Changes in circulating insulin-like growth factor-1 and its binding proteins in yearling rainbow trout during spring under natural and manipulated photoperiods and their relationships with gill Na+, K+-ATPase and body size.

Smoltification in salmonids occurs during spring in response to increasing photoperiod to prepare for marine life. Smoltification is associated with increased hypo-osmoregulatory ability and enhanced growth potential, mediated by growth hormone and insulin-like growth factor (IGF)-1. Rainbow trout is uniquely insensitive to the induction of smoltification-associated changes by photoperiod, such as the activation of gill Na+,K+-ATPase (NKA). We measured the circulating IGF-1 and IGF-binding protein (IGFBP)-2b levels in yearling rainbow trout exposed to natural and manipulated photoperiods during spring and correlated these with gill NKA activity and body size. Although the effect of photoperiod manipulation on body size and circulating IGF-1 and IGFBP-2b was negligible, they were positively correlated with gill NKA activity in fish under simulated natural photoperiod. We next pit-tagged yearling rainbow trout and fed them a restricted ration or to satiation under a natural photoperiod. In April, gill NKA activity was higher in the satiation group than in the restricted group and positively correlated with body size and growth rate. In addition, circulating IGFBP-2b was positively correlated with gill NKA, size and growth, whereas circulating IGF-1 was correlated only with size and growth. The relationship between circulating IGF-1 and growth intensified from May to June, suggesting that the IGF-1-growth relationship was disrupted in April when gill NKA was activated. Two additional IGFBPs were related to growth parameters but not to gill NKA activity. The present study suggests that circulating IGFBP-2b and IGF-1 mediate the size-dependent activation of gill NKA in yearling rainbow trout during spring.

Neural activation in photosensitive brain regions of Atlantic salmon (Salmo salar) after light stimulation.

Photoreceptive inputs to the teleost brain are perceived as image of the visual world and as photo-modulation of neuroendocrine and neuronal signals. The retina and pineal organ are major receptive organs with projections to various parts of the brain, but in the past decades deep brain photoreceptors have emerged as candidates for photoreceptive inputs, either independent or in combination with projections from light sensory organs. This study aimed to test the effects of narrow bandwidth light using light-emitting diodes technology on brain neural activity through putative opsin stimulation in Atlantic salmon. The expression of c-fos, a known marker of neural activity, was compared in situ between dark-adapted salmon parr and following light stimulation with different wavelengths. c-fos expression increased with duration of light stimulation and the strongest signal was obtained in fish exposed to light for 120 minutes. Distinct and specific brain regions were activated following dark to light stimulation, such as the habenula, suprachiasmatic nucleus, thalamus, and hypothalamus. The c-fos expression was overlapping with photoreceptors expressing melanopsin and/or vertebrate ancient opsin, suggesting a potential direct activation by light. Interestingly in the habenula, a distinct ring of vertebrate ancient opsin and melanopsin expressing cells is overlapping with c-fos expression after neural activation. Salmon exposed to different spectra had neural activation in similar brain regions. The most apparent difference was melanopsin expression in the lateral cells of the lateral tuberal nuclus in the hypothalamus, which appeared to be specifically activated by red light. Light-stimulated neuronal activity in the deep brain was limited to subpopulations of neurons, mainly in regions with neuronal modulation activity, retinal and pineal innervations and known presence of nonvisual photoreceptors. The overlapping expression patterns of c-fos and nonvisual opsins support direct light stimulation of deep brain photoreceptors and the importance of these systems in light induced brain activity.

Effects of environmental enrichment on forebrain neural plasticity and survival success of stocked Atlantic salmon.

Fish reared for stocking programmes are severely stimulus deprived compared with their wild conspecifics raised under natural conditions. This leads to reduced behavioural plasticity and low post-release survival of stocked fish. Environmental enrichment can have positive effects on important life skills, such as predator avoidance and foraging behaviour, but the neural mechanisms underpinning these behavioural changes are still largely unknown. In this study, juvenile Atlantic salmon (Salmo salar) were reared in an enriched hatchery environment for 7 weeks, after which neurobiological characteristics and post-release survival were compared with those of fish reared under normal hatchery conditions. Using in situ hybridization and qPCR, we quantified the expression of brain-derived neurotrophic factor (bdnf) and the neural activity marker cfos in telencephalic subregions associated with relational memory, emotional learning and stress reactivity. Aside from lower expression of bdnf in the Dlv (a region associated with relational memory) of enriched salmon, we observed no other significant effects of enrichment in the studied regions. Exposure to an enriched environment increased post-release survival during a 5 month residence in a natural river by 51%. Thus, we demonstrate that environmental enrichment can improve stocking success of Atlantic salmon parr and that environmental enrichment is associated with changes in bdnf expression in the fish's hippocampus-equivalent structure.

Phylogenetic Reclassification of Vertebrate Melatonin Receptors To Include Mel1d.

The circadian and seasonal actions of melatonin are mediated by high affinity G-protein coupled receptors (melatonin receptors, MTRs), classified into phylogenetically distinct subtypes based on sequence divergence and pharmacological characteristics. Three vertebrate MTR subtypes are currently described: MT1 (MTNR1A), MT2 (MTNR1B), and Mel1c (MTNR1C / GPR50), which exhibit distinct affinities, tissue distributions and signaling properties. We present phylogenetic and comparative genomic analyses supporting a revised classification of the vertebrate MTR family. We demonstrate four ancestral vertebrate MTRs, including a novel molecule hereafter named Mel1d. We reconstructed the evolution of each vertebrate MTR, detailing genetic losses in addition to gains resulting from whole genome duplication events in teleost fishes. We show that Mel1d was lost separately in mammals and birds and has been previously mistaken for an MT1 paralogue. The genetic and functional diversity of vertebrate MTRs is more complex than appreciated, with implications for our understanding of melatonin actions in different taxa. The significance of our findings, including the existence of Mel1d, are discussed in an evolutionary and functional context accommodating a robust phylogenetic assignment of MTR gene family structure.

Proactive avoidance behaviour and pace-of-life syndrome in Atlantic salmon.

Individuals in a fish population differ in key life-history traits such as growth rate and body size. This raises the question of whether such traits cluster along a fast-slow growth continuum according to a pace-of-life syndrome (POLS). Fish species like salmonids may develop a bimodal size distribution, providing an opportunity to study the relationships between individual growth and behavioural responsiveness. Here we test whether proactive characteristics (bold behaviour coupled with low post-stress cortisol production) are related to fast growth and developmental rate in Atlantic salmon, Salmo salar. Boldness was tested in a highly controlled two-tank hypoxia test were oxygen levels were gradually decreased in one of the tanks. All fish became inactive close to the bottom at 70% oxygen saturation. At 40% oxygen saturation level a fraction of the fish actively sought to avoid hypoxia. A proactive stress coping style was verified by lower cortisol response to a standardized stressor. Two distinct clusters of bimodal growth trajectories were identified, with fast growth and early smoltification in 80% of the total population. There was a higher frequency of proactive than reactive individuals in this fast-developing fraction of fish. The smolts were associated with higher post-stress plasma cortisol than parr, and the proactive smolts leaving hypoxia had significant lower post-stress cortisol than the stayers. The study demonstrated a link between a proactive coping and fast growth and developmental ratio and suggests that selection for domestic production traits promotes this trait cluster.

Transient photoreception in the hindbrain is permissive to the life history transition of hatching in Atlantic halibut.

In nonmammalian vertebrates, photoreception takes place in the deep brain already early in development, but knowledge is lacking about the functions of these nonvisual photoreceptive systems. Prior to hatching, Atlantic halibut has a transient bilateral cluster of photoreceptive cells in the hindbrain. The cluster is imbedded in a neuronal network projecting to the narrow belt of hatching glands in the yolk sac. In halibut, hatching is inhibited in light and activated by transfer to darkness and c-fos analysis during hatching shows that the hindbrain cluster and hatching glands have neural activation. Unexpectedly, the hindbrain cluster expresses dual photopigments, vertebrate ancient opsin and melanopsin. Evolutionarily, these opsins are believed to belong to different classes of photopigments found in rhabdomeric and ciliary photoreceptors. The concept that an organism develops transient light sensitivity to target critical aspects of life history transitions as hatching provides a fascinating landscape to investigate the timing of other biological events.

Allostatic Load and Stress Physiology in European Seabass (Dicentrarchus labrax L.) and Gilthead Seabream (Sparus aurata L.).

The present study aimed to compare effects of increasing chronic stress load on the stress response of European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) to identify neuroendocrine functions that regulate this response. Fish were left undisturbed (controls) or exposed to three levels of chronic stress for 3 weeks and then subjected to an acute stress test (ACT). Chronic stress impeded growth and decreased feed consumption in seabass, not in seabream. In seabass basal cortisol levels are high and increase with stress load; the response to a subsequent ACT decreases with increasing (earlier) load. Basal cortisol levels in seabream increase with the stress load, whereas the ACT induced a similar response in all groups. In seabass and seabream plasma α-MSH levels and brain stem serotonergic activity and turnover were similar and not affected by chronic stress. Species-specific molecular neuro-regional differences were seen. In-situ hybridization analysis of the early immediate gene cfos in the preoptic area showed ACT-activation in seabream; in seabass the expression level was not affected by ACT and seems constitutively high. In seabream, expression levels of telencephalic crf, crfbp, gr1, and mr were downregulated; the seabass hypothalamic preoptic area showed increased expression of crf and gr1, and decreased expression of mr, and this increased the gr1/mr ratio considerably. We substantiate species-specific physiological differences to stress coping between seabream and seabass at an endocrine and neuroendocrine molecular level. Seabass appear less resilient to stress, which we conclude from high basal activities of stress-related parameters and poor, or absent, responses to ACT. This comparative study reveals important aquaculture, husbandry, and welfare implications for the rearing of these species.

Comparison between Atlantic salmon Salmo salar post-smolts reared in open sea cages and in the Preline raceway semi-closed containment aquaculture system.

The use of closed containment (CCS) or semi-closed containment systems (S-CCS) for Atlantic salmon Salmo salar aquaculture is under evaluation in Norway. One such system is the Preline S-CCS, a floating raceway system that pumps water from 35 m depth creating a constant current through the system. Exposing fish to moderate water currents is considered aerobic exercise and it is often perceived as positive for fish welfare, growth, food utilization, muscle development and cardiac health. The present study compared fish reared in the Preline S-CCS and in a reference open pen. Samples were taken in fresh water before being transferred to the seawater systems and after 1, 2 and 4 months in seawater and analysed for growth, mortality, muscle development and plasma insulin-like growth factor I (IGF-I) levels. Moreover, gene transcription were determined in the skeletal muscle [igf-I, insulin-like growth factor 1 receptor a (igf1ra) and insulin-like growth factor 1 binding protein 1a (igf1bp1a)] and cardiac transcription factors [myocyte-specific enhancer factor 2C (mef2c), gata4 and vascular endothelial growth factor (vegf)]. While the results suggest that post-smolts in Preline S-CCS were smaller than reference fish, fish from Preline S-CCS have less accumulated mortality at the end of the experiment and showed 2.44 times more small muscle fibres than the reference group fish after 4 months in seawater. These results confirmed what was previously observed in the second generation of Preline. Similar levels of big muscle fibres between Preline S-CCS and reference suggest a similar hypertrophy of muscle fibres even with lower IGF-I expression in the Preline S-CCS. Cardiac gene transcription suggests cardiac hypertrophy was observed after 4 months in seawater in the Preline S-CCS group. Altogether, Preline S-CCS is a promising technology able to produce more robust S. salar with a faster growth and lower mortality in the subsequent standard open cage system growth period.

Early life stress induces long-term changes in limbic areas of a teleost fish: the role of catecholamine systems in stress coping.

Early life stress (ELS) shapes the way individuals cope with future situations. Animals use cognitive flexibility to cope with their ever-changing environment and this is mainly processed in forebrain areas. We investigated the performance of juvenile gilthead seabream, previously subjected to an ELS regime. ELS fish showed overall higher brain catecholaminergic (CA) signalling and lower brain derived neurotrophic factor (bdnf) and higher cfos expression in region-specific areas. All fish showed a normal cortisol and serotonergic response to acute stress. Brain dopaminergic activity and the expression of the α2Α adrenergic receptor were overall higher in the fish homologue to the lateral septum (Vv), suggesting that the Vv is important in CA system regulation. Interestingly, ELS prevented post-acute stress downregulation of the α2Α receptor in the amygdala homologue (Dm3). There was a lack of post-stress response in the ß2 adrenergic receptor expression and a downregulation in bdnf in the Dm3 of ELS fish, which together indicate an allostatic overload in their stress coping ability. ELS fish showed higher neuronal activity (cfos) post-acute stress in the hippocampus homologue (Dlv) and the Dm3. Our results show clear long-term effects on limbic systems of seabream that may compromise their future coping ability to environmental challenges.

How do individuals cope with stress? Behavioural, physiological and neuronal differences between proactive and reactive coping styles in fish.

Despite the use of fish models to study human mental disorders and dysfunctions, knowledge of regional telencephalic responses in non-mammalian vertebrates expressing alternative stress coping styles is poor. As perception of salient stimuli associated with stress coping in mammals is mainly under forebrain limbic control, we tested region-specific forebrain neural (i.e. mRNA abundance and monoamine neurochemistry) and endocrine responses under basal and acute stress conditions for previously characterised proactive and reactive Atlantic salmon. Reactive fish showed a higher degree of the neurogenesis marker proliferating cell nuclear antigen (pcna) and dopamine activity under basal conditions in the proposed hippocampus homologue (Dl) and higher post-stress plasma cortisol levels. Proactive fish displayed higher post-stress serotonergic signalling (i.e. higher serotonergic activity and expression of the 5-HT1A receptor) in the proposed amygdala homologue (Dm), increased expression of the neuroplasticity marker brain-derived neurotropic factor (bdnf) in both Dl and the lateral septum homologue (Vv), as well as increased expression of the corticotropin releasing factor 1 (crf1 ) receptor in the Dl, in line with active coping neuro-profiles reported in the mammalian literature. We present novel evidence of proposed functional equivalences in the fish forebrain with mammalian limbic structures.

The Ontogeny and Brain Distribution Dynamics of the Appetite Regulators NPY, CART and pOX in Larval Atlantic Cod (Gadus morhua L.).

Similar to many marine teleost species, Atlantic cod undergo remarkable physiological changes during the early life stages with concurrent and profound changes in feeding biology and ecology. In contrast to the digestive system, very little is known about the ontogeny and the localization of the centers that control appetite and feed ingestion in the developing brain of fish. We examined the expression patterns of three appetite regulating factors (orexigenic: neuropeptide Y, NPY; prepro-orexin, pOX and anorexigenic: cocaine- and amphetamine-regulated transcript, CART) in discrete brain regions of developing Atlantic cod using chromogenic and double fluorescent in situ hybridization. Differential temporal and spatial expression patterns for each appetite regulator were found from first feeding (4 days post hatch; dph) to juvenile stage (76 dph). Neurons expressing NPY mRNA were detected in the telencephalon (highest expression), diencephalon, and optic tectum from 4 dph onward. CART mRNA expression had a wider distribution along the anterior-posterior brain axis, including both telencephalon and diencephalon from 4 dph. From 46 dph, CART transcripts were also detected in the olfactory bulb, region of the nucleus of medial longitudinal fascicle, optic tectum and midbrain tegmentum. At 4 and 20 dph, pOX mRNA expression was exclusively found in the preoptic region, but extended to the hypothalamus at 46 and 76 dph. Co-expression of both CART and pOX genes were also observed in several hypothalamic neurons throughout larval development. Our results show that both orexigenic and anorexigenic factors are present in the telencephalon, diencephalon and mesencephalon in cod larvae. The telencephalon mostly contains key factors of hunger control (NPY), while the diencephalon, and particularly the hypothalamus may have a more complex role in modulating the multifunctional control of appetite in this species. As the larvae develop, the overall progression in temporal and spatial complexity of NPY, CART and pOX mRNAs expression might be correlated to the maturation of appetite control regulation. These observations suggest that teleost larvae continue to develop the regulatory networks underlying appetite control after onset of exogenous feeding.

Effects of Two Sublethal Concentrations of Mercury Chloride on the Morphology and Metallothionein Activity in the Liver of Zebrafish (Danio rerio).

Mercury (Hg) is a highly hazardous pollutant widely used in industrial, pharmaceutical and agricultural fields. Mercury is found in the environment in several forms, elemental, inorganic (iHg) and organic, all of which are toxic. Considering that the liver is the organ primarily involved in the regulation of metabolic pathways, homeostasis and detoxification we investigated the morphological and ultrastructural effects in Danio rerio liver after 96 h exposure to two low HgCl2 concentrations (7.7 and 38.5 µg/L). We showed that a short-term exposure to very low concentrations of iHg severely affects liver morphology and ultrastructure. The main effects recorded in this work were: cytoplasm vacuolization, decrease in both lipid droplets and glycogen granules, increase in number of mitochondria, increase of rough endoplasmic reticulum and pyknotic nuclei. Pathological alterations observed were dose dependent. Trough immunohistochemistry, in situ hybridization and real-time PCR analysis, the induction of metallothionein (MT) under stressor conditions was also evaluated. Some of observed alterations could be considered as a general response of tissue to heavy metals, whereas others (such as increased number of mitochondria and increase of RER) may be considered as an adaptive response to mercury.

A comparative study of the response to repeated chasing stress in Atlantic salmon (Salmo salar L.) parr and post-smolts.

When Atlantic salmon parr migrate from fresh water towards the sea, they undergo extensive morphological, neural, physiological and behavioural changes. Such changes have the potential to affect their responsiveness to various environmental factors that impose stress. In this study we compared the stress responses in parr and post-smolt salmon following exposure to repeated chasing stress (RCS) for three weeks. At the end of this period, all fish were challenged with a novel stressor and sampled before (T0) and after 1h (T1). Parr had a higher growth rate than post-smolts. Plasma cortisol declined in the RCS groups within the first week suggesting a rapid habituation/desensitisation of the endocrine stress axis. As a result of the desensitised HPI axis, RCS groups showed a reduced cortisol response when exposed to the novel stressor. In preoptic area (POA) crf mRNA levels were higher in all post-smolt groups compared to parr. 11ßhsd2 decreased by RCS and by the novel stressor in post-smolt controls (T1), whereas no effect of either stress was seen in parr. The grs were low in all groups except for parr controls. In pituitary, parr controls had higher levels of crf1r mRNA than the other parr and post-smolt groups, whilst pomcb was higher in post-smolt control groups. Overall, 11ßhsd2 transcript abundance in parr was lower than post-smolt groups; after the novel stressor pomcs, grs and mr were up-regulated in parr control (T1). In summary, we highlight differences in the central stress response between parr and post-smolt salmon following RCS.

Fatty acids linked to cardiovascular mortality are associated with risk factors.

BACKGROUND: Although saturated fatty acids (FAs) have been linked to cardiovascular mortality, it is not clear whether this outcome is attributable solely to their effects on low-density lipoprotein cholesterol (LDL-C) or whether other risk factors are also associated with FAs. The Western Alaskan Native population, with its rapidly changing lifestyles, shift in diet from unsaturated to saturated fatty acids and dramatic increase in cardiovascular disease (CVD), presents an opportunity to elucidate any associations between specific FAs and known CVD risk factors. OBJECTIVE: We tested the hypothesis that the specific FAs previously identified as related to CVD mortality are also associated with individual CVD risk factors. METHODS: In this community-based, cross-sectional study, relative proportions of FAs in plasma and red blood cell membranes were compared with CVD risk factors in a sample of 758 men and women aged ≥35 years. Linear regression analyses were used to analyze relations between specific FAs and CVD risk factors (LDL-C, high-density lipoprotein cholesterol, triglycerides, C-reactive protein, systolic blood pressure, diastolic blood pressure, heart rate, body mass index, fasting glucose and fasting insulin, 2-hour glucose and 2-hour insulin). RESULTS: The specific saturated FAs previously identified as related to CVD mortality, the palmitic and myristic acids, were adversely associated with most CVD risk factors, whereas unsaturated linoleic acid (18:2n-6) and the marine n-3 FAs were not associated or were beneficially associated with CVD risk factors. CONCLUSIONS: The results suggest that CVD risk factors are more extensively affected by individual FAs than hitherto recognized, and that risk for CVD, MI and stroke can be reduced by reducing the intake of palmitate, myristic acid and simple carbohydrates and improved by greater intake of linoleic acid and marine n-3 FAs.

Stress in Atlantic salmon: response to unpredictable chronic stress.

Combinations of stressors occur regularly throughout an animal's life, especially in agriculture and aquaculture settings. If an animal fails to acclimate to these stressors, stress becomes chronic, and a condition of allostatic overload arises with negative results for animal welfare. In the current study, we describe effects of exposing Atlantic salmon parr to an unpredictable chronic stressor (UCS) paradigm for 3 weeks. The paradigm involves exposure of fish to seven unpredictable stressors three times a day. At the end of the trial, experimental and control fish were challenged with yet another novel stressor and sampled before and 1 h after that challenge. Plasma cortisol decreased steadily over time in stressed fish, indicative of exhaustion of the endocrine stress axis. This was confirmed by a lower cortisol response to the novel stressor at the end of the stress period in chronically stressed fish compared with the control group. In the preoptic area (POA) and pituitary gland, chronic stress resulted in decreased gene expression of 11ßhsd2, gr1 and gr2 in the POA and increased expression of those genes in the pituitary gland. POA crf expression and pituitary expression of pomcs and mr increased, whereas interrenal gene expression was unaffected. Exposure to the novel stressor had no effect on POA and interrenal gene expression. In the pituitary, crfr1, pomcs, 11ßhsd2, grs and mr were down-regulated. In summary, our results provide a novel overview of the dynamic changes that occur at every level of the hypothalamic-pituitary gland-interrenal gland (HPI) axis as a result of chronic stress in Atlantic salmon.

Functional divergence of type 2 deiodinase paralogs in the Atlantic salmon.

Thyroid hormone (TH) is an ancestral signal linked to seasonal life history transitions throughout vertebrates. TH action depends upon tissue-localized regulation of levels of active TH (triiodothyronine, T3), through spatiotemporal expression of thyroid hormone deiodinase (dio) genes. We investigated the dio gene family in juvenile Atlantic salmon (Salmo salar) parr, which prepare for seaward migration in the spring (smoltification) through TH-dependent changes in physiology. We identified two type 2 deiodinase paralogs, dio2a and dio2b, responsible for conversion of thyroxine (T4) to T3. During smoltification, dio2b was induced in the brain and gills in zones of cell proliferation following increasing day length. Contrastingly, dio2a expression was induced in the gills by transfer to salt water (SW), with the magnitude of the response proportional to the plasma chloride level. This response reflected a selective enrichment for osmotic response elements (OREs) in the dio2a promoter region. Transcriptomic profiling of gill tissue from fish transferred to SW plus or minus the deiodinase inhibitor, iopanoic acid, revealed SW-induced increases in cellular respiration as the principal consequence of gill dio2 activity. Divergent evolution of dio2 paralogs supports organ-specific timing of the TH-dependent events governing the phenotypic plasticity required for migration to sea.

Apolipoprotein E isoforms 3/3 and 3/4 differentially interact with circulating stearic, palmitic, and oleic fatty acids and lipid levels in Alaskan Natives.

Lifestyle changes in Alaskan Natives have been related to the increase of cardiovascular disease and metabolic syndrome in the last decades. Variation of the apolipoprotein E (Apo E) genotype may contribute to the diverse response to diet in lipid metabolism and influence the association between fatty acids in plasma and risk factors for cardiovascular disease. The aim of this investigation was to analyze the interaction between Apo E isoforms and plasma fatty acids, influencing phenotypes related to metabolic diseases in Alaskan Natives. A sample of 427 adult Siberian Yupik Alaskan Natives was included. Fasting glucose, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, Apo A1, and Apo B plasma concentrations were measured using reference methods. Concentrations of 13 fatty acids in fasting plasma were analyzed by gas chromatography, and Apo E variants were identified. Analyses of covariance were conducted to identify Apo E isoform and fatty acid main effects and multiplicative interactions. The means for body mass index and age were 26 ± 5.2 and 47 ± 1.5, respectively. Significant main effects were observed for variation in Apo E and different fatty acids influencing Apo B levels, triglycerides, and total cholesterol. Significant interactions were found between Apo E isoform and selected fatty acids influencing total cholesterol, triglycerides, and Apo B concentrations. In summary, Apo E3/3 and 3/4 isoforms had significant interactions with circulating levels of stearic, palmitic, oleic fatty acids, and phenotypes of lipid metabolism in Alaskan Natives.

The effects of environmental enrichment and age-related differences on inhibitory avoidance in zebrafish (Danio rerio Hamilton).

The inhibitory avoidance paradigm allows the study of mechanisms underlying learning and memory formation in zebrafish (Danio rerio Hamilton). For zebrafish, the physiology and behavior associated with this paradigm are as yet poorly understood. We therefore assessed the effects of environmental enrichment and fish age on inhibitory avoidance learning. Fish raised in an environmentally enriched tank showed decreased anxiety-like behavior and increased exploration. Enrichment greatly reduced inhibitory avoidance in 6-month (6M)- and 12-month (12 M)-old fish. Following inhibitory avoidance, telencephalic mRNA levels of proliferating cell nuclear antigen (pcna), neurogenic differentiation (neurod), cocaine- and amphetamine-regulated transcript 4 (cart4), and cannabinoid receptor 1 (cnr1) were lower in enriched-housed fish, while the ratios of mineralocorticoid receptor (nr3c2)/glucocorticoid receptor α [nr3c1(α)] and glucocorticoid receptor ß [nr3c1(ß)]/glucocorticoid receptor α [nr3c1(α)] were higher. This was observed for 6M-old fish only, not for 24-month (24 M) old fish. Instead, 24 M-old fish showed delayed inhibitory avoidance, no effects of enrichment, and reduced expression of neuroplasticity genes. Overall, our data show strong differences in inhibitory avoidance behavior between zebrafish of different ages and a clear reduction in avoidance behavior following housing under environmental enrichment.

Early steps in the European eel (Anguilla anguilla)-Vibrio vulnificus interaction in the gills: role of the RtxA13 toxin.

Vibrio vulnificus is an aquatic gram-negative bacterium that causes a systemic disease in eels called warm-water vibriosis. Natural disease occurs via water born infection; bacteria attach to the gills (the main portal of entry) and spread to the internal organs through the bloodstream, provoking host death by haemorrhagic septicaemia. V. vulnificus produces a toxin called RtxA13 that hypothetically interferes with the eel immune system facilitating bacterial invasion and subsequent death by septic shock. The aim of this work was to study the early steps of warm-water vibriosis by analysing the expression of three marker mRNA transcripts related to pathogen recognition (tlr2 and tlr5) and inflammation (il-8) in the gills of eels infected by immersion with either the pathogen or a mutant deficient in rtxA13. Results indicate a differential response that is linked to the rtx toxin in the expression levels of the three measured mRNA transcripts. The results suggest that eels are able to distinguish innocuous from harmful microorganisms by the local action of their toxins rather than by surface antigens. Finally, the cells that express these transcripts in the gills are migratory cells primarily located in the second lamellae that re-locate during infection suggesting the activation of a specific immune response to pathogen invasion in the gill.

Unpredictable chronic stress decreases inhibitory avoidance learning in Tuebingen long-fin zebrafish: stronger effects in the resting phase than in the active phase.

Zebrafish (Danio rerio Hamilton) are increasingly used as a model to study the effects of chronic stress on brain and behaviour. In rodents, unpredictable chronic stress (UCS) has a stronger effect on physiology and behaviour during the active phase than during the resting phase. Here, we applied UCS during the daytime (active phase) for 7 and 14 days or during the night-time (resting phase) for 7 nights in an in-house-reared Tuebingen long-fin (TLF) zebrafish strain. Following UCS, inhibitory avoidance learning was assessed using a 3 day protocol where fish learn to avoid swimming from a white to a black compartment where they will receive a 3 V shock. Latencies of entering the black compartment were recorded before training (day 1; first shock) and after training on day 2 (second shock) and day 3 (no shock, tissue sampling). Fish whole-body cortisol content and expression levels of genes related to stress, fear and anxiety in the telencephalon were quantified. Following 14 days of UCS during the day, inhibitory avoidance learning decreased (lower latencies on days 2 and 3); minor effects were found following 7 days of UCS. Following 7 nights of UCS, inhibitory avoidance learning decreased (lower latency on day 3). Whole-body cortisol levels showed a steady increase compared with controls (100%) from 7 days of UCS (139%), to 14 days of UCS (174%) to 7 nights of UCS (231%), suggestive of an increasing stress load. Only in the 7 nights of UCS group did expression levels of corticoid receptor genes (mr, grα, grß) and of bdnf increase. These changes are discussed as adaptive mechanisms to maintain neuronal integrity and prevent overload, and as being indicative of a state of high stress load. Overall, our data suggest that stressors during the resting phase have a stronger impact than during the active phase. Our data warrant further studies on the effect of UCS on stress axis-related genes, especially grß; in mammals this receptor has been implicated in glucocorticoid resistance and depression.