Dietary ß-glucans differentially modulate immune and stress-related gene expression in lymphoid organs from healthy and Aeromonas hydrophila-infected rainbow trout (Oncorhynchus mykiss).

Although ß-glucans stimulating effects have already been demonstrated on the immune system of numerous animal species, available data remain relatively variable and more research should be done regarding the complexity of underlying mechanisms. In this context, the present study aimed to evaluate the stress and immune-related effects of dietary ß-glucans (i.e. Macrogard®) by considering a number of influencing factors such as the dose (0, 0.1, 0.2 and 0.5% in food), feeding duration (15 versus 30 days), tissue (blood, kidney, spleen, gills) and infection status (healthy or infected). Blood parameters (lysozyme, ACH50 activities, leucocyte populations) and mRNA expression level of several immune- and stress-related genes (TFN-α1, IL-1ß, IL10, COX-2, TGF-ß, MC2R, HSP70) were measured. Our results suggest that spleen may be a highly responsive organ to dietary ß-glucans both in healthy or infected fish, and that this organ may therefore significantly contribute to the immune reinforcement induced by such immunostimulatory diet. Our study further reveals that overdoses of ß-glucans and/or prolonged medication can lead to a non-reactive physiological status and, consequently, to a poor immune response. All in all, the current data emphasizes the need for further extensive research in the field of dietary ß-glucans as a preventive method for farmed fish protection.

Physiological and immune response of juvenile rainbow trout to dietary bovine lactoferrin.

Lactoferrin, a large multifunctional glycoprotein, is involved in many physiological functions but its immunomodulatory pathways are not well characterized in fish. The objective of the present study was to investigate the temporal effect of dietary bovine lactoferrin (BLf) at low (0.1%) and high (1%) on immunological organs of rainbow trout juveniles. BLf diets did not affect specific growth rate, haematocrit, splenic index, spleen respiratory burst activity as well as humoral (mIgM) and neutrophils (MPO) gene expressions after short term - 35 days (D35) and long term nutrient test - 51 days (D51) of feeding. Both low and high BLf doses induced enhanced level of plasma alternative complement activity, plasma total immunoglobulin on D35 and D51, lymphocyte plus thrombocyte cell proportion on D35 and monocyte cell proportion in total blood leukocyte cells on D51. On D51 but not on D35, BLf diets upregulated the expression of inflammatory genes in kidney for il-1 at the low BLf dose, il-8 at both BLf doses and il-6 at the high BLf dose in spleen, and il-10 at both BLf doses in kidney. Moreover, the expression of T helper (cd4-2α; cd4-2ß) genes was significantly upregulated only on D51 by both BLf doses in both spleen and kidney tissues. On D51, controls and BLf treated fish were intraperitoneally injected with A. salmonicida achromogenes. The expression of 13 immune genes was evaluated at 44 h post-injection (D54). The expression of lysozyme gene was upregulated by both BLf doses after bacterial infection both in spleen and kidney. The expression of mcsfrα (spleen) and tgf-ß1 (kidney) was also modulated by both BLf doses. Low and high BLf doses enhanced disease resistance of rainbow trout juveniles with the cumulative survival rate of 36% and 38% respectively while those of the control was 19% after 14 days challenged with bacteria. The results indicate that BLf diets activated the humoral immunity, associated to blood leukocyte cells of rainbow trout after short term BLf administration, and the long term BLf administration was necessary for sensitizing other lymphoid organs such as in spleen and kidney. Only after long term test, BLf diets induced significantly higher levels of innate and adaptive immune gene expressions than those of the control. Dietary BLf activated more markedly the expression of innate immune genes than the adaptive ones; this upregulation of some immune genes could explain the high disease resistance observed in rainbow trout juveniles fed BLf.

Osmoregulatory and immunological status of the pond-raised striped catfish (Pangasianodon hypophthalmus S.) as affected by seasonal runoff and salinity changes in the Mekong Delta, Vietnam.

In Vietnam, the production of striped catfish Pangasianodon hypophthalmus S. reached more than 1.2 millions of tons in 2014 and is mainly gathered in the Mekong Delta (South Vietnam). A survey was carried out during the dry season 2013 (March-April) and rainy season 2014 (July-August) in 12 fish farms of the lower (Tra Vinh Province) and higher (Can Tho Province) Mekong River Delta. This study allowed drawing up current key osmoregulatory and innate immune parameters of striped catfish in aquaculture depending on the geographical location, particularly the proximity to the River Mekong Estuary and the seasonal surface runoff. In the dry season, plasma osmolality was positively correlated with salinity. Gill Na+K+ ATPase dropped in the rainy season, while kidney Na+K+ ATPase remained stable. Abundance of immune blood cells, especially thrombocytes and monocytes, tended to increase in farms located in tidal area. Production of reactive oxygen species in the spleen, kidney lysozyme activity and plasma complement activity did not vary whatever the season or the proximity to the estuary. Plasma lysozyme activity was 50-fold higher than in kidney and increased in the rainy season as well as in tidal sites. Kidney complement activity decreased in the dry season, especially in tidal sites. In conclusion, regarding key osmoregulatory and immune factors, striped catfish farms located in the Mekong Delta may be affected by seasonal and regional salinity and runoff fluctuations.

Chronic hyperosmotic stress interferes with immune homeostasis in striped catfish (Pangasianodon hypophthalmus, S.) and leads to excessive inflammatory response during bacterial infection.

Hyperosmotic stress has often been investigated from osmoregulation perspectives while the effects of such stress on the immune capacity remain largely unexplored. In this study, striped catfish were submitted to three salinity profiles (freshwater, low saline water, saline water) during 20 days, followed by infection with a virulent bacteria, Edwardsiella ictaluri, responsible for the enteric septicaemia of catfish. Osmoregulatory (plasma osmolality, gill Na(+)K(+)ATPase), immune (blood cells, lysozyme activity, complement activity, respiratory burst) parameters and mortality rate were investigated. In addition, abundances of heat shock protein 70 and high mobility group box 1 were explored. With elevated salinity, plasma osmolality severely increased while gill Na(+)K(+)ATPase slightly increased. Salinity alone stimulated the number of granulocytes, lysozyme activity and respiratory burst but depleted the number of thrombocytes. Salinity in combination with infection stimulated the number of monocytes and ACH50. On the contrary, erythrocytes, hematocrit, heat shock protein 70 and high mobility group box 1 did not significantly vary with salinity profiles. Then, salinity induced earlier onset on mortalities after E. ictaluri inoculation whereas cumulative mortality reach 79.2%, 67.0% and 91.7% respectively in freshwater, low saline water and saline water. In conclusion, salinity stimulates several immune functions in striped catfish but prolonged exposure to excessive hyperosmotic condition may lead to excessive inflammatory response and death.

In vivo response of some immune and endocrine variables to LPS in Eurasian perch (Perca fluviatilis, L.) and modulation of this response by two corticosteroids, cortisol and 11-deoxycorticosterone.

In fish, the endocrine system, especially corticosteroids pathway, strongly interacts with immune system. On the other hand, in vivo co-stimulation of both systems is not well documented. To better understand this interaction, we decided to evaluate the in vivo effects of both stimulation of the immune system and co-stimulation of both systems in Eurasian perch juveniles. Fish were injected either with 10mgkg(-1) LPS, or with a combination of LPS and 0.8mgkg(-1) cortisol or LPS and 0.08mgkg(-1) 11-deoxycorticosterone (DOC) and sampled 1, 3 or 7days after injection. LPS affected the immune system by increasing plasma lysozyme activity and blood neutrophils populations. During the same time-course, LPS decreased the proportion of a mixture of lymphocytes and thrombocytes in blood and TNF-α expression in spleen. Cortisol modulated the LPS-mediated response in TNF-α mRNA expression levels in spleen. Contrary to LPS alone, the association of LPS with DOC modulated the abundance of complement component 3 (C3) mRNA in spleen. On the other hand, LPS altered the corticotropic axis by decreasing mRNA expression levels of all corticosteroid receptors and of 11ß-HSD-2 in spleen. Both corticosteroids injected were not able to balance these LPS-induced suppressive effects on corticosteroid receptors and 11ß-HSD-2 expression levels in spleen. Contrary to LPS alone, the association of LPS with DOC modulated GR-1b expression in gills. These results indicated that LPS is a strong modulator of the corticosteroid receptors expression in spleen. Furthermore, we report for the first time a LPS-induced decrease of the mineralocorticoid receptor expression. Finally, corticosteroids were able to modulate the LPS-mediated response at the transcriptional level.

First evidence of the possible implication of the 11-deoxycorticosterone (DOC) in immune activity of Eurasian perch (Perca fluviatilis, L.): comparison with cortisol.

Cortisol, the main corticosteroid in fish, is frequently described as a modulator of fish immune system. Moreover, 11-deoxycorticosterone (DOC) was shown to bind and transcriptionally activate the mineralocorticoid receptor and may act as a mineralocorticoid in fish. Immune modulations induced by intraperitoneal injections of these two corticosteroids were assessed in Eurasian perch juveniles. Cortisol and DOC were injected at 0.8 mg kg(-1) and 0.08 mg kg(-1) body weight respectively. Cortisol increased plasma lysozyme activity 72 h post-injection, C-type lysozyme expression in spleen from 1 to 72 h post-injection, and favoured blood neutrophils at the expense of a mixture of lymphocytes and thrombocytes. Moreover, 6 h after injection, cortisol reduced expression levels of the pro-inflammatory cytokine TNF-α in spleen. DOC had no effects on the immune variables measured in plasma, but increased expression levels of C-type lysozyme and apolipoprotein A1 mRNA in both gills and spleen. Meanwhile, DOC stimulated its putative signalling pathway by increasing expression of mineralocorticoid receptor and 11ß-hydroxysteroid dehydrogenase-2 in spleen. These results confirmed the role of cortisol as an innate, short term immune stimulator. For the first time, DOC is described as a possible immune stimulator in fish.

Physiological and proteomic responses to single and repeated hypoxia in juvenile Eurasian perch under domestication--clues to physiological acclimation and humoral immune modulations.

We evaluated the physiological and humoral immune responses of Eurasian perch submitted to 4-h hypoxia in either single or repeated way. Two generations (F1 and F5) were tested to study the potential changes in these responses with domestication. In both generations, single and repeated hypoxia resulted in hyperglycemia and spleen somatic index reduction. Glucose elevation and lysozyme activity decreased following repeated hypoxia. Complement hemolytic activity was unchanged regardless of hypoxic stress or domestication level. A 2D-DIGE proteomic analysis showed that some C3 components were positively modulated by single hypoxia while C3 up- and down-regulations and over-expression of transferrin were observed following repeated hypoxia. Domestication was associated with a low divergence in stress and immune responses to hypoxia but was accompanied by various changes in the abundance of serum proteins related to innate/specific immunity and acute phase response. Thus, it appeared that the humoral immune system was modulated following single and repeated hypoxia (independently of generational level) or during domestication and that Eurasian perch may display physiological acclimation to frequent hypoxic disturbances.

Evidence that elevated water temperature affects the reproductive physiology of the European bullhead Cottus gobio.

Climate change is predicted to increase the average water temperature and alter the ecology and physiology of several organisms including fish species. To examine the effects of increased water temperature on freshwater fish reproduction, adult European bullhead Cottus gobio of both genders were maintained under three temperature regimes (T1: 6-10, T2: 10-14 and T3: 14-18°C) and assessed for gonad development (gonadosomatic index-GSI and gonad histology), sex steroids (testosterone-T, 17ß-estradiol-E2 and 11-ketotestosterone-11-KT) and vitellogenin (alkali-labile phosphoprotein phosphorus-ALP) dynamics in December, January, February and March. The results indicate that a 8°C rise in water temperature (T3) deeply disrupted the gonadal maturation in both genders. This observation was associated with the absence of GSI peak from January to March, and low levels of plasma sex steroids compared with T1-exposed fish. Nevertheless, exposure to an increasing temperature of 4°C (T2) appeared to accelerate oogenesis with an early peak value in GSI and level of plasma T recorded in January relative to T1-exposed females. In males, the low GSI, reduced level of plasma 11-KT and the absence of GSI increase from January to March support the deleterious effects of increasing water temperature on spermatogenesis. The findings of the present study suggest that exposure to elevated temperatures within the context of climate warming might affect the reproductive success of C. gobio. Specifically, a 4°C rise in water temperature affects gametogenesis by advancing the spawning, and a complete reproductive failure is observed at an elevated temperature of 8°C.

Health of farmed fish: its relation to fish welfare and its utility as welfare indicator.

This brief review focuses on health and biological function as cornerstones of fish welfare. From the function-based point of view, good welfare is reflected in the ability of the animal to cope with infectious and non-infectious stressors, thereby maintaining homeostasis and good health, whereas stressful husbandry conditions and protracted suffering will lead to the loss of the coping ability and, thus, to impaired health. In the first part of the review, the physiological processes through which stressful husbandry conditions modulate health of farmed fish are examined. If fish are subjected to unfavourable husbandry conditions, the resulting disruption of internal homeostasis necessitates energy-demanding physiological adjustments (allostasis/acclimation). The ensuing energy drain leads to trade-offs with other energy-demanding processes such as the functioning of the primary epithelial barriers (gut, skin, gills) and the immune system. Understanding of the relation between husbandry conditions, allostatic responses and fish health provides the basis for the second theme developed in this review, the potential use of biological function and health parameters as operational welfare indicators (OWIs). Advantages of function- and health-related parameters are that they are relatively straightforward to recognize and to measure and are routinely monitored in most aquaculture units, thereby providing feasible tools to assess fish welfare under practical farming conditions. As the efforts to improve fish welfare and environmental sustainability lead to increasingly diverse solutions, in particular integrated production, it is imperative that we have objective OWIs to compare with other production forms, such as high-density aquaculture. However, to receive the necessary acceptance for legislation, more robust scientific backing of the health- and function-related OWIs is urgently needed.

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch, Perca fluviatilis.

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated. Plasma cortisol and glucose levels increased markedly 1h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1-72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses. Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.

Early-life infection with a bacterial pathogen increases expression levels of innate immunity related genes during adulthood in zebrafish.

Early-life exposure to different stressors can lead to various consequences on fish health status in later life development. To evaluate the effects of Aeromonas salmonicida achromogenes infection in the early-life on immunity in adulthood, zebrafish were either early-infected at 18 days post-fertilization (dpf), chronically infected from 18 to 35 dpf, or late infected at 35 dpf and then grown up to 61 dpf to be re-infected with the pathogen. The age of first infection was shown to influence both, level and timing of the immune gene expressions, especially for inflammation-related genes. In addition, evidence for an innate immune memory in zebrafish primarily infected with the pathogen at 35 dpf and re-infected at 61dpf provide new insights to consolidate the concept of a "trained" innate immunity in fish.

Physiological and proteomic responses to corticosteroid treatments in Eurasian perch, Perca fluviatilis: Investigation of immune-related parameters.

The comparative effects of cortisol and 11-deoxycorticosterone (DOC), two major corticosteroids in fish, have yet received little attention in teleosts. We evaluated the proteomic and immune responses of Eurasian perch to chronic corticosteroid treatments. We implanted immature perch with cortisol (80mg/kg) or DOC (4mg/kg) and measured the proportions of blood leucocytes, immune indices in the plasma, spleen and liver (complement and lysozyme activity, total immunoglobulin and immune gene expression in the tissues) and differential proteome expression (corticosteroid versus control) in the liver and the spleen on days 2, 4 and 14 post-treatment. Implantation of cortisol decreased the ratio of blood leucocytes and depressed Ig levels in both organs while DOC modulated the proportion of leucocyte sub-populations (increase in lymphocytes and decrease in granulocytes). In contrast, the innate humoral immunity was not strongly influenced by any of corticosteroid implants. The only immune parameter that was significantly affected was lysozyme, after DOC treatment. A number of proteins were differentially regulated by these hormones and some were identified in the liver (21 for cortisol and 8 for DOC) and in the spleen (10 for cortisol and 10 for DOC). None of the proteins was directly linked to immunity, except the natural killer enhancing factor, which was repressed by cortisol in the spleen. Our results also confirm that the proteins involved in energetic and glucose metabolism are affected by corticosteroids. Furthermore, these corticosteroids differently regulate immune status in Eurasian perch and they primarily impact leucocytes, as opposed to innate immune function.

Do sewage treatment plant discharges substantially impair fish reproduction in polluted rivers?

Sewage treatment plants are frequently associated with the release of xenobiotics and, consequently, with alterations of the reproductive function induced by many of these substances in aquatic organisms. In order to assess the impacts of sewage treatment plant (STP) discharges in polluted rivers, two sentinel species (gudgeon Gobio gobio and stoneloach Barbatula barbatula) were caught during their reproductive cycle upstream and downstream two STPs (STP1--Goffontaine, STP2--Wegnez). Gonadosomatic index, histological (testicular and ovarian stages, atretic follicles, intersexuality) and endocrine (sex steroids, aromatase activity, alkali-labile phosphorus) parameters were assayed. In brief, the results revealed no systematic significant differences (p<0.05) between upstream and downstream sites, whatever the STP, species or sampling period. However, stoneloach females displayed some signs of reproductive impairment and endocrine disruption downstream STP1 (reduced GSI, oocyte diameter and ALP concentrations, increased proportion of atretic follicles) and STP2 (changes in gonadal aromatase activity and plasma levels of 11-KT and T). Few significant changes were observed for gudgeon males and females while there were no significant differences between upstream and downstream sites for stoneloach males. Moreover, plasma E(2) concentrations recorded in gudgeon males sampled in all sites were as high as in females and this was confirmed by high ALP levels. Besides, spermatogenesis of gudgeon males was delayed in STP1 upstream and downstream sites compared to the corresponding sites in STP2. These observations for gudgeon males do not seem related to STP discharge but to a probable estrogenicity of the river. Therefore, as shown by the results, stoneloach seemed more sensitive than gudgeon to STP discharges. In the present study, sewage treatment plant discharges do not substantially impair fish reproduction. In this respect, caution is required when generalising negative impacts of STP discharges.

Synergic stress in striped catfish (Pangasianodon hypophthalmus, S.) exposed to chronic salinity and bacterial infection: Effects on kidney protein expression profile.

UNLABELLED: Hyperosmotic stress has often been investigated from osmoregulation perspectives while the effects of such stress on the immune capacity remain largely unexplored. In this study, striped catfish were submitted to a gradual hyperosmotic stress (up to 20ppt), followed by an infection with a virulent strain of an intracellular bacteria, Edwardsiella ictaluri. Physiological parameters (plasma osmolarity, gill NaK ATPase) have been investigated. Moreover, a label free quantitative proteomics workflow was used to study how salinity affects the proteome of kidney of no infected and infected fish. The flow consisted in initial global profiling of relative peptide abundances (by LC/MS, peak area quantification based on extracted ion currents) followed by identification (by MS/MS). Differentially expressed proteins were analysed in DAVID interface to isolate functional pathways involved in the response to bacterial infection and which are influenced by environmental salinity changes. In response to hyperosmotic stress, plasma osmolarity increased while gill Na(+)K(+) ATPase slightly increased. In kidney proteome, 2024 proteins were identified, among which 496 proteins were differentially expressed. Hierarchical clustering analysis leads to isolation of 4 main clusters. Several pathways and functional categories, mostly related to cell metabolism, response to stress, cell structure, immunity and ion homeostasis were highlighted and discussed. SIGNIFICANCE: The striped catfish is a highly worldwide commercialized species and is mainly produced in the Mekong Delta (Vietnam), which is currently subjected to sea level rise and increasing salt intrusions in these aquacultures resources. Moreover, salt is commonly used as antiseptic in aquacultures to treat microbial diseases. To date, the effect of prolonged exposure to hyperosmotic condition have been mainly looking at changes regarding the growth and the osmoregulation while the effect of such stress on the immune capacity and susceptibility to pathogen have been poorly investigated. This study aims to highlight molecular changes in kidney occurring during acclimation to hyperosmotic conditions, particularly those involved in fish immune defences.