other mammals and fish. We found that PoIL-1RI is widely expressed in the tissues studied and shows a significant immune response after stimulation with bacteria and pathogen-associated molecular patterns (PAMPs) both in vitro and in vivo. After PoIL-1RI was overexpressed in olive flounder embryonic cell line (FEC), pro-inflammatory cytokines (IL -1β, IL -6, IL -8, TNF-α) and interferon (IFN-α, IFN-γ) were significantly upregulated. And we found that after overexpressing PoIL-1RI in FEC, the antibacterial ability of FEC was significantly stronger than that of the control group, and we found that overexpression of PoIL-1RI gene significantly increased the activity of NF-κB signaling pathway. These results suggest that PoIL-1RI plays an important role in innate immune response. • PoIL-1RI has three Ig domains and one TIR domain. • PoIL-1RI is involved in the inflammatory response. • PoIL-1RI participates in the antibacterial effect. • PoIL-1RI can significantly activate the NF-kB signaling pathway. [ABSTRACT FROM AUTHOR]

In farmed animals, individualities have demonstrated links to health and welfare. However, differential modulation of orexigenic, anorexigenic neuroendocrine and endocrine factors in different individuals with different individualities remain unclear. Therefore, we investigated the contribution of the hypothalamic-pituitary-interrenal (HPI), anorexigenic and orexigenic signal molecules to the regulation of appetite in different individuals of adult olive flounder exposed to social isolation stress. We employed behavior assays to separate flounder into three behavioural types (proactive- 14%, intermediate- 30% and reactive- 56%). We found marked elevation in plasma corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and cortisol, and a stronger suppression of feeding post-stress in reactive than other groups. Reactive also had significantly higher CRH and urotensin (UI) mRNA levels in the preoptic area, hypothalamus, hind brain, spinal cord and caudal neurosecretory system (CNSS) than the rest of the groups. Similarly, brain anorexigenic cholecystokinin (CCK), serotonin (5-HT1A) and glucocorticoid receptor (GR) mRNA levels in the preoptic area, telencephalon, hypothalamus, hind brain and CNSS were all higher in reactive than other groups. Contrary, reactive had lower neuropeptide Y (NPY), ghrelin and prolactin mRNA levels in the preoptic area, telencephalon, hypothalamus and CNSS than other groups. There was no significant difference between proactive and intermediate in all measured parameters. Overall, the results demonstrate that social isolation stress is differentially modulated in different individuals and those CRH-related peptides, anorexigenic and orexigenic signalling molecules contribute to divergent stress response and regulation of appetite in P. olivaceus. [Display omitted] • The plasma stress hormones and gene expression are higher in reactive than intermediate and proactive groups. • The expression of anorexigenic factors in CNS are higher in reactive than intermediate and proactive groups. • The expression of orexigenic factors in CNS are lower in reactive than intermediate and proactive groups. • The appetite regulation is mediated by both stressor and individuality in fish. [ABSTRACT FROM AUTHOR]

• Adrenaline serves as an excitatory neuromodulator on Dahlgren cells. • Adrenaline promotes firing activity of Dahlgren cells through α1 and β3 adrenergic receptors. • The CNSS CRH, UI and UII mRNA were up-regulated with adrenaline. Adrenaline is one of the most important neurotransmitters in the central nervous system and is produced during stress. In this study, we investigated the modulatory role of adrenaline and adrenergic receptors on the neuroendocrine Dahlgren cells in the caudal neurosecretory system (CNSS) of olive flounder. Ex vivo electrophysiological recordings revealed that adrenaline significantly increased the firing frequency and altered the firing pattern of Dahlgren cells. Moreover, treatment with adrenaline led to a significant upregulation of ion channels and major hormone secretion genes in CNSS at the mRNA levels. Additionally, treatment with adrenaline resulted in a significantly elevation in the expression levels of α1- and β3-adrenergic receptors. Furthermore, the β3-adrenergic receptor antagonist exerts a significant inhibitory effect on adrenaline-induced enhancement firing activities of Dahlgren cells, whereas the α1-adrenergic receptor antagonist displays a comparatively weaker inhibitory effect. Additionally, the enhanced firing activity induced by adrenaline could be effectively suppressed by both α1- and β3-adrenergic receptor antagonists. Taken together, these findings provide strong evidence in favor of the excitatory effects of adrenaline through α1 and β3 adrenergic receptors in CNSS to stimulate the secretion of stress-related hormones, β3-adrenergic receptor plays a more dominant role in the modulation of firing activities of Dahlgren cells by adrenaline and thereby regulates the stress response in olive flounder. [ABSTRACT FROM AUTHOR]

Abstract Personality is defined as consistency in an individual's behavioral responses towards a given situation over time. Previous studies reported the presence of bold personality (BP) and shy personality (SP) in the olive flounder (Paralichthys olivaceus). Links between personality and the stress response have been studied in teleost fish; however, the response of SP and BP flounder to salinity stress is unknown. In this study, salinity tolerance of larvae at 25 days after hatch (DAH) and juvenile (50 DAH) flounders was investigated at salinities of 3, 5, 25, 45 and 50 parts per thousand (ppt). On 25 DAH, at salinities of 5 and 25 ppt, both SP and BP flounder keep 80% survival rate, while BP flounder larvae had higher survival rates at salinity of 45 ppt when compared with SP flounder. On 50 DAH, salinity tolerance of SP and BP flounder greatly improved, with 70% survival at salinitie of 3, 5 and 25 ppt but low survival rates (35%) at 50 ppt. At 45 ppt, BP exhibited slightly higher survival rate (80%) than SP flounder (60%) after a 96 h challenge. In addition, juvenile BP flounder (8 months) had lower plasma cortisol level, were more motivated to feed and showed more active responses during net confinement. Furthermore, Na+/K+/ATPase α1 (NKAα1) mRNA expression showed no significant difference between SP and BP flounder in SW, but was up-regulated after transfer to FW in SP and BP flounder. BP flounder expressed higher levels of gill Na+/K+/2Cl− cotransporter (NKCC1) mRNA than SP flounder in seawater (SW), while there were no differences between groups after transfer to freshwater (FW). Taken together, BP flounder had a low plasma cortisol level and an elevated appetite, as well as hyper-osmoregulatory ability. Selection for BP flounder in aquaculture is potentially important to improve animal welfare and increase production efficiency. Highlights • Personality of bold and shy is observed in olive flounder. • Flounder larvae of bold personality have higher survival rate at high salinity. • Flounder larvae of bold personality have lower cortisol levels in plasma. • Adult bold flounder show higher level expression of gill NKCC1 mRNA than shy flounder. [ABSTRACT FROM AUTHOR]

The neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) plays an essential role in the regulation of neural activity via multiple receptors. Here, we investigated the functional role of serotoninergic input on the Dahlgren cell population in the caudal neurosecretory system (CNSS) of olive flounder. In this study, the effect of 5-HT on the firing activity of Dahlgren cells was explored in terms of changes in firing frequency and firing pattern using multicellular recording electrophysiology ex vivo , and the role of several 5-HT receptor subtypes in the regulation was determined. The results revealed that 5-HT increased the firing frequency in a concentration-dependent manner and altered the firing pattern of Dahlgren cells. The effect of 5-HT on the firing activity of Dahlgren cells was mediated through the 5-HT 1A and 5-HT 2B receptors, selective agonists of both receptors effectively increased the firing frequency of Dahlgren cells, and selective receptor antagonists could also effectively inhibit the increase in firing frequency caused by 5-HT. In addition, the mRNA levels of major signaling pathway-related genes, ion channels, and major secretion hormone genes were significantly upregulated in CNSS after treatment with 5-HT. These findings demonstrate that 5-HT acts as an excitatory neuromodulator on Dahlgren cells and enhances neuroendocrine activity in CNSS. [Display omitted] • 5-HT acts as an excitatory neuromodulator on Dahlgren cells in a concentration-dependent manner. • 5-HT promotes firing activity of Dahlgren cells by 5-HT 1A and 5-HT 2B receptors. • The expression of genes related to CNSS secretions, ion channel expression, and cAMP and IP3/Ca2+ signaling were upregulated with 5-HT stimulation. [ABSTRACT FROM AUTHOR]

A neuromodulatory role for glutamate has been reported for magnocellular neuroendocrine cells in mammalian hypothalamus. We examined the potential role of glutamate as a local intercellular messenger in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) in the euryhaline flounder Paralichthys olivaceus . In pharmacological experiments in vitro, glutamate (Glu) caused an increase in electrical activity of Dahlgren cells, recruitment of previously silent cells, together with a greater proportion of cells showing phasic (irregular) activity. The glutamate substrate, glutamine (Gln), led to increased firing frequency, cell recruitment and enhanced bursting activity. The glutamate effect was not blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801, or the GluR1/GluR3 (AMPA) receptor antagonist IEm1795-2HBr, but was blocked by the broad-spectrum α-amino-3-hydroxy- 5- methyl-4-isoxazo-lepropionic acid (AMPA) receptor antagonist ZK200775. Our transcriptome sequencing study revealed three AMPA receptor (GluR1, GluR2 and GluR3) in the olive flounder CNSS. Quantitative RT-PCR revealed that GluR2 receptor mRNA expression was significant increased following dose-dependent superfusion with glutamate in the CNSS. GluR1 and GluR3 receptor mRNA expression were decreased following superfusion with glutamate. L-type Ca 2+ channel mRNA expression had a significant dose-dependent decrease following superfusion with glutamate, compared to the control. In the salinity challenge experiment, acute transfer from SW to FW, GluR2 receptor mRNA expression was significantly higher than the control at 2 h. These findings suggest that GluR2 is one of the mechanisms which can medicate glutamate action within the CNSS, enhancing electrical activity and hence secretory output. [ABSTRACT FROM AUTHOR]

Urotensin II (UII) is a kind of fish somatostatins cyclic peptide, which was originally extracted from the caudal neurosecretory system (CNSS). The system of UII and UII receptor (UIIR) has been reported to have multiple physiological regulatory functions, such as cardiovascular control, osmoregulation, and lipid metabolism. However, the effect of UII and UIIR on the ovarian development has not been covered. This study investigated the expression pattern of UII and UIIR in the ovarian follicles and explored their impact on ovarian development in olive flounder Paralichthys olivaceus. The results showed that the highest UII and UIIR mRNA levels were observed at stage II and stage III follicles during ovarian development, respectively. In situ hybridization revealed that a strong signal of UII was expressed in the oocyte nuclei of stage II follicles, however, UIIR was found in the follicle cells and oocyte cytoplasm of stage II and stage III follicles. Similarly, immunohistochemistry found positive signal of UII was detected in the oocyte nuclei of stage II follicles. The results from in vitro culture of olive flounder follicles suggested the expression of UII and UIIR mRNA levels significantly increased by 10 IU/ml human chorionic gonadotropin (hCG) for 9 h. Furthermore, the transcriptional expression of UII and UIIR was not statistically significantly changed by 17α, 20β-dihydroxy-4-pregnen-3-one (DHP). These results firstly suggested that UII and UII receptor may play vital roles in regulating ovarian growth in olive flounder. [Display omitted] [ABSTRACT FROM AUTHOR]

In animals, personalities have demonstrated links to behavioral traits, health and disease susceptibility, and welfare. These performance differences are more obvious under environmental stress. Previous studies have shown that animal personality is heritable. However, it is still unclear whether there will be differences in the performance traits of the offspring of flounders with different personalities (OFDP). In this study, we measured the weight and length of OFDP at 25 days-post-hatch (DPH) and 50 DPH. The offspring of bold individuals (OB) were heavier than the offspring of shy individuals (OS). Temperature tolerance (4 °C, 18 °C and 28 °C) and disease resistance (bacteria half lethal concentration) of larvae at 25 DPH and 50 DPH of OB and OS were investigated, and OB had a higher survival rate than OS at both 4 °C, 18 °C and Vibrio anguillarum (Va) exposure. 20 microsatellite markers related to growth rate, temperature tolerance, and disease resistance traits were used to analyze the variability of microsatellites and the possible relationship with OFDP. 16 microsatellites were correlated with the personality of flounder, and they had significant differences between OB and OS. The polymorphic microsatellite loci with the most significant correlation with growth, heat tolerance, and disease resistance were selected and sequenced. The microsatellite sequence comparison indicated that the microsatellite molecular composition of the OB was related to some better performance traits. These results add to the knowledge base available to olive flounder aquaculture and suggest that OB presents advantages for production purposes and provides valuable insights for fish personality selective breeding. • Bold flounder's offspring grow faster and have stronger environmental adaptability. • The microsatellites linked to the performance traits are related to personality. • The microsatellite composition of the bold flounder's offspring is more advantageous. • Parental personality can influence offspring performance traits in olive flounder. [ABSTRACT FROM AUTHOR]

In farmed animals, individuality have demonstrated links to performance traits, health and disease susceptibility, and animal welfare. This research aims at exploring whether there are differences in the secretion of hormones in the caudal neurosecretory system of different individuailty, and investigating whether it is related to physiological behaviors. In the experiment we selected through multiple behavioral tests two types of olive flounders, bold individuals (BI) and shy individuals (SI), and found that they differed in behavior and physiology. The standard metabolic rate, maximum metabolic rate, and absolute aerobic scope of BI were markedly higher than those of SI. Additionally, the swimming speed of BI was also higher than that of SI in the natural photoperiod. BI and SI showed distinct coping styles to deal with acute stress. Overall, the number of Dahlgren cells secreting UI, the relative UI and CRH mRNA expression in the caudal neurosecretory system (CNSS) of SI was relatively higher than that in BI. By contrast, the number of Dahlgren cells secreting UII and the mRNA expression of UII was lower than that of BI. Through the correlation analysis, it was found that there were some differences in hormone secretion among different individuality groups, which indicated individuality affected hormone production and the number of secretion cells and existed correlation with respiratory metabolism, spontaneous behavior, appetite. It means differences in the regulation mechanism of the flounders in BI and SI. • Bold individuals have higher aerobic capacity, spontaneous activity and appetite than shy individuals. • CRH, UI or UII mRNA expression in CNSS are negatively or positively correlated with metabolism, activity and appetite. • Individuality influences hormone secretion in CNSS. [ABSTRACT FROM AUTHOR]

In farmed animals, personalities have demonstrated links to performance traits, health and welfare, disease susceptibility, and to coping with environmental stress. The teleost caudal neurosecretory system (CNSS) of the posterior spinal cord is involved in the regulation of the osmoregulatory and stress responses and can directly sense a hypothermal challenge via a transient receptor potential ion channel. Here we investigated the global transcriptomic response of the CNSS of shy and bold individuals of the olive flounder (Paralichthys olivaceus) acclimated to 18 °C and transferred to 12 °C (low temperature treatment, LT) or 24 °C (high temperature treatment, HT) for 2 h (acute) or 8 days (chronic) in relation to fish maintained at 18 °C (control). Overall, differential transcriptomic responses were stronger in bold individuals and induced by acute HT and chronic LT treatments. Acute HT induced innate immunity, circadian rhythm, and cellular stress response pathways and, specifically in bold individuals, a heightened response of molecular chaperones of cellular response to heat which may be related to the mechanisms of rescue of downregulated RNA splicing processes. Chronic LT also caused downregulation of innate immunity pathways as well as pathways related to cell division. In addition, the CNSS of bold individuals was enriched in pathways related to regulation of cell cycle, adaptive immune response, and apoptosis, while cellular metabolism pathways were downregulated in shy individuals. The putative temperature sensor genes Trpv1 and Trpa1 were upregulated, respectively, after acute HT and chronic LT treatments, but there was no indication of a specific neurosecretory response of the CNSS. While several of the observed transcriptomic responses to temperature appear to be shared by other tissues and species, it is also clear that bold and shy behavioural personalities show marked different responses, with bold individuals the most responsive. These results add to the knowledge base available on olive flounder aquaculture, indicating a differential role of the CNSS in the response to temperature change according to behavioural personalities. [Display omitted] • Acute high and chronic low temperature greatly modified CNSS gene expression • Acute high temperature induced innate immunity, cellular stress response pathways • Chronic low temperature downregulated innate immunity and cell division pathways • Temperature sensor genes were induced by acute high and chronic low temperature • The CNSS gene expression response was greater in bold compared to shy individuals [ABSTRACT FROM AUTHOR]

Urotensin I (UI), a member of the corticotropin-releasing hormone family of peptides, regulates a diverse array of physiological functions, including appetite regulation, defensive behavior and stress response. In this study, firstly, the tissue-specific distribution of UI mRNA in olive flounder (Paralichthys olivaceus) was characterized and we found that UI mRNA was highly expressed in caudal neurosecretory system (CNSS) tissue. Secondly, alignment analysis found that a conserved cAMP response binding (CREB) site and a TATA element were located in the proximal promoter of UI gene. In addition, treatment of forskolin activatated cAMP-CREB pathway and induced the up-regulation of UI mRNA in cultured CNSS, suggesting the role of CREB in regulating the UI mRNA expression. Furthermore, plasma UI concentration and UI mRNA in CNSS showed obvious daily rhythm, with higher values in the daytime while lower values in the nighttime. Thirdly, using bold personality (BP) and shy personality (SP) flounder as an animal model, we found that flounder exhibited significantly higher locomotor activity in the nighttime than in the daytime (P < 0.001), and BP flounder showed significantly higher locomotor activity (P < 0.001) compared with SP flounder both in the daytime and nighttime. Analysis of feeding behavior revealed that BP flounder showed a shorter latency to feed and more attacks to prey. Furthermore, the qPCR and immunohistochemistry results showed that BP flounder expressed significantly lower level of UI mRNA and protein in CNSS tissue. Collectively, our study suggested that the UI plays an important role in locomotor activity and appetite regulation, which provides a basis for understanding the mechanism of defensive behavior and animal personality in flounder. [Display omitted] • The urotensin I (UI) mRNA was highly expressed in CNSS tissue of olive flounder. • The plasmatic UI concentration and UI mRNA in CNSS showed obvious daily rhythm. • Flounder with bold personality exhibited higher locomotor activity and more feeding behavior compared with shy flounder. [ABSTRACT FROM AUTHOR]

[Display omitted] • GABA caused a decrease in electrical activity of Dahlgren cells. • GABA modulates Dahlgren cell activity via GABA A receptors. • GABA A receptor mRNA increased after stimulation with GABA. γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. We investigated its potential role as a neurotransmitter in the neuroendocrine Dahlgren cell population of the caudal neurosecretory system (CNSS) of the flounder Paralichthys olivaceus. The application of GABA in vitro resulted in a decrease in electrical activity of Dahlgren cells, followed by an increase of the number of silent cells, together with a decreased firing frequency of all three activity patterns (tonic, phasic, bursting). GABA A receptor agonist etomidate decreased Dahlgren cell firing activity, in a similar way to GABA. The response to GABA was blocked by the GABA A receptor antagonist bicuculline. GABA A receptor gamma2 subunit (Gabrg2) and chloride channel (Clcn2) mRNA expression were significantly upregulated in the CNSS after GABA superfusion. These data suggest that GABA may modulate CNSS activity in vivo mediated by GABA A receptors. [ABSTRACT FROM AUTHOR]

Animal individuality is proposed to describe individual differences in behavior. Individuals may exhibit consistent differences in behavioral traits within a population, and these traits are heritable. It has also been known that animal individuality can be attributed to both genetic and environmental effects. We previously observed the presence of bold individuals (BI) and shy individuals (SI) in olive flounder (Paralichthys olivaceus). However, the individuality distribution and the behavioral phenotype of offspring with different individualities remain unclear. In this study, the temperature and acidity challenges of larvae at 25 days-post-hatch (DPH) and juvenile (50 DPH) of bold and shy flounder offspring were investigated at pH = 7.3, 7.7, 8.1 and temperature at 4, 18, 28 °C. On 25 DPH, bold offspring (BO) showed higher survival rates with acidity at pH = 7.3 and 4 °C challenge. Three behavioral phenotypes (bold, middle and shy individuals, BI, MI and SI) were observed in both juvenile (8 MPH) bold and shy flounder offspring. The percentage of SI offspring from the shy maternal line (SS) was higher than BI offspring from the bold maternal line (BB) (0.75 vs. 0.58). BI from both BO (BB) and SO (SB) were more motivated to feed and active during net confinement than SI (BS and SS). Furthermore, BB and SB exhibited higher metabolic rates and actively responded to predator stressor, while BS and SS showed a divergent tendency. However, the metabolism of the offspring seemed to be partially affected by their maternal line. Taken together, our study indicates that screening of animal individuality in population would improve animal welfare and increase the output efficiency of flounder in aquaculture. • Bold offspring larvae had stronger environmental adaptability than shy ones during early development. • Offspring from shy maternal lines had higher heritability than bold ones. • Bold individuals from both bold and shy offspring exhibited higher metabolic rates, and actively responded to predator stress. • Physiological traits of offspring were partially affected by their maternal lines. [ABSTRACT FROM AUTHOR]

Corticotropin-releasing hormone (CRH) has been implicated in multiple physiological processes, such as circadian rhythms, food intake and anxiety-like behavior. In telelost fishes, CRH is predominantly expressed in the caudal neurosecretory system (CNSS), a much low level in hypothalamus and gonads, while undetectable levels in other tissues. However, the mechanisms governing this tissue-specific expression remain unknown. In this study, firstly, we investigated the expression pattern of CRH mRNA in different tissues of olive flounder (Paralichthys olivaceus). Secondly, we found that flounder exhibited low locomotor activity in the daytime, concomitant with the highest CRH mRNA expression in CNSS at noon. Thirdly, we examined whether epigenetic mechanisms through DNA methylation are involved in tissue-specific expression of CRH mRNA. Promoter methylation of the CRH gene was assessed through bisulphate sequencing methods. In the proximal promoter, almost no methylation was detected in the muscle, hypothamus and CNSS. However, different methylation was detected in the distal promoter of CRH. The methylation was 63% in CNSS, while that in hypothalamus and muscle was 85% and 96%, respectively. Lastly, bioinformatics analysis revealed that a conserved AP-1 binding site (5-TCACTGA-3) was located in the proximal promoter of CRH gene. In vivo experiment, lipopolysaccharide (LPS) intraperitoneally injection in the flounder up-regulated c-Fos and CRH mRNA in CNSS (P < 0.01). In CNSS tissue culture experiment, forskolin treatment significantly induced the expression of c-Fos, c-Jun and CRH mRNA (P < 0.01). Collectively, our data provide the evidence that distal promoter methylation and c-Fos signal pathway are involved in transcriptional regulation of CRH expression in flounder. Unlabelled Image • Flounder exhibited lower locomotor activity in the daytime than at night, and showed the daily rhythm of CRH mRNA in CNSS. • The distal promoter of CRH gene in the muscle, hypothalamus and CNSS exhibited different patterns of DNA methylation. • The stimulation of c-Fos gene by LPS or forskolin induced up-regulation of CRH mRNA expression in CNSS. [ABSTRACT FROM AUTHOR]

• Taurine caused a decrease in electrical activity of Dahlgren cells. • Both the antagonist of GlyR and GABA A R can block the effect of taurine on Dahlgren cells. • GlyR and GABA A R mRNA increased after stimulation with taurine. • Taurine modulates Dahlgren cell activity via GlyR and GABA A R. Taurine plays role in neural development and physiological functions such as endocrine regulation in the central nervous system (CNS), and it is one of the most abundant free amino acid there. We investigated its potential effect as a neurotransmitter in the group of neuroendocrine Dahlgren cells at flounder Paralichthys olivaceus caudal neurosecretory system (CNSS). The application of taurine in vitro led to a reduction in electrical activity of Dahlgren cells, followed by a rise in the number of silent cells, at the same time the frequency of all three activity patterns (tonic, phasic, bursting) in Dahlgren cells was reduced. Both strychnine (a glycine receptor antagonist) and bicuculline (a GABA A receptor antagonist) can block the response to taurine separately. Transcriptome sequencing analysis showed the existence of glycine receptor (GlyR) and GABA A receptor (GABA A R) in the flounder CNSS, and the GlyR, GABA A R, and Cl- channel mRNA expression were significantly raised after taurine superfusion according to quantitative RT-PCR results. These data indicate that taurine may mediate Dahlgren cell population of CNSS activity in vivo through GlyR and GABA A R, thereby, regulating stress-response. [ABSTRACT FROM AUTHOR]