Lluis Tort

Stress and immune modulation in fish

Stress is an event that most animals experience and that induces a number of responses involving all three regulatory systems, neural, endocrine and immune. When the stressor is acute and short-term, the response pattern is stimulatory and the fish immune response shows an activating phase that specially enhances innate responses. If the stressor is chronic the immune response shows suppressive effects and therefore the chances of an infection may be enhanced. In addition, coping with the stressor imposes an allostatic cost that may interfere with the needs of the immune response. In this paper the mechanisms behind these immunoregulatory changes are reviewed and the role of the main neuroendocrine mechanisms directly affecting the building of the immune response and their consequences are considered.

Crowding stress induces changes in serum haemolytic and agglutinating activity in the gilthead sea bream Sparus aurata

Sea bream Sparus aurata were subjected to crowding for 3 weeks and the levels of plasma cortisol and glucose were determined in order to ascertain the occurrence of stress. At the same time, selected indicators of the immune response were monitored through blood lymphocyte counting and selected humoral responses such as the haemagglutination activity (HA) of serum towards rabbit erythrocytes (RaRBC) and the alternative complement pathway (ACP) levels. The results show an initial moderate increase in cortisol followed by hyperglycaemia and recovery to basal levels. A pattern of immunodepression was detected afterwards as shown by decreases in ACP levels after 5 days and haemagglutination titre and circulating lymphocytes after 9 days. Nevertheless, ACP levels showed a significant increase after 9 days, and HA titres and circulating lymphocyte numbers also increased after 15 days. Normal values were recovered after 15 days for lymphocytes and ACP and 21 days for agglutination. Results are discussed in view of the incidence of stress on indicative parameters of endocrine and immune systems.

Effect of vitamin E and C dietary supplementation on some immune parameters of gilthead seabream (Sparus aurata) juveniles subjected to crowding stress

Abstract High stocking density is an aquaculture-related situation which causes crowding stress in fish and potentially affecting the immune system. Protection against disease can be induced through immunostimulation, enhancing defence mechanisms. Vitamins C and E are among the most important nutrients influencing the immune system, although the effect of these vitamins in the amelioration of stress in fish has not been studied in detail. An experiment was carried out to study the effect dietary supplementation of vitamin C or vitamin E on the immune system of fish under a crowding stress situation in a commercial farm. A commercial diet for this species was supplemented with vitamin C or vitamin E and these diets were assayed at high stocking density (over 40 kg m−3) and the results were compared with those from fish fed the commercial diet and held at farm conditions (20 kg m−3). Some biological and immunological parameters were studied at the end of the experimental period. Fish held at high stocking density showed lower final weight than fish held at lower stocking density. These also showed higher plasma cortisol levels (over 6 ng cortisol ml−1 plasma for all the diets assayed) than those fish held at lower density (1.13 ng cortisol ml−1 plasma). High stocking density produced higher serum lysozyme activity values in fish fed the commercial diet compared with those fish held at lower density (67.49 and 36.84 units ml−1, respectively). This effect was not observed in fish held at high stocking density but fed vitamin C and vitamin E supplemented diets (lysozyme activity: 21.97 and 19.71 units ml−1, respectively). High stocking density also appeared to decrease serum alternative complement activity. In those fish fed the vitamin E supplemented diet, levels were similar to fish held at low stocking density, suggesting the role of this vitamin in this defense mechanism.

Cortisol and finfish welfare

Previous reviews of stress, and the stress hormone cortisol, in fish have focussed on physiology, due to interest in impacts on aquaculture production. Here, we discuss cortisol in relation to fish welfare. Cortisol is a readily measured component of the primary (neuroendocrine) stress response and is relevant to fish welfare as it affects physiological and brain functions and modifies behaviour. However, we argue that cortisol has little value if welfare is viewed purely from a functional (or behavioural) perspective—the cortisol response itself is a natural, adaptive response and is not predictive of coping as downstream impacts on function and behaviour are dose-, time- and context-dependent and not predictable. Nevertheless, we argue that welfare should be considered in terms of mental health and feelings, and that stress in relation to welfare should be viewed as psychological, rather than physiological. We contend that cortisol can be used (with caution) as a tractable indicator of how fish perceive (and feel about) their environment, psychological stress and feelings in fish. Cortisol responses are directly triggered by the brain and fish studies do indicate cortisol responses to psychological stressors, i.e., those with no direct physicochemical action. We discuss the practicalities of using cortisol to ask the fish themselves how they feel about husbandry practices and the culture environment. Single time point measurements of cortisol are of little value in assessing the stress level of fish as studies need to account for diurnal and seasonal variations, and environmental and genetic factors. Areas in need of greater clarity for the use of cortisol as an indicator of fish feelings are the separation of (physiological) stress from (psychological) distress, the separation of chronic stress from acclimation, and the interactions between feelings, cortisol, mood and behaviour.

Background colour influence on the stress response in cultured red porgy Pagrus pagrus

Red porgy Pagrus pagrus were placed and maintained in white, grey and black background fibreglass tanks for 2 weeks. Additionally, fish kept in white and black background tanks were then subjected to crowding stress. After 2, 9, 16 and 23 days, blood samples were taken and plasma cortisol, alpha melanocyte stimulating hormone (α-MSH) and glucose values were analysed and compared with values from uncrowded fish from white or black tanks. Measurements of plasma cortisol and α-MSH in unstressed red porgy from white, grey and black tanks revealed no significant differences among the three groups. However, the results show that background colour markedly affects the in vitro interrenal sensitivity to both α-MSH and ACTH, as interrenal cells from black adapted fish had become virtually unresponsive to both secretagogues. Crowded fish on a black background showed a prominent increase in plasma cortisol after 2 days, which was followed by a recovery. In fish crowded on a white background however, the increase of cortisol was lower but was maintained through the entire experiment. Plasma α-MSH levels increased at 23 days as a consequence of crowding; this increase was also dependent on the background, being more prominent in fish placed in white background tanks. Thus, in the red porgy, a white background appears to modify the stress response, particularly in the long term.

Serum haemolytic and agglutinating activity as indicators of fish immunocompetence: their suitability in stress and dietary studies

Gilthead sea bream, Sparus aurata, were subjected to different stressful situations and the haemolytic activity mediated by the alternative complement and the agglutinating serum activity were tested as indicators of immunocompetence. The results show that both parameters were significantly compromised after chronic or repeated acute stress as well as in groups fed with vitamin E or Ω3 highly unsaturated fatty acids (HUFA) deficient diets and in infected fish. These results suggest that both haemolytic and agglutinating activity are suitable indicators for immunocompetence as they are non-specific responses induced by non-specific stressors. In addition, a comparison is performed with other immune indicators subjected to the same stressors.

Non-specific immune responses in the red porgy Pagrus pagrus after crowding stress

Abstract Red porgy (Pagrus pagrus) were exposed to crowding stress for 23 days. The responses measured were plasma glucose and cortisol concentrations, basic haematological variables and non-specific responses of the immune system: haemolytic activity of serum based on the alternative complement pathway (ACP), lysozyme and haemagglutinating activity of serum, total immunoglobulin concentration, and number of circulating lymphocytes. The results showed an initial moderate increase in cortisol followed by hyperglycaemia, and a further decrease of both variables over the following days. Immunodepression was apparent as shown by decreases in ACP levels and circulating lymphocytes after 2 days and haemagglutination titre after 9 days. Later samples showed a recovery of basal levels for lymphocyte numbers and haemagglutination titre and a significant increase of lysozyme levels after 16 days. Haemolytic levels did not recover at the end of the experiment. Results are discussed in view of the incidence of stress on indicative parameters of endocrine and immune systems in this species of recent interest for aquaculture.

Cholinergic and Adrenergic Tones in the Control of Heart Rate in Teleosts. How Should They be Calculated

Cholinergic and adrenergic tones were calculated for three different teleost fish species: Gadus morhua, Labrus bergylta, and Sparus aurata using atropine as a muscarinic receptor antagonist and either sotalol or propranolol as β-adrenoceptor antagonists. Depending on the order of administration of atropine and the two β-adrenoceptor antagonists, it was observed that propranolol but not sotalol enhanced cholinergic tone. Thus, if propranolol is used to determine autonomic cardiac influences, it has to be injected after atropine and not before. Differences in intrinsic heart rate were observed between treatments in two of the three species studied, suggesting the activity of a non-cholinergic non-adrenergic mechanism in heart rate control in fish. Different models to calculate cholinergic and adrenergic tones are discussed. The additive model described by other authors is appropriate provided that no interaction exists between cholinergic and adrenergic influences. We demonstrate no interaction in the species studied in this experiment. Finally, a modification of the additive model that uses R-R interval instead of heart rate in the computation is proposed. This is justified with a computer simulation in terms of the linearity of the response given the reciprocal relationship between R-R interval and heart rate. comp biochem physiol 118A;1:131-139, 1997.

Stress-related hormones modulate cytokine expression in the head kidney of gilthead seabream (Sparus aurata)

Neuro-endocrine and immune systems closely interact in fish, and their regulation is crucial for the maintenance of good health of cultured fish. We have used the seabream head kidney to study whether stress-related hormones can modulate the immune response. For this purpose, the effects of adrenaline, adrenocorticotropic hormone (ACTH) and cortisol on the expression of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and the anti-inflammatory cytokine TGF-beta1 were determined by means of quantitative real-time PCR on isolated head kidney cells. ACTH (150 ng mL(-1)) caused an acute increase of TNF-alpha and IL-6 mRNA levels as well as an inhibition of IL-1beta expression. The expression of the anti-inflammatory cytokine TGF-beta1 was also increased, although in a lower extent. Adrenaline (1 muM) early effects were only clear inhibiting IL-1beta expression but not TNF-alpha, IL-6 or TGF-beta1 mRNA levels, while a longer exposure to the hormone inhibited all cytokines. Moreover, cortisol (50 and 100 ng mL(-1)) reduced the expression of all cytokines in a dose-dependent manner. Bacterial lipopolysaccharide (LPS) stimulated IL-1beta expression and inhibited that of the anti-inflammatory TGF-beta1, although it was ineffective on TNF-alpha and IL-6. In addition, adrenaline and cortisol decreased the LPS-stimulated IL-1beta expression, further demonstrating their previously reported anti-inflammatory effects. The combination of ACTH and LPS, on the other hand, did not affect LPS-stimulated IL-1beta expression but was effective increasing TNF-alpha expression. Taking all these results in consideration, we conclude that the expression of pro- and anti-inflammatory cytokines in the seabream head kidney is highly influenced by stress-related hormones, thus indicating an important role for the endocrine system in the modulation of the immune response in teleost fish.

Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes

We measured Ca2+ uptake by the sarcoplasmic reticulum (SR) in trout ventricular myocytes, measuring indo 1 fluorescence in permeabilized cells or ionic currents in single myocytes subjected to voltage clamp. Titration of the SR Ca2+ pumps with thapsigargin gave a pump site density of 454 pmol/mg cell protein. Lowering the temperature from 20°C to 10 or 5°C reduced the SR Ca2+ uptake rate in permeabilized myocytes by 50 and 63%, respectively. Surprisingly, Ca2+ leak from the SR also decreased with decreasing temperatures. Exposure of single myocytes to 10 mM caffeine (Caf) induced a cell contracture and an inward ionic current. Neither contracture nor current decreased significantly after rest periods of 120 and 320 s. The inward current was due to Ca2+ extrusion by the Na+/Ca2+exchanger (NCX), and the time integral of the exchange current ( I NCX) was used to calculate the SR Ca2+ content. This gave a steady-state SR Ca2+content of 22.5 ± 2.8 amol Ca2+/pF or 750 μM. When the SR was loaded by depolarizing the cell to +50 mV, the Ca2+ content increased with increasing length of the depolarization, reaching a maximum of 52.0 ± 5.9 amol Ca2+/pF. When the cell was depolarized to different voltages for 3 s, a subsequent Caf-induced I NCXincreased with increasing voltage. At +100 mV, the Ca2+ content was 36.6 ± 3.8 amol/pF, giving a maximal SR Ca2+uptake rate of 12.2 ± 1.2 amol Ca2+ ⋅ pF-1 ⋅ s-1or 417 μM/s. We conclude that maximal SR Ca2+ content and Ca2+ uptake rates can be estimated using specific SR Ca2+ loading protocols. Contrary to the general assumption that contraction in lower vertebrates depends largely on transsarcolemmal Ca2+ fluxes, we found that although the L-type Ca2+ current is insufficient to fully activate contraction, the SR is capable of participating in the regulation of the cytosolic Ca2+ during the excitation-contraction coupling in trout ventricular myocytes.