Hypothalamic-pituitary-interrenal Research Articles

The role of phenotypic plasticity and corticosterone in coping with pond drying conditions in yellow-bellied toad (Bombina variegata, Linnaeus 1758) tadpoles.

Amphibian larvae inhabiting temporary ponds often exhibit the capacity to accelerate development and undergo metamorphosis in challenging conditions like desiccation. However, not all species exhibit this ability, the yellow-bellied toad (Bombina variegata) is one such example. The underlying mechanisms behind the inability to accelerate development under desiccation remain largely unexplored. The hypothalamic-pituitary-interrenal (HPI) axis and corticosterone (CORT), which act synergistically with thyroid hormone, are thought to facilitate metamorphosis in response to desiccation stress. In this study, we aimed to investigate whether modification in the HPI axis, particularly CORT levels, contributes to the absence of adaptive plasticity in B. variegata under desiccation stress. The study design included four treatments: high water level, high water level with exogenous CORT, low water level, and low water level with metyrapone (a CORT synthesis inhibitor). The main objective was to evaluate the effects of these treatments on whole-body corticosterone levels, life history, morphological traits, and oxidative stress parameters during the prometamorphic and metamorphic climax developmental stages. While low water level had no effect on total corticosterone levels, larval period, body condition index, and metamorphic body shape, it negatively affected metamorph size, mass, and growth rate. Our findings suggest that constant exposure to desiccation stress over generations may have led to modifications in the HPI axis activity in B. variegata, resulting in adaptation to changes in water level, evident through the absence of stress response. Consequently, CORT may not be a relevant stress indicator in desiccation conditions for this species.

Structural Characterization and Antidepressant-like Effects of Polygonum sibiricum Polysaccharides on Regulating Microglial Polarization in Chronic Unpredictable Mild Stress-Induced Zebrafish.

Polysaccharides are one of the main active ingredients of Polygonum sibiricum (PS), which is a food and medicine homolog used throughout Chinese history. The antidepressant-like effects of PSP and its underlying mechanisms remain elusive, especially the regulation of microglial polarization. The current study determined the chemical composition and structural characteristics of PSP. Then, the chronic unpredictable mild stress (CUMS) procedure was carried out on the zebrafish for 5 weeks, and PSP was immersed for 9 days (1 h/d). The body weight of zebrafish was monitored, and behavioral tests, including the novel tank test and light and dark tank test, were performed to evaluate the antidepressant-like effects of PSP. Then, the function of the hypothalamic-pituitary-interrenal (HPI) axis, the levels of peripheral inflammation, neuronal and blood-brain barrier damage in the mesencephalon and telencephalon, and the mRNA expression of M1/M2 phenotype genes in the brain were examined. PSP samples had the typical structural characteristics of polysaccharides, consisting of glucose, mannose, and galactose, with an average Mw of 20.48 kDa, which presented porous and agglomerated morphologies. Compared with untreated zebrafish, the depression-like behaviors of CUMS-induced zebrafish were significantly attenuated. PSP significantly decreased the levels of cortisol and pro-inflammatory cytokines and increased the levels of the anti-inflammatory cytokines in the body of CUMS-induced depressive zebrafish. Furthermore, PSP remarkably reversed the neuronal and blood-brain barrier damage in the mesencephalon and telencephalon and the mRNA expression of M1/M2 phenotype genes in the brain. These findings indicated that the antidepressant-like effects of PSP were related to altering the HPI axis hyperactivation, suppressing peripheral inflammation, inhibiting neuroinflammation induced by microglia hyperactivation, and modulating microglial M1/M2 polarization. The current study provides the foundations for future examinations of PSP in the functional foods of emotional regulation.

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

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

Cortisol Quantification for Assessing Stress-Induced Changes in Zebrafish Larvae.

The stress response, mainly mediated by cortisol, plays a critical role in the regulation of physiological and behavioral homeostasis through a variety of mechanisms. Different aquatic animal models have been widely employed to understand the pathobiology of stress and stress-related brain disorders. The early life stress can affect the hypothalamic-pituitary-interrenal (HPI) axis and induce cellular and molecular impairments that impact the brain functioning later in life. However, these alterations have been poorly explored mainly due to the lack of suitable models. In this chapter, the vortex flow stimulation, an acute stress that causes a forced swimming and activates the HPI axis, is described and its correlations with behavioral outputs reported. To this end, the early life stages of zebrafish are used as animal models for modeling stress disorders experimentally. The behavioral despair model can be employed as an initial screening tool for assessing neural circuit activation and motor alterations. Taken together, the implementation of this strategy in this animal model allows the analysis of stress responses in a simple manner and its correlation with neural circuitries and motor alterations.

Exploring transcriptional and post-transcriptional epigenetic regulation of crf and 11βhsd2 in rainbow trout brain during chronic social stress

Using dominance hierarchies in juvenile rainbow trout (Oncorhynchus mykiss) as a model of chronic social stress in fish, we explored whether epigenetic transcriptional and post-transcriptional mechanisms are involved in the gene expression of corticotropin-releasing factor (crf) and 11β-hydroxysteroid dehydrogenase (11βhsd2), key factors involved in the regulation of the endocrine stress axis response. In juvenile rainbow trout pairs, subordinate individuals display sustained elevation of circulating cortisol concentrations. Cortisol production is controlled by the hypothalamic-pituitary-interrenal (HPI) axis in fish and initiated by CRF release from the preoptic area (POA). Given that crf is modulated during chronic social stress, and that such stress has been implicated in the epigenetic regulation of crf in other taxa, we probed a role for epigenetic regulation of crf transcript abundance in chronically stressed rainbow trout. We also investigated the regulation of the cortisol-metabolising enzyme 11βhsd2 in the POA, which is upregulated in subordinates. The potential involvement of DNA methylation and microRNAs (miRNAs) in the regulation of crf transcript abundance was investigated during social stress in the POA of fish, as was the potential involvement of miRNAs in 11βhsd2 regulation. Although transcript abundances of crf were elevated in subordinate fish after 4 days, DNA methylation profiles within putative promoter sequences upstream of the crf gene were not significantly affected by chronic stress. An inverse relationship between crf and its predicted posttranscriptional regulator miR-103a-3p in the POA suggests that miRNAs may be involved in mediating the effects of chronic social stress on key components of the endocrine stress axis.

Temperature- and genotype-dependent stress response and activation of the hypothalamus-pituitary-interrenal axis during temperature-induced sex reversal in pejerrey Odontesthes bonariensis, a species with genotypic and environmental sex determination

In the pejerrey Odontesthes bonariensis (Atheriniformes, Atherinopsidae), exposure to high and low temperatures during the critical period of sex determination (CPSD) induce testicular and ovarian differentiation, respectively, regardless of the presence or not of the sex determining gene amhy, which is crucial for testis formation only at intermediate, sexually neutral temperatures. In this study we explored the existence of genotype-specific signaling of Crh (Corticotropin Releasing Hormone) family genes and their associated carrier protein, receptors, and other stress-related genes in response to temperature during the CPSD and the potential involvement of the central nervous system via the hypothalamus-pituitary-interrenal (HPI) axis in the sex determination of this species. The Crh family genes crhb, uts1, ucn3, the receptor crhr1 and the stress-related genes gr1, gr2, nr3c2 were transiently upregulated in the heads of pejerrey larvae during the CPSD by high temperature alone or in combination with other factors. Only crhr2 transcript abundance was not influenced by temperature but independently by time and genotype. In most cases, mRNA abundance was higher in the XX heads compared to that of XY individuals. The mRNAs of some of these genes were localized in the hypothalamus of pejerrey larvae during the CPSD. XX larvae also showed higher whole-body cortisol titers than the XY, downregulation of cyp19a1a and upregulation of the testis-related genes amhy/amha in trunks (gonads) and were 100% masculinized at the high temperature. In contrast, at the low temperature, crhbp and avt were upregulated in the heads, particularly the former in XY larvae. cyp19a1a and amhy/amha were up- and downregulated, respectively, in the gonads, and fish were 100% feminized. Signaling via the HPI axis was observed simultaneously with the first molecular signs of ongoing sex determination/differentiation in the gonads. Overall, the results strongly suggest a temperature-dependent, genotype-specific regulatory action of the brain involving the Crh family of stress-related genes on the process of environmental sex determination of pejerrey.

Probiotic Lactobacillus rhamnosus alleviates the neurotoxicity of microcystin-LR in zebrafish (Danio rerio) through the gut-brain axis

Microcystin-LR (MCLR) is one of the most toxic cyanobacterial toxins and is harmful to the central nervous system of fish. Probiotic additives can improve neuroendocrine function in fish. Although both MCLR and probiotics aim at the nervous system, whether they interact with each other and the mechanisms remain unexplored. In the present study, 4-month-old zebrafish were exposed to 0, 2.2, and 22 μg/L of MCLR for 28 days with or without the probiotic L. rhamnosus. We found that MCLR exposure could inhibit the swimming speed of zebrafish, while the presence of L. rhamnosus mitigated this abnormality. To elucidate the mechanism of how L. rhamnosus alleviates MCLR-induced neurotoxicity, we examined the bioaccumulation of MCLR, changes in neurotransmitters, immune biochemical indicators, and hormone content of the hypothalamic-pituitary-interrenal (HPI) axis in zebrafish along the gut-brain axis. Our results showed L. rhamnosus could reverse the abnormal swimming behavior and eventually alleviate neurotoxicity in zebrafish by modulating intestinal and brain neural signaling, neuroinflammation, and HPI axis responses. This study provides implications for the application of probiotics in the aquaculture industry.

Molecular mechanisms of the stress-induced regulation of the inflammatory response in fish

Stressors in the environment of aquatic organisms can profoundly affect their immune system. The stress response in fish involves the activation of the hypothalamus-pituitary-interrenal (HPI) axis, leading to the release of several stress hormones, among them glucocorticoids, such as cortisol, which bind and activate corticosteroid receptors, namely the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). These receptors are highly expressed on immune cells, thereby allowing stress to have a potent effect that is classically considered to suppress immune function. In this review, we highlight the conserved structure and function of GR and MR among vertebrates and describe their role in modulating inflammation by regulating the expression of pro-inflammatory and anti-inflammatory genes. In particular, the involvement of MR during inflammation is reviewed, which in many studies has been shown to be immune-enhancing. In recent years, the use of zebrafish as a model organism has opened up new possibilities to study the effects of stress on inflammation, making it possible to investigate knockout lines for MR and/or GR, in combination with transgenic models with fluorescently labeled leukocyte subpopulations that enable the visualization and manipulation of these immune cells. The potential roles of other hormones of the HPI axis, such as corticotrophin-releasing hormone (Crh) and adrenocorticotropic hormone (Acth), in immune modulation are also discussed. Overall, this review highlights the need for further research to elucidate the specific roles of GR, MR and other stress hormones in regulating immune function in fish. Understanding these mechanisms will contribute to improving fish health and advancing our knowledge of stress signalling.

Transgenerational effects of microplastics on Nrf2 signaling, GH/IGF, and HPI axis in marine medaka Oryzias melastigma under different salinities

Little information is available on the toxicity of microplastics (MPs) under different salinities in aquatic organisms. Consequently, the effects of larvae exposure to 180 μg/L MPs with 5.0 μm diameter on growth, antioxidant capacity and stress response were investigated in exposed F1 larvae and unexposed F2 larvae in marine medaka Oryzias melastigma at 5 ‰ and 25 ‰ salinities. Poor growth performance of F1 and F2 larvae and F1 adult fish was merely found under high salinity, as well as changes in the growth hormone/insulin-like growth factor-I (GH/IGF). Although malondialdehyde (MDA) content and antioxidant capacity remained constant in F1 larvae under high salinity, MPs increased MDA content and reduced antioxidant capacity in F2 larvae. Contrarily, MDA and antioxidant capacity increased in F1 and F2 larvae under low salinity. The mRNA expression levels of genes in the NF-E2-related factor 2 (Nrf2) pathway were dysregulated. Cortisol levels in the whole body increased in F1 larvae and recovered to the control level under low salinity while cortisol levels declined in F1 larvae and increased in F2 larvae under high salinity, which was related to the transcriptional regulation of the hypothalamus–pituitary–interrenal (HPI) axis genes. To summary, the present study determined the toxic effects of MPs on growth, antioxidant capacity, and stress response by disturbing Nrf2, HPI, and GH/IGF signaling in exposed larvae and unexposed offspring of marine medaka in a salinity-dependent manner. For the first time, our results highlight the interference effects of salinity on MPs toxicity in fish.

Combined effects of crowding stress and low salinity on GH/IGF axis, antioxidant response, and HPI axis in largemouth bass (Micropterus salmoides) larvae

The present study aimed to determine if low salinity could mitigate negative effects of crowding stress on freshwater largemouth bass (Micropterus salmoides). To the end, larvae were subjected to crowding stress (CS) or low salinity (LS, 0.5‰), alone or in combination for 28 days. Growth, antioxidant capacity and stress response were investigated. CS significantly reduced body weight, total antioxidant capacity (TAC) and catalase (CAT) activity in the brain and head kidney, TAC and superoxide dismutase (SOD) activity in the liver, and increased cortisol levels in the whole body, suggesting negative effects of CS on fish. Contrarily, LS promoted body weight, body length and condition factor, and reduced MDA contents in the liver and head kidney and whole-body cortisol levels. Increases in TAC and CAT activity in the brain, SOD and CAT activities in the liver, and TAC, SOD and CAT activities in the head kidney were also observed in the larvae subjected to LS. The results indicated that LS promoted growth and antioxidant capacity and reduced stress response, which was also observed in fish subjected to CS. In order to elucidate the related molecular mechanisms, gene expression in the growth hormone/insulin-like growth factor-I (GH/IGF) axis, NF-E2-related factor 2 (Nrf2) pathway, and hypothalamus–pituitary–interrenal (HPI) axis were examined. We found that CS + LS up-regulated growth hormone release hormone gene (ghrh), growth hormone gene (gh), and hormone receptor gene (ghra) in the brain and ghra and insulin-like growth factor genes (igf1 and igf2b) in the liver compared with CS alone. The mRNA levels of Nrf2 pathway related genes were sharply enhanced by CS but were maintained relative constant by CS + LS in the brain and liver compared with the control. In the head kidney, CS + LS up-regulated Nrf2 pathway related genes compared with CS alone. HPI axis related genes tended to be up-regulated by CS but remain unchanged by CS + LS compared with the control. LS and CS + LS down-regulated both genes encoding cytochrome P450 enzymes (cyp17a1 and cyp11c1) which are rate-limiting enzymes for cortisol synthesis compare with the control and CS, respectively. Taken together, the present results for the first time provide a feasible suggestion for culture of largemouth bass larvae at high stocking density in saline waters.

LED light spectra differently affected growth, antioxidant capacity, stress response, GH/IGF and HPI axis in largemouth bass (Micropterus salmoides) larvae

This study aimed to investigate the effects of white (control), blue, green and red light-emitting diodes (LEDs) spectra on the growth, antioxidant and stress response, and mRNA levels in the growth hormone/insulin-like growth factor-I (GH/IGF) axis, NF-E2-related factor 2 (Nrf2) pathway and hypothalamus–pituitary–interrenal (HPI) axis in largemouth bass (Micropterus salmoides) larvae. The blue and green LEDs significantly increased body weight and condition factor, which may be explained by increased mRNA expression of GH/IGF axis genes that encode insulin-like growth factor, growth hormone, hormone release hormone, and growth hormone receptors. The promoted antioxidant capacity was observed in the brain of fish subjected to blue LEDs and liver of fish subjected to blue and green LEDs, which may be associated with increased expression of nrf2 and its downstream genes. Both blue and green LEDs did not significantly affect cortisol levels but down-regulated several HPI axis genes. Unlike blue and green LEDs, red LED increased malondialdehyde (MDA) levels, reduced the activities of superoxide dismutase (SOD) and catalase (CAT), and total antioxidant capacity (TAC), and down-regulated Nrf2 signaling in the head kidney. Cortisol levels increased, and HPI axis was down-regulated in fish exposed to red LEDs. Taken together, blue and green LEDs had beneficial effects on growth, antioxidant capacity, and stress response via physiological and transcriptional regulation of GH/IGF axis, Nrf2 and HPI axis signaling, which may help to improve the welfare of largemouth bass in indoor recirculating water aquaculture facilities.

Micro-polystyrene plastic and benzo[α]pyrene exposure affects the endocrine system and causes physiological stress in Carassius auratus

Microplastics, owing to their hydrophobic properties and the various chemicals used in their production, can act as carriers of persistent organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs). In this study, we exposed the goldfish Carassius auratus to benzo[α]pyrene (BaP, 10 μg/L), a representative PAH, and micro-polystyrene plastic (MP; 10 and 100 beads/L), of size 1.0 μm, as a single or complex environmental stressor, and evaluated the stress response and the resulting DNA damage. The expression of CRH and ACTH mRNA in the pituitary gland and hypothalamus, of the hypothalamus–pituitary–interrenal (HPI) axis, increased significantly after 6 h of exposure. Plasma cortisol levels showed a similar trend to the expression of stress-regulating genes along the HPI axis, and a significant increase was observed in the combined exposure groups (BaP + LMP [low-concentration MP] and BaP + HMP [high-concentration MP]) compared to those in the single exposure group. H2O2 concentration and CYP1A1 and MT mRNA expression levels in the liver were significantly higher in the combined exposure groups compared with in the single exposure groups. In situ hybridization revealed a similar pattern of MT mRNA expression, and many signals were observed in the BaP + HMP group. Furthermore, the BaP + HMP group showed more DNA damage, and the degree of DNA damage increased with exposure time for all experimental groups, except for the control group. Therefore, exposure to BaP and MP alone can induce stress in goldfish; however, when a combination of both substances is provided, their synergistic effect leads to increased stress and DNA damage. MP was confirmed to be a more serious stress-inducing factor in goldfish than BaP, based on the expression levels of stress-regulating genes along the HPI axis.

Early life stage exposure to cadmium and zinc within hour affected GH/IGF axis, Nrf2 signaling and HPI axis in unexposed offspring of marine medaka Oryzias melastigma

Information on transgenerational effects of cadmium (Cd) and zinc (Zn) within hour of exposure is scarce. To the end, larvae of marine medaka Oryzias melastigma at 0 day-post-hatching (dph) were subjected to LC50 for 96-h of Cd or Zn for 0.5 and 6 h, and then transferred into clear water for 95 days until the generation of offspring larvae at 25 dph. Growth, antioxidant capacity and stress response in offspring larvae were examined. Exposure to Zn for 0.5 h or Cd for 0.5 h and 6 h promoted growth performance and reduced total antioxidant capacity (TAC) and activities of superoxide dismutase (SOD) and catalase (CAT). Malondialdehyde (MDA) and cortisol levels declined in larvae following Zn exposure for 0.5 h, whereas Cd exposure increased MDA content and did not affect cortisol levels. These physiological changes could be partially explained by transcription of genes in the hormone/insulin-like growth factor-I (GH/IGF) axis, NF-E2-related factor 2 (Nrf2) signaling, and hypothalamus–pituitary–interrenal (HPI) axis. For example, Zn exposure for 0.5 h up-regulated genes encoding growth hormone (gh) and insulin-like growth factor binding protein (igfbp1) and down-regulated mRNA levels of nrf2, Kelch-like-ECH-associated protein 1 gene (keap1a), keap1b, sod1, mineralocorticoid receptor (mr), corticotropin-releasing hormone receptor (crhr1), corticotropin-releasing hormone binding protein (crhbp), cytochrome P450 (cyp11a1, cyp17a1) and hydroxysteroid dehydrogenase (hsd3b1). Cd exposure for 0.5 and 6 h up-regulated growth hormone release hormone (ghrh) and igfbp1, down-regulated nrf2 and keap1a, and did not affect mRNA levels of HPI axis genes. Taken together, this study demonstrated that short-term metal exposure during larvae phase had positive and negative effects on offspring even after a long recovery.

Changes in cortisol and corticosteroid receptors during dynamic salinity challenges in Mozambique tilapia

In estuarine environments, euryhaline fish maintain a narrow range of internal osmolality despite daily changes in environmental salinity that can range from fresh water (FW) to seawater (SW). The capacity of euryhaline fish to maintain homeostasis in a range of environmental salinities is primarily facilitated by the neuroendocrine system. One such system, the hypothalamic-pituitary-interrenal (HPI) axis, culminates in the release of corticosteroids such as cortisol into circulation. Cortisol functions as both a mineralocorticoid and glucocorticoid in fish because of its roles in osmoregulation and metabolism, respectively. The gill, a key site for osmoregulation, and the liver, the primary storage site for glucose, are known targets of cortisol’s actions during salinity stress. While cortisol facilitates acclimation to SW environments, less is known on its role during FW adaptation. In this study, we characterized the responses of plasma cortisol, mRNA expression of pituitary pro-opiomelanocortin (pomc), and mRNA expression of liver and gill corticosteroid receptors (gr1, gr2, and mr) in the euryhaline Mozambique tilapia (Oreochromis mossambicus) under salinity challenges. Specifically, tilapia were subjected to salinity transfer regimes from steady-state FW to SW, SW to FW (experiment 1) or steady state FW or SW to tidal regimen (TR, experiment 2). In experiment 1, fish were sampled at 0 h, 6 h, 1, 2, and 7 d post transfer; while in experiment 2, fish were sampled at day 0 and day 15. We found a rise in pituitary pomc expression and plasma cortisol following transfer to SW while branchial corticosteroid receptors were immediately downregulated after transfer to FW. Moreover, branchial expression of corticosteroid receptors changed with each salinity phase of the TR, suggesting rapid environmental modulation of corticosteorid action. Together, these results support the role of the HPI-axis in promoting salinity acclimation, including in dynamically-changing environments.

Negative feedback regulation in the hypothalamic-pituitary-interrenal axis of rainbow trout subjected to chronic social stress

The formation of dominance hierarchies in pairs of juvenile rainbow trout (Oncorhynchus mykiss) results in subordinate individuals exhibiting chronically elevated plasma cortisol concentrations. Cortisol levels reflect a balance between cortisol production, which is coordinated by the hypothalamic-pituitary-interrenal (HPI) axis in teleost fish, and negative feedback regulation and hormone clearance, which act to lower cortisol levels. However, the mechanisms contributing to the longer-term elevation of cortisol levels during chronic stress are not well established in fishes. The current study aimed to determine how subordinate fish maintain elevated cortisol levels, by testing the prediction that negative feedback and clearance mechanisms are impaired by chronic social stress. Plasma cortisol clearance was unchanged by social stress based on a cortisol challenge trial, hepatic abundance of the cortisol-inactivating enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11βHSD2), and tissue fate of labelled cortisol. The capacity for negative feedback regulation in terms of transcript and protein abundances of corticosteroid receptors in the preoptic area (POA) and pituitary appeared stable. However, changes in 11βHSD2 and mineralocorticoid receptor (MR) expression suggest subtle regulatory changes in the pituitary that may alter negative feedback. The chronic cortisol elevation observed during social subordination likely is driven by HPI axis activation and compounded by dysregulated negative feedback.

Acetic acid-induced pain elicits stress-, and camouflage-related responses in zebrafish: Modulatory effects of opioidergic drugs on neurobehavioral phenotypes

While pain results from the activation of nociceptors following noxious stimuli, mounting evidence links pain- and stress-related responses in mammals. In zebrafish, the activation of hypothalamic-pituitary-interrenal (HPI) axis may also regulate body pigmentation (the camouflage response). Here, we aimed to investigate a putative relationship between pain-, stress-, and camouflage-related parameters in adult zebrafish. To answer this question, we assessed whether intraperitoneal acetic acid injection can activate the HPI axis, measuring whole-body cortisol and the camouflage response as physiological endpoints in the presence or absence of morphine or naloxone, an opioid antagonist. Acetic acid induced a stereotypic circling behavior in the top of the tank, accompanied by abdominal writhing-like response, a specific phenotype that reflects local nociceptive effect. Both whole-body cortisol levels and camouflage response increased in the acetic acid group, while morphine prevented these responses, and naloxone antagonized morphine-induced effects. Moreover, we observed positive correlations between representative behavioral, physiological and skin coloration endpoints, and a “pain index” was proposed to summarize phenotypic profile of zebrafish under different pharmacological manipulations. Collectively, these findings suggest a coordinated activation of pain, camouflage- and stress-related pathways following acetic acid injection in zebrafish. Our data also support that camouflage response represents a novel and relevant biomarker for future probing pain and stress neurobiology, with a robust sensitivity to opioidergic drugs.

The perturbation of parabens on the neuroendocrine system in zebrafish larvae

Parabens, as the synthetic preservatives, have caused universal environmental contamination and human exposure. Whether parabens could disturb neuroendocrine system was still ambiguous. In this study, the effects of four commonly-used parabens, i.e. methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP) and butyl paraben (BuP), were tested on the neuroendocrine system of zebrafish larvae by investigating the swimming behavior, the related hormones and biomarkers in the hypothalamic-pituitary-interrenal (HPI) axis. The results showed that all test chemicals significantly reduced the swimming distance and mean velocity of zebrafish larvae. The adrenocorticotropic hormone (ACTH) levels in zebrafish larvae were significantly increased, while the cortisol levels were obviously decreased by paraben exposure. The transcriptional analysis showed that the expressions of the target genes including gr, mr and crhr2 in the HPI axis were mostly down-regulated. The exploration of the initial molecular event showed that parabens could bind with the glucocorticoid receptor (GR) and trigger its transactivation, according to MDA-kb2 luciferase assay and molecular docking analysis. The interaction of parabens with the GR included the hydrogen bond and hydrophobic interaction. The findings herein revealed the potential deleterious effects of parabens on the neuroendocrine system of zebrafish larvae, thus accumulating the in vivo toxicological data on this kind of food preservatives.

Loss of behavioral stress response in blind cavefish reduces energy expenditure.

The stress response is essential for animal self-defense and survival. However, species may exhibit stress response variation depending on their specific environmental and selection pressures. Blind cavefish dwell in cave environments, which differ markedly in stressors and resource availability compared to surface aquatic environments. However, whether blind cavefish exhibit differences in stress response as an adaptation to their cave environments remains unclear. Here, we investigated differences in stress response in six closely related Triplophysa species, including three blind cavefish (T. longibarbata, T. jiarongensis, and T. rosa) and three normal-sighted river fish (T. nasobarbatula, T. dongsaiensis, and T. bleekeri). Results showed that blind cavefish exhibited a range of distinct behavioral responses compared to sighted river fish, including greater levels of activity, shorter duration of freezing, absence of erratic movements or thrashing behavior, and opposite behavioral trends over time. Furthermore, the cavefish species demonstrated attenuated increases in metabolic rate in response to stressors related to novel environments. Cave-dwelling T. rosa also exhibited lower basal hypothalamic-pituitary-inter-renal (HPI) axis-related gene expression levels and stress hormone concentrations compared to river-dwelling T. bleekeri. These results suggest that blind cavefish may have lost their behavioral stress response, potentially mediated by a reduction in basal activity of the HPI axis, thus enabling the conservation of energy by reducing unnecessary expenditure in energy-limited caves.

Physical and social enrichment influences the adaptability-related behaviors of black rockfish Sebastes schlegelii: An effect mediated by social behaviors, HPI axis and neurogenesis

Environmental enrichment is a promising method to increase fish ecological fitness in hatchery release and to improve fish welfare in the aquaculture industry. However, little is known about the effects of physical and social enrichment on fish behavioral development and the relevant regulatory mechanisms. To address this knowledge gap, a typical aquaculture fish, black rockfish Sebastes schlegelii, was exposed to contrasting environments (i.e., physically enriched environment vs. barren environment, and socially low-density environment vs. high-density environment) for six weeks. Subsequently, the adaptability-related behaviors and social interactions during the captive period were determined, and stress-related physiological parameters and neurogenesis-related gene markers were measured. In general, the fish from an enriched environment expressed significantly higher sheltering propensity and anti-predator ability while less risk-taking frequency than the fish from a barren environment. And meanwhile, the fish from a low-density environment presented significantly less risk-taking behavior and more anti-predator responses than high-density fish. A structurally barren environment significantly increased fish aggressive frequency and locomotor activity compared with an enriched environment, whereas the social environment did not affect fish social interactions. For physiological status, the hypothalamus-pituitary-interrenal (HPI) axis and corticosteroid receptor system activities of barren fish were significantly higher than those of enriched fish, and meanwhile, the high-density fish also presented significantly higher physiological stress than low-density fish. For neurogenesis processes, an enriched environment did not affect fish brain cell proliferation but increased telencephalon neuronal differentiation ratio and immature neuron number. In contrast, a low-density environment significantly stimulated cell proliferating activity in telencephalon but did not affect brain neuronal differentiation activity. These novel results verified the conspicuous effects of physical and social environments on the adaptability-related behaviors in aquaculture fish, and finally, we proposed a novel regulatory pathway for fish behavioral plasticity, i.e., environmental stimuli - social interactions - HPI axis - corticosteroid receptor system - neurogenesis - adaptability-related behaviors. These results may lay the foundations for restoring fishery resources and for improving fish welfare, add novel knowledge to fish biology and environmental enrichment framework, and help deeply understand the mechanisms of environmental effects on marine animal behaviors.

Loss‐of‐function mutations in melanocortin-1 receptor modulate immune response in teleost fishes

The melanocortin system is an ancient neuroendocrine system conserved from teleosts to mammals. The melanocortin system is a set of complex neuroendocrine signaling pathways involved in numerous physiological processes, and particularly associated with the hypothalamic-pituitary-interrenal (HPI) axis response. The melanocortin 1 receptor (MC1R) is the central melanocortin receptor involved in pigmentation in vertebrates, including fish. In order to assess the immune role of MC1R, this study used a homozygous Mc1r knockout zebrafish. Hence, skin cortisol levels, variations in the blood leucocyte population, as well as the expression levels of immune genes in various tissues of wild-type TU strain (Tübingen, Nüsslein-Volhard Lab) (WT) and homozygous mc1r knockout zebrafish (mc1rK.O.) stimulated with LPS was carried out. Results show that the mc1rK.O. mutant fish produce lower levels of cortisol in mucus and fewer macrophages in blood after exposure to LPS compared to control fish. Regarding the expression of immune genes, mutant fish show a significant increase in the expression of the anti-inflammatory interleukin il10. These results suggest that the mc1rK.O. mutant fish may follow an alternative mechanism among the immune responses, where macrophages seem to have an anti-inflammatory function, attenuating nitric oxide (NO) production and providing an advantage through the mitigation of excessive or strong inflammatory reactions. Nonetheless, a lower number of this cell type could imply a reduced phagocytic potential in the face of an infection. At the same time, lower cortisol levels in the mc1rK.O. mutant fish could be an advantage as for the lower susceptibility to stress and the physiological and metabolic consequences of high cortisol levels.