Unstressed Fish Research Articles

Dietary tryptophan intervention counteracts stress-induced transcriptional changes in a teleost fish HPI axis during inflammation

Immune nutrition is currently used to enhance fish health by incorporating functional ingredients into aquafeeds. This study aimed to investigate the connections between tryptophan nutrition and the network that regulates the communication pathways between neuroendocrine and immune systems in European seabass (Dicentrarchus labrax). When tryptophan was supplemented in the diet of unstressed fish, it induced changes in the hypothalamic-pituitary-interrenal axis response to stress. Tryptophan-mediated effects were observed in the expression of anti-inflammatory cytokines and glucocorticoid receptors. Tryptophan supplementation decreased pro-opiomelanocortin b-like levels, that are related with adrenocorticotropic hormone and cortisol secretion. When stressed fish fed a tryptophan-supplemented diet were subjected to an inflammatory stimulus, plasma cortisol levels decreased and the expression of genes involved in the neuroendocrine response was altered. Modulatory effects of tryptophan dietary intervention on molecular patterns seem to be mediated by altered patterns in serotonergic activity.

Effects of rearing density and in-tank structure on the stress response in juvenile Chinook salmon (Oncorhynchus tshawytscha)

Current salmonid hatchery practices typically include high rearing densities without structure within the water column. In the present study, we assessed the effects of low (conservation hatchery) and high (production hatchery) rearing densities on the plasma cortisol response during simulated transport stress and whether density might interact with in-tank structure to further mitigate the stress response. After 10 months of rearing in their respective treatments, we subjected fish to a transportation simulation stressor that encompassed fish being caught, bucketed, and crowded at a lowered water depth in a smaller tank for 3 h, then caught and bucketed to a novel tank of similar size and flow. We sampled stressed and unstressed fish for plasma cortisol determination over a 23 h time period. Additionally, we determined resting cortisol secretion from head kidneys collected from unstressed fish at time 0 h from each rearing treatment to determine if resting cortisol secretion pattern was affected by the rearing conditions. There was no significant difference in size of the fish at the time of sampling; overall, they averaged 24.7 g in weight and 136.2 mm in fork length. Fish reared with in-tank structure at low density had the lowest plasma cortisol concentration during the stressor, while fish reared without in-tank structure at high density had the highest plasma cortisol concentration in response to the stressor. In-tank structure, high-density reared fish had less plasma cortisol than no-structure high-density fish, but was elevated over low-density in-tank structure fish. Rearing condition had no meaningful effect on resting cortisol secretion by the interrenals. We speculate that low density with in-tank structure rearing would ultimately lead to the highest survival rates when hatchery Chinook salmon are released into the wild due to their reduced plasma cortisol concentration in response to a stressor.

Chronic stress negatively impacts wound healing, welfare, and stress regulation in internally tagged Atlantic salmon (Salmo salar).

The desire to understand fish welfare better has led to the development of live monitoring sensor tags embedded within individuals for long periods. Improving and understanding welfare must not come at the cost of impaired welfare due to a tag's presence and implantation process. When welfare is compromised, the individual will experience negative emotions such as fear, pain, and distress, impacting the stress response. In this study, Atlantic salmon (Salmo salar) underwent surgical implantation of a dummy tag. Additionally, half of this group was introduced to daily crowding stress. Both groups and an untagged group were followed for 8weeks using triplicate tanks per group. Sampling took place once a week, and where stress was given, it was conducted 24h before sampling. Stress-related measurements were taken to understand if tagging caused chronic stress and explore the chronic stress response and its impact on wound healing. Primary stress response hormones measured included CRH, dopamine, adrenocorticotropic hormone, and cortisol. Secondary stress response parameters measured included glucose, lactate, magnesium, calcium, chloride, and osmolality. Tertiary stress response parameters measured included weight, length, and five fins for fin erosion. Wound healing was calculated by taking the incision length and width, the inflammation length and width, and the inside wound length and width. The wound healing process showed that stressed fish have a larger and longer-lasting inflammation period and a slower wound healing process, as seen from the inside wound. The tagging of Atlantic salmon did not cause chronic stress. In contrast, daily stress led to an allostatic overload type two response. ACTH was elevated in the plasma after 4weeks, and cortisol followed elevation after 6weeks, highlighting a breakdown of the stress regulation. Fin erosion was elevated alongside cortisol increase in the stressed group. This data suggests that tagging previously unstressed fish in a controlled environment does not negatively affect welfare regarding stress responses. It also indicates that stress delays wound healing and increases the inflammatory response, highlighting how continued stress causes a breakdown in some stress responses. Ultimately, the tagging of Atlantic salmon can be successful under certain conditions where proper healing is observed, tag retention is high, and chronic stress is not present, which could allow for the possible measurement of welfare indicators via smart-tags.

Effects of Stocking Density, Size, and External Stress on Growth and Welfare of African Catfish (Clarias gariepinus Burchell, 1822) in a Commercial RAS

The effects of semi-intensive (100 kg m−3), intensive (200 kg m−3), and super-intensive (400 kg m−3) stocking densities on the growth and welfare of African catfish (Clarias gariepinus) were investigated under commercial production conditions. Plasma cortisol, glucose, and selected transcripts following a stress challenge, lactate, as well as skin lesions, were analyzed at regular intervals (from 12 g juveniles to 1.5–2.0 kg). The fish grew well, but after 23 weeks, the semi-intensively stocked fish had a mean final weight of 1830.5 g, significantly higher than the super-intensively stocked fish with 1615.4 g, and considerably higher than the intensively stocked fish with 1664.8 g (p > 0.05). Cortisol and glucose responses significantly differed between stressed and unstressed fish, but not between treatment groups. An unforeseen external stressor (nearby demolition noise) caused stress responses among all treatment groups, but was similarly coped with. Mortality ranged between 3.8–9.2%. In the juveniles, skin lesions were reduced under intensive or super-intensive densities, with the least under semi-intensive densities in outgrown fish. Expression profiles of 22 genes were compared in the spleen at semi-intensive and super-intensive densities. The transcript concentrations of most genes remained unchanged, except for slc39a8 and mtf1, which were significantly downregulated in stressed catfish under semi-intensive conditions. We demonstrated that African catfish growth performance and welfare depend on age and stocking density, also reacting to demolition noise. This supports farm management to optimize stocking densities during the grow-out of African catfish in RAS and suggests avoiding external stress.

Leucine did not stimulate growth and accretion in either stressed or unstressed Atlantic salmon

The aim of the current trial was to test whether leucine affected growth and accretion including test any effects on leucine upon stress. Quadruplicate tanks each containing 50 Atlantic salmon (Salmo salar) (mean start body weight of 524 ± 28 g) were fed diets containing 27.3, 30.0, 35.0 and 41.0 g leucine/kg diet for 74 days. Two tanks per dietary group were exposed to a stressor (5 min chasing) three days a week to test whether enriched leucine diet aid coping with chronic stress, while two tanks per dietary groups were left unstressed. The stressed fish consumed less feed and grew less than the unstressed fish, irrespective of diet. Leucine inclusion did not affect protein accretion, but leucine retention declined with increasing dietary leucine in both stressed and unstressed fish. No difference between the stressed or unstressed fish was present. Leucine did not affect relative liver size, but unstressed fish had slightly higher relative liver size compared with stressed fish (p = 0.05). Free leucine in the muscle and liver was not affected by dietary leucine, but unstressed fish had higher concentration of valine and isoleucine in the muscle compared with the stressed fish. Muscle of fish fed elevated leucine had lower mRNA expression of murf1 (p = 0.037) and higher expression of ppara (p = 0.012). Muscle of stressed fish had higher expression of the oxydative genes mnsod (p = 0.049) and catalase (p = 0.037) compared with the fish left unstressed, while in liver, there were no differences of expression of any of the genes tested. In conclusion, diets enriched in leucine had minor effects and neither protein accretion nor growth was affected in either stressed or unstressed fish.

Proteome profile of telencephalon associates attenuated neurogenesis with chronic stress induced mood disorder phenotypes in zebrafish model

Debilitating mental illness like depression and related mood disorders is due to the disruption in circuitry that controls emotion, motivation, and reward, characterized by disparate phenotypes like decrease in socialization, motivation, threshold for threat apprehension, etc. Chronic stress is a major factor in the etiology of these disorders. Here, using a chronic unpredictable stress (CUS) paradigm the characterization of an array of mood disorder phenotypes in adult zebrafish, in comparison to normal control unstressed fish, was achieved using a battery of behavioral assays including novel ones comprising social interaction test, feed approach test, threat response test and novel tank test. For the predictive validity of the model for mood disorders, the mitigative role of a slow (imipramine) and fast (ketamine) acting antidepressant was assessed. The molecular changes associated with CUS-induced mood disorder phenotype was investigated utilizing a high throughput method called isobaric tag for relative and absolute quantification (iTRAQ) in telencephalon, the region critically associated with the processing of emotional information in the fish brain. Out of 222 proteins identified to be significantly altered, 58 were differentially expressed across the stress and antidepressant-treatment groups at more than one fold (in log2) change. Of these proteins, some were implicated in earlier studies on mood disorders such as CABP1, PER2, mTOR, etc. The enrichment of altered proteins by Ingenuity Pathway Analysis (IPA) led us to mTOR and opioid signaling pathways, the top canonical pathways affected in the fish telencephalon. Interestingly, most of the pathways affected converge at the one controlling cell proliferation thus indicating altered neurogenesis, which was validated using immunohistochemistry for cell proliferation markers BrdU, SOX2, and BLBP. The study concludes that molecules that regulate telencephalon neural progenitor cell proliferation or neurogenesis are crucially involved in chronic stress-induced mood disorders by affecting the circuitry that controls emotion and reward.

Development of an immersion challenge model for Streptococcus agalactiae in Nile tilapia (Oreochromis niloticus)

Streptococcus agalactiae, the causative agent of streptococcosis in fish, induces significant mortality in the tilapia aquaculture. To elucidate the pathogenesis of S. agalactiae in tilapia, a simple and feasible challenge model was established in this study. Here, we report the development of an immersion challenge model that mimics a natural outbreak of S. agalactiae. In this model, tilapia were allowed to stress under high temperature, and hypoxia (33.5 ± 0.5°C, 2.0 ± 0.5 mg L-1 dissolved oxygen), and the bacterial exposure increased the mortality. The infection concentration assay showed the cumulative mortality to be 6.67 ± 3.34%, 32.22 ± 1.92%, and 53.3 ± 1.92% for the tilapia in the stressed group (under high temperature and hypoxia after immersed in 6.24×107, 3.12×108 and 1.56×109 CFU mL-1 for 20 min), which were higher than the cumulative mortality (0, 0, and 4.42 ± 1.95%) of the unstressed control group. The infection time assay showed the cumulative mortality to be 6.67 ± 3.34%, 26.67 ± 3.34%, and 35.56 ± 1.93% for the tilapia in the stressed group (under high temperature and hypoxia after immersed with 3.06×108 CFU mL-1 for 5, 20 and 60 min), which were higher than the cumulative mortality (0, 0, and 0%) of the unstressed control group. No mortality was observed in tilapia under stressed water conditions. The high mortality may attributed to the increased bacterial loads at 24 h post infection in the liver (6.81 × 104 vs. 2.63 × 103 CFU mg-1), spleen (1.85 × 106 vs. 8.69 × 104 CFU mg-1), and kidney (3.84 × 105 vs. 2.14 × 104 CFU mg-1) in the stressed fish compared to the unstressed fish. The serum non-specific immune parameters (adenosine deaminase, acetylcholinesterase, catalase, and superoxide dismutase) were also found to be decreased in the stressed fish. In addition, the transcripts of inflammatory-related genes (TNF-α, IL-1β, and IL10) exhibited up-regulation, whereas IgM and IgT exhibited down-regulation in the head kidney of the stressed fish, which could also increase the probability of disease. In conclusion, these findings indicate that the mortality of tilapia increased upon exposure to high temperature and hypoxia with S. agalactiae. The immersion challenge model may provide an insight into the evaluation of pathogenesis and vaccine efficacy.

Down-regulation of corticosteroid receptor in leucocytes of stressed rainbow trout

The relationship between stress and immunosuppression was investigated in peripheral blood leucocytes (PBL) in rainbow trout, with reference to corticosteroid receptor (CR) expression and responses to cortisol- and/or lipopolysaccharide (LPS)-administration. Confinement stress in shallow water resulted in a sustained elevation of plasma cortisol, whereas lysozyme and immunoglobin levels were suppressed. Significant increases in mRNA levels of caspase-6 and insulin-like growth factor (IGF)-I were observed in PBL isolated from stressed fish. Confinement stress also suppressed proinflammatory cytokine, interleukin (IL)-1β, expression in PBL. There were decreasing tendencies for the mRNA levels of CRs in PBL of stressed fish. In-vitro treatment of cortisol and LPS on isolated PBL from unstressed trout increased both IL-1 β and CR mRNA expression. However, in PBL from stressed fish, cortisol and LPS treatment increased IL-1 β but not CR mRNA levels. Proliferative activities estimated as in-vitro incorporation of bromodeoxyuridine (BrdU) were decreased by cortisol in PBL from the unstressed and stressed fish groups; however, LPS-stimulated proliferation was observed only in the unstressed fish. Ratios of apoptotic PBL quantified as cell fragmentation using an automated cell counter were increased by cortisol in both groups; however, LPS-stimulated apoptosis was observed only in the stressed fish. Our study reveals cortisol has immune-suppressive effects in stressed fish, irrespective of CR down-regulation and desensitization. The complexity of immune-endocrine interaction is shown by the stress-induced attenuation of LPS effects.

Reduced stress response in juvenile Chinook Salmon reared with structure

Understanding how rearing environments influence the stress response is relevant to many fish management practices. This is particularly relevant in aquaculture, where it is important to be able to minimize the effects of stress to enhance animal wellbeing. The stress response is also interesting from a basic science perspective, where it is important to understand how environmental differences can influence phenotypic traits and the physiological reaction to stressors. We tested the hypothesis that rearing fish in a more complex environment would reduce their stress response when presented with stressors. Specifically, we tested for the effect of structure in rearing tanks on the stress response of juvenile Chinook Salmon (Oncorhynchus tshawytscha) exposed to a handling and crowding stressor, something like hatchery fish might experience during transportation to new locations. For both rearing treatments (with and without structure), we compared fish that were subjected to the stressor to fish sampled from the respective rearing tank as the control. For the stressed treatment, we subjected fish to net capture followed by three hours in lowered water levels in a novel tank and then moved to a second novel holding tank. This process was designed to mimic typical stressors fish may experience at a hatchery. We analyzed plasma collected before the start of the stressor, during the stressor, and throughout 20 h post-stressor for cortisol in both stressed and unstressed fish. For both rearing treatments, fish that experienced the stressor had elevated plasma cortisol compared to control fish. However, the cortisol levels of fish in the stressed treatment reared with structure were significantly less than fish reared without structure. Additionally, plasma cortisol levels in structure-reared fish returned to baseline by 20 h post-stress while fish reared without structure had not fully recovered. Our findings suggest that increasing complexity of the rearing environment might be advantageous as a way of minimizing effects of handling-type stressors.

Stressed fish don't get hot under the collar

For all creatures, great and small, stress is ubiquitous and unavoidable. Because of the havoc stress can wreak on the body, and the associated changes in behaviour and physiology, stress is of particular concern to creature researchers. ‘As fish biologists we want to ensure our fish are well cared for and mitigate stress as much as possible so that we can observe them behaving as naturally as possible,’ says Nick Jones, from University of St Andrews, UK. So when Jones and some of his fellow researchers came across a paper by Sonia Rey and colleagues (doi:10.1098/rspb.2015.2266), which suggested that fish move into warmer areas to increase their temperature when stressed, they were intrigued. He points out that if this is the case it could be huge, because it could affect how lab animals are cared for and provide insight into stress-related behaviour in nature. Not only that, but an increase in body temperature following stress is common in many endotherms, but otherwise hadn't been observed in ectotherms like fish and amphibians.Jones and an international team of collaborators sought to replicate the study – with a few tweaks – which is more formidable than it sounds. ‘Replication studies seem easy: just follow “the recipe” of the original methods and stick in a few extra controls, but, even still, it took a while to get it right’, says Jones. The original recipe called for confining a group of 12 zebrafish (Danio rerio) to stress them, before releasing them and allowing the fish to swim freely into chambers that were warmer (35°C) or cooler (20°C) than the one into which they were stressed (26°C). The team's secret additional ingredient was to crowd a second group of fish into the stress chamber and then allow them to explore chambers that were all at the same temperature (28°C). However, when the team compared the distributions of the fish that could explore the temperature gradient with the fish that had no temperature choice, they were essentially the same. It seems that the first group of fish were, in fact, not opting for a stress-relieving warm soak after all.Jones's team was also concerned that grouping so many fish together might interfere with the abilities of the individuals to make their own decisions, particularly during times of stress – because zebrafish are highly social – so they cooked up another experiment. This time, they used smaller groups and compared the temperature choices of stressed and unstressed fish when given the option between warm and cool water. Once again, the stressed fish didn't seek out warmth. In fact, they spent less time in the warmer area than the unstressed fish did.Jones suggests that while these findings contradict the results of the previous study, they are not entirely surprising. Ectotherms sometimes lower their body temperature when exhausted – that is, they chill out after strenuous exercise or stress instead of warming up.

Differential response to air exposure in crowded and uncrowded Atlantic cod (Gadus morhua): Consequences for fillet quality

Previous recommendations on bleeding of Atlantic cod (Gadus morhua) to secure proper blood drainage and good muscle quality are based on studies done on un-stressed fish. Therefore, the aim of this study was to investigate the effects of stress by crowding in a net, followed by air exposure before and after slaughter on blood parameters and fillet quality in Atlantic cod. Live fish were either directly or after 4 h of crowding exposed to air for 0, 15 or 30 min prior to or after killing by a blow to the head and bleeding. Blood clotting time, pH, lactate, glucose, and muscle pH were measured. Also, concentrations of haemoglobin in the muscles were measured using Vis/NIR hyperspectral imaging to indicate residual blood. Stress from crowding and air exposure before and after slaughter resulted in increased concentration of haemoglobin in the fillets, with a faster increase in fish crowded and slaughtered after air exposure. Blood clotting time was shorter after 15 min of air exposure, and decreased further with crowding. Blood and muscle pH, and lactate levels were mainly affected by air exposure time. Overall, air exposure had a negative effect on fillet quality, and this effect was strongest and fastest if fish were crowded prior to the air exposure. However, by slaughtering the fish before air exposure, quality can be improved as this delays the increase in the amount of residual blood.

Stress up-regulates oxidative burst in juvenile Chinook salmon leukocytes

When fish perceive stressful scenarios, their hypothalamus-pituitary-interrenal axis is activated resulting in the release of corticotropin releasing hormone, adrenocorticotropic hormone (ACTH), and finally cortisol. The physiologic stress response of fish has most often been linked to the reduced performance of the immune system, with a few exceptions where the immune system is activated. In this report, we tested the hypothesis that oxidative burst activity levels in juvenile Chinook salmon (Oncorhynchus tshawytscha) are altered when the fish is presented with a stressor. Fish were subjected to a stressor for 3 h and then allowed to recover for 20 h following the stressor. Plasma and spleens were collected from euthanized fish before the stressor, at the end of a 3 h stressor, and 23 h after the start of the experiment. Plasma was held at −80 °C until cortisol radioimmunoassay analysis was performed to confirm stress. Spleens were held in Dulbecco's Modified Eagle Medium overnight and analyzed the day following collection. Oxidative burst activity was measured in splenic leukocytes after being stimulated with phorbol 12-myristate 13-acetate. We found a significant increase in activated oxidative burst from fish subjected to the stressor as compared to unstressed fish. Speculation is given to ACTH being the leukocyte priming agent in this experiment rather than the cortisol itself.

Effects of reduced water exchange rate and oxygen saturation on growth and stress indicators of juvenile lumpfish (Cyclopterus lumpus L.) in aquaculture

Lumpfish (Cyclopterus lumpus) is currently used as cleaner fish in Norwegian Atlantic salmon (Salmo salar) farming to control parasitic lice on salmon. The need for high numbers of lumpfish necessitates intensive farming of this species but very limited knowledge is available on the environmental requirements of lumpfish in culture. The present study was therefore carried out to investigate the tolerance of lumpfish to varying water exchange rates and oxygen saturations. Four triplicated treatment groups were established at different water exchange rates, which caused oxygen saturations of 55% (0.7Lmin−1), 69% (1.2Lmin−1), 81% (1.9Lmin−1) and 96% (6Lmin−1). Decreasing water exchange rate and oxygen saturations negatively affected growth, even in the fish groups held at 81% oxygen saturation. The fish group held at 55% oxygen saturation had almost no growth in body mass and showed sign of fin-infections and were terminated halfway through the experiment. A high workload associated with oxygen extraction and reduced appetite is considered the reason for reduced growth at lower oxygen saturations. While plasma cortisol levels in the groups held at 81 and 96% oxygen saturation corresponded to those considered typical for unstressed fish (<10ngmL−1), the levels of plasma cortisol of fish in the 69 and 55% oxygen saturation groups were above 20ngmL−1 in November, indicating a state of chronic stress. A fast, albeit weak, cortisol response to stressors in the lumpfish was confirmed in a separate, 2h long acute handling and hypoxia experiment. Lack of differences between treatments in plasma lactate levels indicates that the lumpfish reduced their food intake and locomotory activity sufficiently to avoid resorting to anaerobic metabolism when exposed to reduced oxygen saturations. It is concluded that the juvenile lumpfish is sensitive to reduced water oxygen saturations and that oxygen saturations below 80% in aquaculture should be avoided.

Effects of dietary yeast inclusion and acute stress on postprandial plasma free amino acid profiles of dorsal aorta-cannulated rainbow trout

In a 4-week experiment, 15 cannulated rainbow trout were fed three diets based on fish meal (FM), Saccharomyces cerevisiae yeast (SC) and Wickerhamomyces anomalus and S. cerevisiae yeast mix (WA). Fish were fed daily, and blood samples were collected on day 7 of each week at 0, 3, 6, 12 and 24 hr after feeding. In the final week, fish were exposed to a 1-min netting stressor. All essential and non-essential plasma amino acid levels except methionine were similar between fish fed diets FM, SC and WA. Plasma methionine and sarcosine were significantly higher in fish fed diets SC and WA, possibly due to the crystalline methionine level, form or feeding regime. Hydroxy-proline and 3-methyl-histidine were higher in fish fed diet FM, which can be explained by the higher levels present in fish meal compared with yeast. In stressed fish, there were no dietary effects on plasma amino acid levels, but significant increases in taurine and cystathionine were found in stressed compared with unstressed fish. These results demonstrate that yeast-based diets produce similar plasma amino acid profiles to fish meal and suggest that yeast may be a suitable fish meal replacement in diets for rainbow trout.

Skin Mucus of Gilthead Sea Bream (Sparus aurata L.). Protein Mapping and Regulation in Chronically Stressed Fish.

The skin mucus of gilthead sea bream was mapped by one-dimensional gel electrophoresis followed by liquid chromatography coupled to high resolution mass spectrometry using a quadrupole time-of-flight mass analyzer. More than 2,000 proteins were identified with a protein score filter of 30. The identified proteins were represented in 418 canonical pathways of the Ingenuity Pathway software. After filtering by canonical pathway overlapping, the retained proteins were clustered in three groups. The mitochondrial cluster contained 59 proteins related to oxidative phosphorylation and mitochondrial dysfunction. The second cluster contained 79 proteins related to antigen presentation and protein ubiquitination pathways. The third cluster contained 257 proteins where proteins related to protein synthesis, cellular assembly, and epithelial integrity were over-represented. The latter group also included acute phase response signaling. In parallel, two-dimensional gel electrophoresis methodology identified six proteins spots of different protein abundance when comparing unstressed fish with chronically stressed fish in an experimental model that mimicked daily farming activities. The major changes were associated with a higher abundance of cytokeratin 8 in the skin mucus proteome of stressed fish, which was confirmed by immunoblotting. Thus, the increased abundance of markers of skin epithelial turnover results in a promising indicator of chronic stress in fish.

Evidence for the involvement of dopamine in stress-induced suppression of reproduction in the cichlid fish Oreochromis mossambicus.

In the present study, we examined whether stress-induced suppression of reproduction is mediated through the catecholaminergic neurotransmitter dopamine (DA) in the female cichlid fish Oreochromis mossambicus. In the first experiment, application of antibody against tyrosine hydroxylase (TH; a marker for DA) in brain sections revealed the presence of intensely stained TH immunoreactive cells in the preoptic area (POA) and nucleus preopticus (NPO) during the previtellogenic phase. These cells showed weak immunoreactivity during the vitellogenic and prespawning phases concomitant with darkly stained luteinising hormone (LH) immunoreactive content in the proximal pars distalis (PPD) of the pituitary gland and fully ripened follicles (stage V) in the ovary of control fish. However, in fish exposed to aquacultural stressors, TH secreting cells remained intensely stained in POA and NPO regions during the prespawning phase, indicating increased synthetic and secretory activity, which was reflected by a significantly higher DA content compared to controls. Increased DA activity as a result of stress was associated with a decrease in the LH immunoreactive content in the PPD and an absence of stage V follicles in the ovary. In the second experiment, administration of DA caused effects similar to those in stressed fish, whereas DA receptor antagonist domperidone (DOM) treatment significantly increased the LH content in the PPD and the number of stage V follicles in unstressed fish. On the other hand, treatment of stressed fish with DOM resulted in dark accumulations of LH immunoreactive content in the PPD accompanied by the presence of stage V follicles in the ovary. Taken together, these results suggest an additional pathway for the inhibitory effects of stress through dopaminergic neurones along the reproductive axis.

Knockout of the Ergothioneine Transporter ETT in Zebrafish Results in Increased 8‐Oxoguanine Levels

Ergothioneine (ET) is expected to provide antioxidative properties due to its double‐bonded sulfur. It is acquired by plants and vertebrates solely through dietary means. However, the precise physiological function of ET remains unclear. Case‐control studies suggest an association of polymorphisms in the human ET transporter (ETT) gene with chronic inflammatory diseases like rheumatoid arthritis [Nat Genet 35, 341‐348], Crohn's disease [Nat Genet 36, 471‐475] and diabetes [Biochem J 25, 171].&#x0D; &#x0D;MethodsHere we have procured, based on retroviral insertion [Genes Dev 13:2713‐24], an ETT knock‐out zebrafish (Danio rerio). The expression profile of ETT in wild‐type zebrafish was analyzed by RT‐PCR and real‐time RT‐PCR and validated by measuring ET tissue content.ResultsStrong signals were obtained with kidney, intestine, skin, brain and eye. In whole‐fish lysates of homozygous knock‐out animals, ET content (0.06 ± 0.01 ng/g wet mass; n=3) was reduced more than 1000‐fold compared to wild‐type (138 ± 61 ng/g wet mass; n = 3). Similarly, all examined organs contained virtually no ET. Carnitine and acetylcarnitine were reduced across all examined tissues of knock‐out fish by a factor of 2.0 ± 0.2, and 1.7 ± 0.1, respectively. Incubation in sublethal concentrations of Pb2+ and Cu2+ led to an increase of the lipid peroxidation markers 4‐hydroxy‐2,3‐trans‐nonenal (HNE) and malondialdehyde (MDA) in knock‐out versus wild‐type zebrafish. Comparison of unstressed fish at the level of small molecules by LC‐MS difference shading [Proc. Natl. Acad. Sci. U.S.A. 102:5256‐61] revealed a 3.8‐fold increase of 8‐oxoguanine in the skin of ETT knockout fish.ConclusionBased on this phenotype we propose that the universal and specific purpose of ET is to eliminate singlet oxygen.

Knockout of the ergothioneine transporter ETT in zebrafish results in increased 8-oxoguanine levels

Ergothioneine (ET) is a natural compound that humans and other vertebrates must absorb from dietary sources. In general, ET is considered an intracellular antioxidant. However, the precise physiological purpose of ET and the consequences of ET deficiency are still unclear. The ergothioneine transporter ETT (human gene symbol SLC22A4) is a highly specific transporter for the uptake of ET. Here, we sought to identify and knock out ETT from zebrafish (Danio rerio) to determine the function of ET. We cloned and assayed three related proteins from zebrafish, only one of which catalyzed the uptake of ET. RT-PCR analysis revealed that the protein is strongly expressed in the skin, brain, kidney, intestine, and eye. In ETT-knockout animals generated by retroviral insertion into exon 1, ET content was reduced by more than 1000-fold compared to the wild type. Thus, ETT is the sole transporter responsible for uptake of ET into zebrafish. ETT-knockout fish did not exhibit obvious differences in morphology or behavior. In whole-fish homogenates, an increase in 4-hydroxy-2,3-trans-nonenal and malondialdehyde was observed, but only after stress caused by incubation with Pb2+ or Cu2+. Comparison of unstressed fish at the level of small molecules by LC–MS difference shading revealed a 3.8-fold increase in 8-oxoguanine (8-oxo-7,8-dihydroguanine) in the skin of ETT-knockout animals. Our knockout represents a new model for examining the consequences of complete absence of ET. Based on the phenotype observed here, we hypothesize that the specific purpose of ET could be to eliminate singlet oxygen.

Variations of physiological and innate immunological responses in goldfish (Carassius auratus) subjected to recurrent acute stress

This study was undertaken to investigate the influence of repeated acute stress on the physiological status and non-specific immune response of goldfish, Carassius auratus. The acute stress was a succession of a 3 min-chasing period followed by a 2 min-air exposure. The goldfish in triplicate tanks were subjected 3 times daily to this stress for one (S3) or three (S9) days. A separate group of unstressed fish was used as control for each sampling time. Blood samples were collected 12, 48 and 120 h after the last stress procedure. Variations of globulin levels, plasma anti-protease and bactericidal activities were not significant in the present study. The haematological parameters and plasma total protein and albumin strongly declined in S9 fish 12 h post-stress compared to control fish. However, plasma cortisol, glucose and lactate levels in both S3 and S9 transiently increased compared to the control fish. Similarly, plasma peroxidase activity transiently increased in both stressed groups 12 h after stress. An increase in plasma lysozyme and complement activities suggested a hormesis-like effect with one-day acute stress improving the immunological response of goldfish while an extension of the stress period to three days impaired physiology and immunity for up to 5 days. This study revealed that recurrent acute stress could immunosuppress goldfish as usually expected of chronic stress.

Blue substrate modifies the time course of stress response in gilthead seabream Sparus aurata

Abstract Environmental enrichment may attenuate stress response of several vertebrates. In the case of fish, although their stress response is well investigated, the effect of structural complexity as a means of environmental enrichment on stress response is not widely studied. The present study aims to investigate the possible effect of structural enrichment (blue gravel substrate) on gilthead seabream Sparus aurata primary and secondary stress responses during confinement and after recovery. Fish (initial mass 20.3 ± 0.22 g; age 0 +) were reared in tanks with blue substrate (BS) or no-substrate (NS) for 75 days. Fish (triplicated groups) were subjected to 90 min of confinement stress and sampled at 30, 60 and 90 min of confinement, as well as at 2, 6 and 24 h after stress (recovery). Triplicate groups of BS and NS fish were left undisturbed (unstressed fish). During rearing BS fish had better growth performance and less aggressive behavior than NS fish. Plasma cortisol levels and brain neurotransmitters of BS and NS fish were increased during confinement. Cortisol reduction (after stress-induced increase) occurred earlier in NS fish (60 min) than BS fish (90 min), while cortisol levels recovered in 2 h in both treatments. Serotonin metabolite 5-HIAA levels and serotonergic activity (5-HIAA/5-HT) were recovered in 6 h in BS fish, while they remained elevated for NS fish. Dopaminergic system response to confinement showed a transient increase in BS fish (60 min), while no effect was observed in NS fish. Concerning secondary stress response, glucose levels of BS fish decreased after 6 h of recovery while NS fish were not affected. On the other hand, NS fish presented a reduction of triacylglycerides (60 and 90 min) during confinement, while BS fish response was not affected. Osmolality and plasma total protein levels increased at 30 min of confinement and returned to unstressed levels in 24 h in both treatments. Present results indicate that the presence of blue substrate did modify the time course of gilthead seabream stress response to confinement. The overall performance of fish reared with blue substrate suggests that they are in better fitness than fish reared in plain tanks. The efficacy of the blue substrate as environmental enrichment enhancing gilthead seabream welfare is further reinforced and strongly encourages for additional investigations.