Body Cortisol Levels Research Articles

Development of Selective Cortisol Biosensors in the Control of Fatigue and Related Diseases

The study of fatigue becomes complex due to several subjective factors related to its causality, making the relationship between the level of fatigue and the concentration of cortisol in an individual characteristic. However, cortisol is identified as the main element released in response to fatigue, and as such, it can be used as a biomarker for its determination and other associated diseases. Inadequate amounts of cortisol are related to non-specific diseases such as diabetes and osteoporosis. The body's cortisol levels fluctuate throughout the day, with the greatest levels in the morning and the lowest in the evening. To correlate these various fluctuations to the degree of fatigue, is required a real-time determination of cortisol levels with an effective sensory device, which overcomes the demand of costly and time-consuming laboratory analysis. Cortisol, as expressed in sweat, has a wide dynamic concentration range that can be used to monitor fatigue during different activities, being useful in preventing diseases and accidents. Currently, cortisol evaluation may be critical as one of the prospective biomarkers for Covid-19-induced significant inflammatory consequences. Some studies show possible severity between the production of pro-inflammatory cytokines related to viral infection by the new Sars-Cov-2 in people with above-average serum cortisol levels. In this regard, the establishment of a simple and low-cost biosensor with rapid diagnosis and in loco application would be beneficial due to the analytical center's independence and the simplicity with patients positive for the Sars-Cov-2 virus or others may be followed and treated. For the cortisol biosensor development, electrochemically exfoliated graphene (EEG) was used as a current collector nanomaterial in combination with chitosan (CS), a biomaterial derived from discarded crustacean shells. The carbon DropSens were modified with nanocomposite containing CS and EEG prepared using an ultrasonication. 21µL of CS_EEG dispersion by drop casting and exposed for 4 hours by white light, in order to dry the nanocomposite and recover all the working electrode. The material not bounded to electrode was then removed after 3 washes of 50µL of Milli-Q water. 10 µL of glutaraldehyde 1 % solution (GA) was dripped on the electrodes and allowed to react for 15 minutes to guarantee greater interaction of the nanocomposite with the antibody. The wet surface of electrodes was then evaluated for the incorporation of monoclonal antibody solution by IgG type specific for cortisol at varying concentrations. A total of 7.5 µL of diluted IgG solution (3.33 ng mL-1) and pure IgG solution were added to the biosensor immune complex (100 ng mL-1) and allowed to act for 30 minutes. Sensory measurements are performed in standard cortisol dilutions in PBS 7.4 in the following concentrations: 1 ng mL-1, 10ng mL -1, 100ng mL-1, 200ng mL-1, 500ng mL-1 and 1 µg. Then, the electrodes with and without antibody were characterized, via electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), FEG microscopy, Goniometry and Zeta Potential. Goniometry assays showed an increase of hydrophilic profile of the nanocomposites after antibody modification conferring a suit surface for biological elements deposition, Fig 1. FTIR and FEG Microscopy provided the interaction of antibody-nanocomposite and antibody- antigen complex, Fig 2 and 3. Infrared analysis showed bands assigned to methyl groups disappeared after antibody adhesion and this condition may relates to the recovery of heavy chains of IgG on the nanocomposite. The increase in capacitance value after each sensory electrochemical measurement provided a cortisol curve calibration, Fig 4. The zeta potential of the immune complex formation showed an increase of values indicating a high interaction of immune system, mainly for GA condition, Fig 5. The cortisol calibration curve was improved by GA intercalation and the detections were enhanced in concentrated antibody condition, Fig 6. Figure 1

Melatonin administration attenuates acute stress by inducing sleep state in zebrafish (Danio rerio)

Melatonin plays a fundamental homeostatic role in basic biological functions, and an anti-stress role has been also proposed for this hormone. This study aimed to evaluate hormonal, enzymatic and behavioral parameters of zebrafish that received administration of melatonin and were submitted to acute stress. A total of 120 wild-type zebrafish were divided into five groups: naïve control (N), negative control group (Stress/C), positive control treated with diazepam (Stress/Diaz), treatment with melatonin at dose 1 (Stress/Melt. 1) and treatment with melatonin at dose 2 (Stress/Melt. 2). The exposure to treatments (diazepam or melatonin) was performed prior to the acute stress protocol, based on a chase by a fishing net during 5 min followed by exposure to the air for 1 min. The body cortisol levels were assessed, as well as oxidative stress (thiobarbituric acid reactive substances, reactive species of oxygen and antioxidant activity), and fish behavior (open field test). Melatonin was able to modulate acute stress effects on zebrafish by inhibiting cortisol increasing levels, reducing locomotor parameters, inducing a sleep state, reducing lipid peroxidation and stimulating antioxidant enzymatic activity.

Early chronic hypoxia does not impact rainbow trout behaviour later in life

In trout aquaculture, tight control of water quality is crucial, especially for young life-history stages. This study examined whether chronic exposure to moderate hypoxia during the fry stage affects the rainbow trout’s behavior during later development. One month after yolk-sac resorption, young trout were exposed for 25 days to hypoxia, obtained by decreasing water flow and bubbling nitrogen gas into the tank (Hypoxia group, H: mean O2: 4.85 mg/L; min: 3.83, max: 6.87 mg/L). A control group was maintained in optimal conditions (C: mean O2: 10.41 mg/L; min: 10.12, max: 10.69 mg/L). At 4 days post-hypoxia, basal (resting) whole body cortisol levels were significantly higher in the H group (166.84 ± 20.67 ng/g) as compared to the C group (103.71 ± 8.78 ng/g), but no post-stress differences were evident (values ∼ 175 ng/g). Long-term effects of hypoxia on various behavioural traits were analysed between 3 and 4 months after the return to normoxia and compared to the C group. The exposure of fry to chronic hypoxia did not impact the fish’s behavioural repertoire (activity, stereotypic behaviour, aggression) in their home tanks after re-acclimation to normoxia. Moreover, basal and post-stress plasma cortisol levels, fear-related behaviours (novel-tank test), and risk-taking behaviours (emergence test) did not differ between the C and H groups. However, this early challenge resulted in an enhanced post-hypoxic growth rate in these fish. Our results show that early exposure to moderate hypoxia does not induce any behavioural changes in rainbow trout after several months of return to normoxia.

Evaluation of the effect of larval stocking density, salinity, and temperature on stress response and sex differentiation in the Dwarf Gourami and Rosy Barb

There is a variety of sexual determination and differentiation mechanisms among teleosts. The phenotypic physiology may be influenced by environmental stress during development as well as genetic factors. In these species, increased levels of the stress hormone cortisol during the undifferentiated labile period correlates with masculinization. In ornamental aquaculture, production of monosex male cohorts is desirable in several species, including the Rosy Barb Pethia conchonius and Dwarf Gourami Trichogaster lalius, which exhibit sexual dichromatism, where the males are more vibrantly colored and therefore more commercially valuable than female conspecifics. Internationally, the use of the synthetic androgen 17α-methyltestosterone (MT) to masculinize cohorts of ornamental fish is common. However, in the U.S., it is currently prohibited to use MT in ornamental aquaculture, putting domestic producers at an economic disadvantage. The current study explored the feasibility and efficacy of using environmental stressors during early larval rearing to influence sexual differentiation and skew populations toward a male phenotype.A suite of experiments were conducted during the first 30 days post hatch with both Rosy Barbs and Dwarf Gouramis. The aim of each experiment was to elevate endogenous cortisol levels in developing larvae using environmental stressors, thus potentially inducing masculinization. Stocking density, salinity, and temperature were manipulated to elicit a natural increase in circulating cortisol. Whole body cortisol levels, survival, growth, and sex ratios of fish in replicate tanks were determined for each experimental treatment. In both species, experimental treatments resulted in differences in growth, survival, and whole-body cortisol levels in specific experiments, but only one experiment resulted in modified sex ratios. The first potential evidence of temperature influence on sex determination was observed in Dwarf Gouramis with a significantly reduced proportion of males found after larval rearing in 30 °C conditions compared to 25 °C. These results provide novel insights into the larval stress physiology of these two species, but also suggest limited sexual plasticity perhaps due to a strong genetic sex determination system.

Fluoxetine Exposure During Sexual Development Disrupts the Stress Axis and Results in Sex- and Time- Dependent Effects on the Exploratory Behavior in Adult Zebrafish Danio rerio.

The antidepressant fluoxetine (FLX), generally the first line of pharmacological treatment in adolescents and pregnant women with affective disorders, is an emerging endocrine disruptor that is also released to the environment through sewage. Recently, we demonstrated that FLX exposure during the first 6 days of life in zebrafish (ZF; Danio rerio) induced a male-specific reduction in the exploratory behavior in the adult ZF that was linked to a reduction in cortisol production that persisted across three generations. Here we investigated sex differences in the behavioral and stress responses following FLX (0.54 and 54 μg⋅L–1) exposure during two periods of sexual development in ZF; early (0–15 days post-fertilization, dpf) and late (15–42 dpf). Our findings revealed that the stress response in females was reduced compared to that of males independent of the treatment. We also found that FLX reduced total body cortisol levels in the adult ZF regardless of sex and window of exposure. The hypocortisol phenotype of our FLX-treated fish was associated with behavioral alterations in the adult fish, which depended on the window of exposure; males were more sensitive to FLX during early development whereas females were affected during late development. A sexually dimorphic behavioral response induced by the low cortisol phenotype was observed in the FLX-treated ZF; females had higher exploratory activity whereas the males had reduced behavior. In conclusion, FLX results in sex- and window of exposure-specific effects on the behavioral activities in adult ZF. These findings highlight the importance of sex differences and timing on the long-term effects of antidepressant treatments. Knowledge of the sex-specific effects of antidepressants and the importance of early life exposure to chemical stressors may help us understand the impact of highly prescribed drugs such as FLX on the fetus from FLX-treated pregnant women as well as aquatic species in environments receiving sewage effluents.

Synthesis of the N-methyl Derivatives of 2-Aminothiazol-4(5H)-one and Their Interactions with 11βHSD1-Molecular Modeling and in Vitro Studies.

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is an enzyme that affects the body's cortisol levels. The inhibition of its activity can be used in the treatment of Cushing's syndrome, metabolic syndrome and type 2 diabetes. In this study, we synthesized new derivatives of 2-(methylamino)thiazol-4(5H)-one and tested their activity towards inhibition of 11β-HSD1 and its isoform - 11β-HSD2. The results were compared with the previously tested allyl derivatives. We found out that methyl derivatives are weaker inhibitors of 11β-HSD1 in comparison to their allyl analogs. Due to significant differences in the activity of the compounds, molecular modeling was performed, which was aimed at comparing the interactions between 11β-HSD1 and ligands differing by substituent at the amine group (allyl vs. methyl). Modeling showed that the absence of the allyl group can lead to the rotation of whole ligand molecule which affects its interaction with the enzyme.

Size‐dependent stress response in juvenile Arctic charr (Salvelinus alpinus) under prolonged predator conditioning

Predator conditioning can be used to improve post-release antipredator recognition of hatchery-reared salmonids. However, possible negative stress-related effects of prolonged predator conditioning on juvenile fish physiology are poorly understood. We studied the effects of prolonged (91 days) predator odour exposure on whole-body cortisol level and spleen size in six full-sib families of juvenile hatchery-bred Arctic charr (Salvelinus alpinus). Chemical cues from water containing charr-fed pikeperch (Sander lucioperca) were used as the predator exposure stimuli and lakewater was used as a chemical control. Our study revealed that juvenile body cortisol levels post-predator conditioning were affected by treatment, fish size and their interaction. Importantly, among the smaller (i.e. slowest growing) charr, the predator-exposed fish had higher cortisol levels than control fish, while the opposite pattern was true for the larger fish. These results suggest that chemical cues from charr-fed predators induce a prolonged stress response in juvenile charr. As prolonged predation exposure seems to elevate stress levels in a size-dependent manner, the larger, faster growing fish could possibly have intrinsically lower stress responses to predation threats than smaller, slower growing fish. Possible coupling between stress sensitivity and growth requires further attention due to the likely implications for the management of unintended domestication among captive-reared salmonids.

Cortisol plays a role in the high environmental ammonia associated suppression of the immune response in zebrafish

Exposure to high environmental ammonia (HEA) levels increases the vulnerability of fishes to parasitic, viral and bacterial diseases. We tested the hypothesis that elevated plasma cortisol levels play a role in the HEA-mediated immunosuppression in fishes. To this end, we tested the effect of exogenous cortisol treatment on the lipopolysaccharide (LPS)-induced immune response in zebrafish (Danio rerio). Also, to test whether glucocorticoid receptor (GR) signaling is involved in HEA-mediated immunosuppression, zebrafish were treated with mifepristone, a GR antagonist, and the LPS-induced immune response assessed after HEA exposure. We evaluated a panel of important immunity-related genes including interleukin 1β (il1b) and suppressor of cytokine signaling (socs-1a, 2, 3) and acute phase response genes [serum amyloid A (saa), transferrin (tfa), leukocyte cell-derived chemotaxin 2-like (lect2l), haptoglobin (hp), hepcidin (=hepatic anti-microbial peptide hamp), and complement component 3b (c3b)] by real-time quantitative PCR. Our results demonstrate that exogenous cortisol administration as well as elevated cortisol levels in response to HEA exposure modulate mRNA transcript levels of key mediators of the innate immune response in zebrafish. Mifepristone treatment reduced whole body cortisol levels and eliminated the HEA-mediated changes in transcript abundance of socs1a, il1b, as well as APR genes. Together, these results suggest that the HEA effect on the innate immune response is in part mediated by cortisol signaling, while the mode of action, including the receptors involved remains to be elucidated.

Exploring the efficacy of immersion analgesics in zebrafish using an integrative approach

Home Office guidelines recommend the use of analgesics for all protected species, including fish during scientific procedures likely to result in pain, suffering, distress and lasting harm. In larger fish species some drugs have shown efficacy in terms of perioperative analgesia when administered intramuscularly or at the site of tissue damage. However, this is impractical in very small species (<1g) and when large numbers need to be treated. Immersion is a commonly used route of administering anaesthesia in fish and may be a less invasive alternative to administering analgesia. Therefore, this study examined the efficacy of three water soluble analgesic drugs (buffered acetylsalicylic acid, butorphanol tartrate and lidocaine) provided pre- and post-surgery (tail fin clip) through addition to the tank water. The effectiveness of these drugs was measured as the reduction of changes in pain-related post-surgical behaviour and since pain is inherently stressful, HPI axis activity, quantified as whole body cortisol levels. To determine uptake of each drug, whole body concentrations were determined by HPLC-mass spectroscopy. Administration of both lidocaine and acetylsalicylic acid resulted in a significant reduction of post-nociceptive behavioural patterns, compared with animals exposed to saline solution. There was no effect of treatment on whole body cortisol. Finally, uptake was verified for lidocaine with whole-body concentrations showing clear dose dependency, whereas the other two compounds were not detectable. As the only drug with both a behavioural sparing effect and pharmacokinetic validation in this study, low-dose lidocaine immersions can be recommended for the first time to aid perioperative analgesia and provide a valuable refinement for reducing the impact of invasive procedures in zebrafish thereby improving welfare.

Cortisol regulates sodium homeostasis by stimulating the transcription of sodium-chloride transporter (NCC) in zebrafish (Danio rerio)

In mammals, sodium/hydrogen exchanger (NHE) and sodium-chloride cotransporter (NCC) are expressed in renal tubules, and exhibit functional redundancy and mutual compensation in Na+ uptake. In teleosts, the gills of the adult and skin of the embryonic stage function as external kidneys, and ionocytes are responsible for ionoregulation in these tissues. NHE- and NCC-expressing ionocytes mutually cooperate to adjust Na+ uptake, which is analogous to the activity of the mammalian kidney. Cortisol is a hormone that controls Na+ uptake through regulating NCC expression and activity in mammals; however, cortisol-mediated control of NCC expression is little understood in non-mammalian vertebrates, such as teleosts. It is essential for our understanding of the evolution of such regulation to determine whether cortisol has a conserved effect on NCC in vertebrates. In the present study, we treated zebrafish embryos with low Na+ medium (LNa, 0.04 mM Na+) for 3 d to stimulate the mRNA expression of nhe3b, ncc, and cyp11b1 (a cortisol-synthesis enzyme) and whole body cortisol level. Exogenous cortisol treatment (20 mg/l, 3 d) resulted in an elevation of whole-body Na+ content, ncc expression, and the density of ncc-expressing cells in zebrafish larvae. In loss-of-function experiments, microinjection of glucocorticoid receptor (gr) morpholino (MO) suppressed sodium content, ncc expression, and the density of ncc-expressing cells, but injection of mr MO had no such effects. In addition, exogenous cortisol treatment and gr MO injection also altered ncc expression and the density of ncc-expressing cells in gcm2 morphant larvae. Taken together, cortisol and GR appear to regulate Na+ absorption through stimulating ncc expression and the differentiation of ncc-expressing ionocytes, providing new insights into the actions of cortisol on Na+ uptake.

Effects of cypermethrin on survival, morphological and biochemical aspects of rohu (Labeo rohita) during early development.

The present study was conducted to investigate the effect of sub-lethal doses of Cypermethrin (CYP) on the survival and morphological and biochemical aspects of rohu (Labeo rohita) during early developmental stages. Newly fertilized eggs were incubated in the presence of sub-lethal concentration of CYP (20% of LC50 i.e., 8.43 μg L(-1)) in a well designed flow through system. Increased mortality was found with increase in exposure time. Deformities like eroded yolk and margins, elongated yolk sac, larvae with short tail, no eyes or larvae with zigzag movement and paralyzed larvae were observed in CYP treated group from blastula to fry stage. The activities of antioxidant like Catalase (CAT), Peroxidase (POD) and Lactoperoxidase (LPO) were decreased from 24 to 96 h in the control group whereas increased in CYP treated group. The Glutathione reducatse (GR) activity was also increased with time in both the control and treated groups; however, the activity was significantly higher in CYP treated group. Similarly, the whole body cortisol level showed an increasing trend with time in both control and treated groups. However, in CYP exposed group the cortisol level was considerably higher after 24 h exposure but statistically comparable to the control group after 96 h. It can be concluded from the present results that CYP even at very low concentration adversely affects the early development of L. rohita and enhances mortality. The obtained results reveal that CYP may affect the wild population of fish by inducing oxidative stress and modulating stress response during early ontogenesis.

Maternal transfer of bisphenol A impacts the ontogeny of cortisol stress response in rainbow trout

Bisphenol A (BPA) is widespread in the aquatic environment, and early life-stage exposure to this chemical affects growth and development in fish. We tested the hypothesis that BPA accumulation in eggs, mimicking maternal transfer, impacts the development and function of the hypothalamus-pituitary-interrenal (HPI) axis in rainbow trout (Oncorhynchus mykiss). To mimic maternal transfer, oocytes were loaded with 0 (control), 1, 4 and 40ng BPA embryo−1. We measured the temporal changes in whole body cortisol content, transcript levels of corticosteroidogenesis-related genes and glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) protein expressions during early development. In addition, trout larvae were subjected to an acute stressor at key developmental stages (at hatch, just prior to first feed and post hatch) and whole body cortisol levels measured to assess the functional integrity of the HPI axis. The developmental profile of whole body cortisol content was altered in the 1 and 40ng BPA groups compared to the control group. Also, the two key rate-limiting steps in steroidogenesis, the steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage (P450scc) enzyme transcript levels were disrupted in the larvae originating from BPA accumulated eggs prior to first feed. Whole body GR and MR protein expressions were higher in the 40ng BPA group compared to other groups prior to first feed. Stressor-induced elevation in whole body cortisol levels were evident at 65 days post-fertilization (dpf) and later, but not at 42 dpf in trout larvae. This cortisol response to a stressor exposure was attenuated in the 4 and 40ng BPA groups at 65 dpf, but not at 140 dpf. Together, accumulation of BPA in eggs, mimicking maternal transfer of this chemical, disrupts the ontogeny of cortisol stress response in trout larvae and may compromise the target tissue responsiveness to cortisol stimulation during early development.

Atrazine and its degradates have little effect on the corticosteroid stress response in the zebrafish

The present study examined the effects of atrazine on basal and forced swimming induced changes in whole body cortisol content in adult zebrafish. Zebrafish were exposed to graded concentrations of atrazine or the atrazine degradates deisopropylatrazine (DIA), deethylatrazine (DEA) and diamino-s-chlorotriazine (DACT) for up to 10days. Some fish were sampled for the measurement of whole body cortisol levels under basal conditions while others were sampled after being subjected to a 20min swimming challenge in order to quantify stress induced cortisol levels. In one experiment, zebrafish were subjected to two bouts of forced swimming 3h apart to test whether prior atrazine exposure affects the ability of the fish to respond appropriately to a repeated stressor. The results demonstrated that controls not exposed to atrazine and zebrafish exposed to atrazine or the atrazine degradates at nominal concentrations of up to 100μg/L consistently exhibited increased whole body cortisol content in response to the swimming challenge. Separate analyses revealed few changes in basal or stress induced cortisol levels following atrazine exposure. Overall, these data suggest that atrazine and some of its degradates at the concentrations tested have minimal effects on the cortisol mediated stress response in the zebrafish.

Acclimation of Zebrafish to Transport Stress

Welfare of fish is commonly neglected when they are transported. This study examines the effect of a 72-h mock transport on certain aspects of the stress physiology of two groups of zebrafish-the first transported in water enriched with a nitrifying bacterial consortium and the second in water without the enrichment. Zebrafish were examined at different time points-before packing (BP), immediately after packing them in transport bags (AP), at the end of transport (AT), and 72 h thereafter (PT)-to assess the primary (cortisol) and secondary (glucose) stress responses. In addition, the relevant genes in hypothalamic-pituitary-interrenal (HPI) axis (crf in brain, mc2r, star, cyp11c1, and hsd11b2 in kidney), including that of mineralocorticoid receptor (mr in kidney), were studied. Procedures during packing caused an increase in whole body cortisol levels of both fish groups. Only in the fish transported without the bacterial consortium, an increase in the levels of whole body cortisol as well as blood glucose was observed at the end of the transport. At the same time point and in the same fish group, the transcripts of mr and hsd11b2 were enhanced, probably to cope with the stress and to maintain homeostasis. The mRNA levels of the other genes in the HPI stress axis (crf, mc2r, star, and cyp11c1) were not significantly altered. Zebrafish transported in water enriched with the bacterial consortium exhibited a speedier stress acclimation. Nevertheless, only through in-depth studies the beneficial effect of the consortium can be confirmed.

Cortisol regulates Na+ uptake in zebrafish, Danio rerio, larvae via the glucocorticoid receptor

Unlike other freshwater fish previously examined, zebrafish are capable of increasing their rate of Na+ uptake during chronic exposure to acidic water (pH 4). In the present study, the potential role of cortisol in the induction of Na+ uptake during acid-exposure was investigated. When zebrafish larvae (4days post-fertilization) were treated with waterborne cortisol, the rate of Na+ uptake was significantly increased; this effect was blocked by co-incubating larvae with RU-486, an antagonist selective for the glucocorticoid receptor (GR). A similar induction in Na+ uptake, which was also blocked by RU-486, was observed when larvae were treated with dexamethasone, a selective GR agonist. Conversely, treating larvae with aldosterone, a selective agonist for the mineralocorticoid receptor (MR) had no effect on Na+ uptake. Acid-exposure increased whole body cortisol levels and translational knockdown of GR using antisense morpholinos prevented the full induction of Na+ uptake during exposure to acidic water, further confirming the role of cortisol and GR in Na+ uptake stimulation. Using immunohistochemistry, GR was localized to ionocytes known to be responsible for Na+ uptake (HR-cells). Knockdown of Rhcg1, an apical membrane ammonia channel or Na+/H+ exchanger 3b (NHE3b), proteins known to play an important role in facilitating Na+ uptake in acidic water, prevented the stimulatory effects of cortisol treatment on Na+ uptake, suggesting that cortisol regulates Na+ uptake by stimulating an Rhcg1–NHE3b “functional metabolon”.

Whole body cortisol and expression of HSP70, IGF-I and MSTN in early development of sea bass subjected to heat shock

Whole body cortisol levels were determined during early larval developmental stages of sea bass ( Dicentrarchus labrax) subjected to a heat shock with the aim to investigate the correlation between the stress event and the activation of the hypothalamic–pituitary–interrenal axis. Moreover, the mRNA expression of inducible heat shock protein 70 (HSP70), insulin-like growth factor I (IGF-I) and myostatin (MSTN) was also detected. Whole body cortisol was determined by a radio-immunoassay (RIA) technique whereas the expression of HSP70, IGF-I and MSTN mRNAs was quantified by Real-Time PCR. Cortisol was detectable in all the larvae from hatching but its level increased significantly in larvae submitted to heat shock from 2-day post hatching onwards. An effect of the sole transfer on cortisol levels was detectable at day 10, indicating an increase of the hypothalamic–pituitary–interrenal axis sensitivity from this stage of sea bass development. In animals exposed to heat shock, the expression of inducible HSP70 resulted in a marked increase of mRNA levels already at hatching. This increase was significantly higher from 6 days onwards if compared to controls. Moreover, heat shock resulted in a decrease (although not significant) in IGF-I mRNA expression of stressed larvae if compared to controls. On the contrary, heat shock did not influence the expression of MSTN mRNA in all groups. The results indicate a very early activation of the hypothalamic–pituitary–interrenal axis and in general of the stress response during the development of European sea bass. Moreover, these results suggest the importance of cortisol and inducible HSP70 as bioindicators of stress in aquaculture and confirm the role of IGF-I and MSTN as regulatory factors during development and growth of fish.

Stress response of juvenile flounder ( Paralichthys orbignyanus, Valenciennes 1839), to acute and chronic stressors

This study examined the stress response of flounder, Paralichthys orbignyanus, to acute and chronic stressors present in common aquaculture laboratory practices such as capture, transport and stocking density. Whole body cortisol levels in fish immediately caught and sampled within 3 min after capture were used as an indicator of pre-stress condition. Cortisol were significantly higher in fish caught and transported for 1 h, and in fish acclimated for 15 days in tanks and caught after chasing and netting for 15 min (acute stress conditions). Flounder held at a low density (3.3 kg m −3), had low cortisol levels; however, fish held at a higher density (7.8 kg m −3) resulted in significantly elevated cortisol levels even after 15 days, representing a chronic stress condition. The cortisol stress response of this fish is similar to that shown by other marine teleosts. This study showed that capture, transport, high stocking density and routine husbandry practices have the capacity to stress flounder, causing higher cortisol levels. Rearing density acts as a chronic stressor, and results in marked hierarchical relations of bottom settled over swimming fish. Stress management must be a component of technology development for this species, especially in the case of chronic stress situations.

Gene expression endpoints following chronic waterborne copper exposure in a genomic model organism, the zebrafish, Danio rerio

Although copper (Cu) is an essential micronutrient for all organisms, in excess, waterborne Cu poses a significant threat to fish from the cellular to population level. We examined the physiological and gene expression endpoints that chronic waterborne Cu exposure (21 d) imposes on soft-water acclimated zebrafish at two environmentally relevant concentrations: 8 microg/l (moderate) and 15 microg/l (high). Using a 16,730 65-mer oligonucleotide customized zebrafish microarray chip related to metal metabolism and toxicity to assess the transcriptomic response, we found that 573 genes in the liver responded significantly to Cu exposure. These clustered into three distinct patterns of expression. There was distinct upregulation of a majority of these genes under moderate Cu exposure and a significant downregulation under high Cu exposure. Microarray results were validated by qPCR of eight genes; two genes, metallothionein 2 (mt2) and Na(+)-K(+)-ATPase 1a1 (atp1a1), displayed increased expression under both Cu exposures, indicative of potential genetic endpoints of Cu toxicity, whereas the remaining six genes demonstrated opposing effects at each Cu exposure. Na(+)-K(+)-ATPase enzyme activity decreased during Cu exposure, which may be linked to Cu's competitive effects with Na(+). Whole body cortisol levels were significantly increased in Cu-exposed fish, which prompted an analysis of the promoter region of all significantly regulated genes for glucocorticoid (GRE) and metal (MRE) response elements to dissociate metal- and stress-specific gene responses. Of the genes significantly regulated, 30% contained only a GRE sequence, whereas 2.5% contained only a consensus MRE. We conclude that the indirect effects of Cu exposure regulate gene expression to a much greater degree than the direct effects.

Using Efinol®L during transportation of marbled hatchetfish,Carnegiella strigata(Günther)

The objective of this experiment was to test the efficacy of a probiotic (Efinol®L) during transportation of marbled hatchetfish, Carnegiella strigata. Wild specimens were captured from a small stream and transported for 24 h in plastic fish boxes with a probiotic (10 mg L−1) and probiotic-free water. The boxes were sampled at 3, 12 and 24 h of transport. At the end of the experiment, the survival rate was close to 100% in both treatments. Dissolved oxygen diminished with time in both treatments, but the probiotic group had significantly higher levels. Conductivity, pH and ammonia increased significantly during the transport, demonstrating higher levels in the probiotic-free group. Fish from both treatments presented very high net Na+ and K+ effluxes after 3 h of transport. At 24 h, net K+ effluxes in fish of the probiotic treatment reached values close to zero and a significantly lower Na+ efflux was observed. Cortisol levels in both treatments at 3 and 12 h were significantly higher than that in control samples. Higher body cortisol levels were observed in the probiotic-free group than that in the probiotic group at 3 and 12 h. The results demonstrate that addition of a probiotic during fish transport improves water quality and leads to fish presenting a lower stress response intensity.

Inclusion of the glucocorticoid receptor in a hypothalamic pituitary adrenal axis model reveals bistability

BackgroundThe body's primary stress management system is the hypothalamic pituitary adrenal (HPA) axis. The HPA axis responds to physical and mental challenge to maintain homeostasis in part by controlling the body's cortisol level. Dysregulation of the HPA axis is implicated in numerous stress-related diseases.ResultsWe developed a structured model of the HPA axis that includes the glucocorticoid receptor (GR). This model incorporates nonlinear kinetics of pituitary GR synthesis. The nonlinear effect arises from the fact that GR homodimerizes after cortisol activation and induces its own synthesis in the pituitary. This homodimerization makes possible two stable steady states (low and high) and one unstable state of cortisol production resulting in bistability of the HPA axis. In this model, low GR concentration represents the normal steady state, and high GR concentration represents a dysregulated steady state. A short stress in the normal steady state produces a small perturbation in the GR concentration that quickly returns to normal levels. Long, repeated stress produces persistent and high GR concentration that does not return to baseline forcing the HPA axis to an alternate steady state. One consequence of increased steady state GR is reduced steady state cortisol, which has been observed in some stress related disorders such as Chronic Fatigue Syndrome (CFS).ConclusionInclusion of pituitary GR expression resulted in a biologically plausible model of HPA axis bistability and hypocortisolism. High GR concentration enhanced cortisol negative feedback on the hypothalamus and forced the HPA axis into an alternative, low cortisol state. This model can be used to explore mechanisms underlying disorders of the HPA axis.