Sea water acclimation of rainbow trout (Oncorhynchus mykiss) modulates the mucosal transcript immune response induced by Vibrio anguillarum and Aeromonas salmonicida vaccine, and prevents further transcription of stress-immune genes in response to acute stress.

Mucosal tissues appear to be more important in fish than in mammals due to living in a microbial-rich aquatic milieu, yet the complex interaction between the immune and the neuroendocrine system in these tissues remains elusive. The aim of this work was to investigate the mucosal immune response in immunized rainbow trout vaccinated with Alpha ject vaccine (bivalent), kept in fresh water (FW) or transferred to seawater (SW), and to evaluate their response to acute stress (chasing). Acute stress resulted in higher levels of plasma cortisol (Sham+Stress and Vaccine+Stress). A similar response was observed in skin mucus, but it was lower in Vaccine+Stress compared with stressed fish. With a few exceptions, minimal alterations were detected in the transcriptomic profile of stress-immune gene in the skin of vaccinated and stressed fish in both FW and SW. In the gills, the stress elicited activation of key stress-immune components (gr1, mr, ß-ar, hsp70, c3, lysozyme, α-enolase, nadph oxidase, il1ß, il6, tnfα, il10 and tgfß1) in FW, but fewer immune changes were induced by the vaccine (nadph oxidase, il6, tnfα, il10 and igt) in both SW and FW. In the intestine, an array of immune genes was activated by the vaccine particularly those related with B cells (igm, igt) and T cells (cd8α) in FW with no stimulation observed in SW. Therefore, our survey on the transcriptomic mucosal response demonstrates that the immune protection conferred by the vaccine to the intestine is modulated in SW. Overall, our results showed: i) plasma and skin mucus cortisol showed no additional stress effect induced by prolonged SW acclimation, ii) the stress and immune response were different among mucosal tissues which indicates a tissue-specific response to specific antigens/stressor. Further, the results suggest that the systemic immune organs may be more implicated in infectious events in SW (as few changes were observed in the mucosal barriers of immunized fish in SW) than in FW.

Comparative study of stress and immune-related transcript outcomes triggered by Vibrio anguillarum bacterin and air exposure stress in liver and spleen of gilthead seabream (Sparus aurata), zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss).

The stress and immune-related effects of short-term (1, 6 and 24 h) air exposure stress (1 min), bath vaccination with Vibrio anguillarum bacterin, and both stressors combined were evaluated in liver and spleen of Sparus aurata, Danio rerio and Onchorhynchus mykiss. Expression profiles of immune (interleukin 1 beta: il1ß; tumor necrosis factor alpha: tnfα; interleukin 10: il10; tumor growth factor beta: tgfß1; immunoglobulin M: igm; lysozyme: lys; complement protein c3: c3) and stress-related genes (glucocorticoid receptor: gr; heat shock protein 70: hsp70; and enolase) were analysed by RT-qPCR. Cortisol level was assessed by radioimmunoassay. The gene expression patterns in liver and spleen were found to be differentially regulated in a time- and organ-dependent manner among species. In seabream, a higher il1ß-driven inflammatory response was recorded. In zebrafish, air exposure stress but not bath vaccination alone modulated most of the changes in liver and spleen immune transcripts. Stressed and vaccinated trout showed an intermediate pattern of gene expression, with a lower upregulation of immune-related genes in liver and the absence of changes in the expression of hsp70 and enolase in spleen (as it was observed in seabream but not in zebrafish). Following air exposure, cortisol levels increased in plasma 1 h post-stress (hps) and then decreased at 6 hps in O. mykiss and D. rerio. By contrast, in S.aurata the cortisol level remained higher at 6 hps suggesting a greater degree of responsiveness to this stressor. When fish were exposed to combined air exposure plus bath vaccination cortisol levels were also augmented at 1 and 6 hps in O. mykiss and S.aurata and restored to basal level at 24 hps, whereas in D. rerio the response was higher in response to the combination of both stressors. In addition, V. anguillarum bacterin vaccination triggered cortisol secretion only in D. rerio, suggesting a greater responsiveness of D. rerio hypothalamic-pituitary-interrenal axis. Overall, comparing the tissue transcription responsiveness, liver was found to be more implicated in the response to handling stress compared to spleen. These results also indicate that a species-specific response accounts for the deviations of stress and immune onset in the liver and spleen in these fish species.

Modulatory in vitro effect of stress hormones on the cytokine response of rainbow trout and gilthead sea bream head kidney stimulated with Vibrio anguillarum bacterin.

In fish, the stress response and their consequences in the immune system have been widely described. Recently, a differential cytokine regulation between rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) was reported after treatment with stress hormones together with their receptor antagonists. Nevertheless, there is no evidence of whether antagonists for stress hormone receptors may influence the interaction between hormones and cytokines after bacterial administration. Thus, the aim of our study was to evaluate the cytokine expression in the presence of stress hormones (cortisol, ACTH, adrenaline), hormone receptor antagonists and inactivated Vibrio anguillarum bacterin in rainbow trout and gilthead sea bream head kidney primary cell culture (HKPCC). Mifepristone, spironolactone, propranolol and phentolamine were used to block GR, MR, MC2R, and ß-/α-adrenoreceptors. Our results showed an expected increase of the pro-inflammatory and anti-inflammatory response after inactivated V. anguillarum bacterin treatment in both species. Cortisol, ACTH and adrenaline did not modulate the expression of immune-related genes in rainbow trout, while in sea bream cortisol was able to reduce the stimulated gene expression of all cytokines. This effect was only restored to basal expression level in IL-1ß and TNF-α by mifepristone. ACTH reduced both pro-inflammatory and anti-inflammatory cytokine expression, excluding IL-1ß, only in sea bream. Adrenaline enhanced the expression of IL-1ß and TGF-ß1 stimulated by inactivated V. anguillarum in sea bream, and the effect was diminished by propranolol. In sum, our results confirm that the immunoendocrine differences reported at gene expression profile between two teleost species are also observed after exposure to inactivated V. anguillarum bacterin, suggesting that stress hormones would differentially modulate the immune response against pathogens in teleost species.

Cytokine modulation by stress hormones and antagonist specific hormonal inhibition in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) head kidney primary cell culture.

A tight interaction between endocrine and immune systems takes place mainly due to the key role of head kidney in both hormone and cytokine secretion, particularly under stress situations in which the physiological response promotes the synthesis and release of stress hormones which may lead into immunomodulation as side effect. Although such interaction has been previously investigated, this study evaluated for the first time the effect of stress-associated hormones together with their receptor antagonists on the expression of cytokine genes in head kidney primary cell culture (HKPCC) of the freshwater rainbow trout (Oncorhynchus mykiss) and the seawater gilthead sea bream (Sparus aurata). The results showed a striking difference when comparing the response obtained in trout and seabream. Cortisol and adrenocorticotropic hormone (ACTH) decreased the expression of immune-related genes in sea bream but not in rainbow trout and this cortisol effect was reverted by the antagonist mifepristone but not spironolactone. On the other hand, while adrenaline reduced the expression of pro-inflammatory cytokines (IL-1ß, IL-6) in rainbow trout, the opposite effect was observed in sea bream showing an increased expression (IL-1ß, IL-6). Interestingly, this effect was reverted by antagonist propranolol but not phentolamine. Overall, our results confirm the regional interaction between endocrine and cytokine messengers and a clear difference in the sensitivity to the hormonal stimuli between the two species.

Repeated hypoxic episodes allow hematological and physiological habituation in rainbow trout.

Introduction: Under climate change, the increase in temperature in aquatic environments may induce oxygen depletion. In extreme cases, low oxygen may become a limiting factor for fish, thus generating stress. In addition, consecutive hypoxic episodes may complicate the recovery of individuals and hinder their ability to modulate physiological and biochemical responses to maintain homeostasis. Thus, the aim of this study was to determine the hematological and physiological responses of rainbow trout under a condition of repeated hypoxic and manipulation stresses at three different time points. Methods: Every hypoxic episode consisted of exposing the fish to low dissolved oxygen concentrations (2 mgO2/L for 1 h). Following the exposure, the fish were allowed to recover for 1 h, after which they were sampled to investigate hematological and physiological parameters. Results and discussion: The results showed a pattern of habituation reflected by values of hematocrit, hemoglobin, and mean corpuscular volume, indicating a certain ability of rainbow trout to resist this type of repeated hypoxic events, provided that the fish can have some recovery time between the exposures.

Rainbow trout integrated response after recovery from short-term acute hypoxia.

Overcoming a stress situation, such as hypoxia episodes, which involve an allostatic load, will depend on the ability of fish to modulate physiological and biochemical systems to maintain homeostasis. The aim of the study was to determine the integrated stress response after acute hypoxia of the rainbow trout considering the different elements and areas of the stress response: systemic and mucosal, local and global, and from the systemic hypothalamic-pituitary-interrenal axis to skin mucosa. For this purpose, trout were subjected to acute hypoxia (dissolved O2 down to 2 mg/L) for 1 h and then recovered and sampled at 1, 6, and 24 h after reoxygenation. Physiological responses were significantly affected by hypoxic stress and their interaction with time after the challenge, being significant for plasma lactate and cortisol levels, in both plasma and skin mucus. At the central brain level, only trh expression was modulated 1 h after hypoxia which indicates that brain function is not heavily affected by this particular stress. Unlike the brain, the head kidney and skin were more affected by hypoxia and reoxygenation. In the head kidney, an upregulation in the expression of most of the genes studied (gr, il1ß, il6, tgfß1, lysozyme, caspase 3, enolase, hif-1, myoglobin, sod2, gpx, gst, and gsr) took place 6 h after recovery, whereas only hsp70 and il10 were upregulated after 1 h. On the contrary, in the skin, most of the analyzed genes showed a higher upregulation during 1 h after stress suggesting that, in the skin, a local response took place as soon as the stressor was detected, thus indicating the importance of the skin in the building of a stress response, whereas the interrenal tissue participated in a later time point to help prevent further alteration at the central level. The present results also show that, even though the stressor is a physical/environmental stressor, all components of the biological systems participate in the regulation of the response process and the recovery process, including neuroendocrine, metabolism, and immunity.

Comparative Immune- and Stress-Related Transcript Response Induced by Air Exposure and Vibrio anguillarum Bacterin in Rainbow Trout (Oncorhynchus mykiss) and Gilthead Seabream (Sparus aurata) Mucosal Surfaces.

Fish have to face various environmental challenges that may compromise the efficacy of the immune response in mucosal surfaces. Since the effect of acute stress on mucosal barriers in fish has still not been fully elucidated, we aimed to compare the short-term mucosal stress and immune transcriptomic responses in a freshwater (rainbow trout, Oncorhynchus mykiss) and a marine fish (gilthead seabream, Sparus aurata) to bacterial immersion (Vibrio anguillarum bacterin vaccine) and air exposure stress in skin, gills, and intestine. Air exposure and combined (vaccine + air) stressors exposure were found to be inducers of the cortisol secretion in plasma and skin mucus on both species in a time-dependent manner, while V. anguillarum bacterin exposure induced cortisol release in trout skin mucus only. This was coincident with a marked differential increase in transcriptomic patterns of stress- and immune-related gene expression profiles. Particularly in seabream skin, the expression of cytokines was markedly enhanced, whereas in gills the response was mainly suppressed. In rainbow trout gut, both air exposure and vaccine stimulated the transcriptomic response, whereas in seabream, stress and immune responses were mainly induced by air exposure. Therefore, our comparative survey on the transcriptomic mucosal responses demonstrates that skin and gut were generally more reactive in both species. However, the upregulation of immune transcripts was more pronounced in gills and gut of vaccinated trout, whereas seabream appeared to be more stress-prone and less responsive to V. anguillarum bacterin in gills and gut. When fish were subjected to both treatments no definite pattern was observed. Overall, the results indicate that (1) the immune response was not homogeneous among mucosae (2), it was greatly influenced by the specific traits of each stressor in each surface and (3) was highly species-specific, probably as a result of the adaptive life story of each species to the microbial load and environmental characteristics of their respective natural habitats.