C. Fierro-Castro

Differential expression of the corticosteroid receptors GR1, GR2 and MR in rainbow trout organs with slow release cortisol implants

The present study describes the transcriptional levels of the corticosteroid receptors (CRs) GR1, GR2 and MR in the different organs of the rainbow trout (Oncorhynchus mykiss) in response to a slow release of cortisol, throughout a 10-day period. We show that after short term (1 day after cortisol implantation), when the plasma levels of cortisol emulate an acute stress, the GR2 and MR expression levels were upregulated in the brain and head kidney tissues. This result reflects the role of these organs as regulators of the stress response. In general, the rest of the organs, especially gills, intestine, liver, muscle and spleen, showed decreased transcriptional levels of GR1, GR2 and MR, along with the highest plasma cortisol levels. At day 5 after cortisol implantation, when cortisol levels emulate a chronic stress, the most affected organs were gills and skin, where an upregulation of the CRs was found. In the recovery period, when cortisol levels were basal (day 10), we still found changes in the transcriptional levels of the CRs in gills, spleen and gonads. The cortisol increase at days 1 and 5 after implantation is accompanied by high plasma glucose concentrations, supporting the role of cortisol on carbohydrate metabolism. However, after 10days of implantation, glucose returned to control levels suggesting a trade-off on the steady state of the metabolic function. We also observed increased hematocrit and hemoglobin at day 1, indicating a cortisol-induced higher metabolic demand involving an increase in oxygen transport efficiency. Our results demonstrate that increased plasma cortisol induced by a slow-release implant of cortisol mimics the overall effects of stress and affects the expression of the three CRs, generating different transcriptional patterns in a time- and organ-specific manner.

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

Abstract Although &bgr;‐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 &bgr;‐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‐&agr;1, IL‐1&bgr;, IL10, COX‐2, TGF‐&bgr;, MC2R, HSP70) were measured. Our results suggest that spleen may be a highly responsive organ to dietary &bgr;‐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 &bgr;‐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 &bgr;‐glucans as a preventive method for farmed fish protection. HighlightsImmune and stress related gene expression was studied in &bgr;‐glucans fed trout.Spleen is highly responsive to glucan diet, mainly involving proinflammatory cytokines.Overdose of glucan may lead to non‐reactive status and poor immune response.

Assessment of gold nanoparticle effects in a marine teleost (Sparus aurata) using molecular and biochemical biomarkers

Gold nanoparticles (AuNP) are increasingly employed in a variety of applications and are likely to be increasing in the environment, posing a potential emerging environmental threat. Information on possible hazardous effects of engineered nanoparticles is urgently required to ensure human and environmental safety and promote the safe use of novel nanotechnologies. Nevertheless, there is a lack of comprehensive knowledge on AuNP effects in marine species. The present study aimed to assess AuNP effects in a marine teleost, Sparus aurata, by combining endpoints at different biological levels (molecular and biochemical). For that purpose, fish were exposed via water for 96h to 4, 80 and 1600μgL(-1) of AuNP (∼40nm) coated with citrate or polyvinylpyrrolidone (PVP). Results revealed a significant impact of AuNP-PVP in the hepatic expression of antioxidant, immune and apoptosis related genes. Total oxidative status was increased in plasma after exposure to the lowest concentration of AuNP-PVP, although without altering the total antioxidant capacity. Furthermore, AuNP did not induce significant damage in the liver since the activity of neither hepatic indicator (aspartate aminotransferase and alkaline phosphatase) increased. Overall, the present study demonstrated that AuNP, even with a biocompatible coating is able to alter oxidative status and expression of relevant target genes in marine fish. Another important finding is that effects are mainly induced by the lowest and intermediate concentrations of the PVP coated AuNP revealing the importance of different coatings.

Analysis of steroidogenic pathway key transcripts in interrenal cells isolated by laser microdissection (LMD) in stressed rainbow trout

An assessment of the key transcripts expression of the steroidogenesis-related genes in rainbow trout subjected to either acute or chronic stress was performed in both interrenal cells and whole head kidney tissue. The analysis of interrenal cells was possible thanks to the use, for the first time in this specific type of cells, of the technique of laser microdissection (LMD) which allows to isolate specific cells and process them independently of other surrounding cells in the tissue. The results indicated that both acute and chronic stressors induced a significant up-regulation of the steroidogenesis-related genes with a higher but expected degree in the isolated cells. In addition, under acute stress a delay between cortisol levels and transcript expression was found. Under chronic stress a clear relation between plasma cortisol levels, mRNA transcription and interrenal tissue area was observed, since all parameters were concomitantly increased at day 5 after stress. Moreover results indicated that the LMD technique allowed ascertaining with more precision and accuracy whether and when the steroidogenesis-related genes were significantly expressed, disregarding the noise produced by other cells present in the head kidney. Results also showed a typical physiological response in plasma parameters and a positive relationship between plasma cortisol data and transcript abundance in isolated cells. The present results may help to better understand the mechanisms behind the interrenal response to stress challenges in fish.

Evaluation of gemfibrozil effects on a marine fish (Sparus aurata) combining gene expression with conventional endocrine and biochemical endpoints.

The information on the potential hazardous effects of gemfibrozil (GEM) on marine fish is extremely scarce. In the current study, molecular, endocrine and biochemical parameters were assessed in Sparus aurata after 96h waterborne exposure to a GEM concentration range. Hepatic mRNA levels of target genes known to be regulated via peroxisome proliferator-activated receptor α (pparα) in mammals, such as apolipoprotein AI (apoa1) and lipoprotein (lpl) were significantly increased, without a concomitant activation of the ppar pathways. GEM (15μgL(-1)) induced an upregulation in mRNA levels of interleukin 1β (il1β), tumour necrosis factor-α (tnfα) and caspase 3 (casp3), suggesting an activation of proinflammatory processes in S. aurata liver. However, mRNA levels of genes related with the antioxidant defence system and cell-tissue repair were unaltered under the tested experimental conditions. Higher levels of GEM induced a cortisol rise, an indication that it is recognized as a stressor by S. aurata. Cortisol levels and the mRNA levels of il1β, tnfα and casp3 may be suggested as potential biomarkers of GEM effects in marine fish.

Neuroendocrine and Immune Responses Undertake Different Fates following Tryptophan or Methionine Dietary Treatment: Tales from a Teleost Model

Methionine and tryptophan appear to be fundamental in specific cellular pathways involved in the immune response mechanisms, including stimulation of T-regulatory cells by tryptophan metabolites or pro-inflammatory effects upon methionine supplementation. Thus, the aim of this study was to evaluate the immunomodulatory effect of these amino acids on the inflammatory and neuroendocrine responses in juveniles of European seabass, Dicentrarchus labrax. To achieve this, goal fish were fed for 14 days methionine and tryptophan-supplemented diets (MET and TRP, respectively, 2× dietary requirement level) or a control diet meeting the amino acids requirement levels (CTRL). Fish were sampled for immune status assessment and the remaining fish were challenged with intraperitoneally injected inactivated Photobacterium damselae subsp. piscicida and sampled either 4 or 24 h post-injection. Respiratory burst activity, brain monoamines, plasma cortisol, and immune-related gene expression showed distinct and sometimes opposite patterns regarding the effects of dietary amino acids. While neuroendocrine intermediates were not affected by any dietary treatment at the end of the feeding trial, both supplemented diets led to increased levels of plasma cortisol after the inflammatory insult, while brain monoamine content was higher in TRP-fed fish. Peripheral blood respiratory burst was higher in TRP-fed fish injected with the bacteria inoculum but only compared to those fed MET. However, no changes were detected in total antioxidant capacity. Complement factor 3 was upregulated in MET-fed fish but methionine seemed to poorly affect other genes expression patterns. In contrast, fish fed MET showed increased immune cells numbers both before and after immune challenge, suggesting a strong enhancing effect of methionine on immune cells proliferation. Differently, tryptophan effects on inflammatory transcripts suggested an inhibitory mode of action. This, together with a high production of brain monoamine and cortisol levels, suggests that tryptophan might mediate regulatory mechanisms of neuroendocrine and immune systems cooperation. Overall, more studies are needed to ascertain the role of methionine and tryptophan in modulating (stimulate or regulate) fish immune and neuroendocrine responses.

Effects of acute handling stress on short-term central expression of orexigenic/anorexigenic genes in zebrafish

Physiological mechanisms driving stress response in vertebrates are evolutionarily conserved. These mechanisms involve the activation of both the hypothalamic-sympathetic-chromaffin cell (HSC) and the hypothalamic-pituitary-adrenal (HPA) axes. In fish, the reduction of food intake levels is a common feature of the behavioral response to stress but the central mechanisms coordinating the energetic response are not well understood yet. In this work, we explore the effects of acute stress on key central systems regulating food intake in fish as well as on total body cortisol and glucose levels. We show that acute stress induced a rapid increase in total body cortisol with no changes in body glucose, at the same time promoting a prompt central response by activating neuronal pathways. All three orexigenic peptides examined, i.e., neuropeptide y (npy), agouti-related protein (agrp), and ghrelin, increased their central expression level suggesting that these neuronal systems are not involved in the short-term feeding inhibitory effects of acute stress. By contrast, the anorexigenic precursors tested, i.e., cart peptides and pomc, exhibited increased expression after acute stress, suggesting their involvement in the anorexigenic effects.

Modulation of immune genes mRNA levels in mucosal tissues and DNA damage in red blood cells of Sparus aurata by gold nanoparticles

Gold nanoparticles (AuNP) effects on Sparus aurata were evaluated on skin, gills and intestine by assessing the expression of immune genes and in peripheral blood evaluating genetic damage. Fish were exposed to 0.5 and 50 μg/L AuNP for 96 h. Results showed that exposure to 50 μg/L AuNP induced an upregulation in the expression of innate immune genes in gills (c3, lys, il1β, tnfα, il6, il10 and tgfβ) and intestine (il1β, tnfα and il6). Furthermore, mRNA levels of hsp70 and hsp90 were increased in gills after exposure to 0.5 μg/L AuNP, when compared to 50 μg/L. Present data demonstrated the sensitivity of gills and intestines to AuNP exposure supporting their use in the study of fish responses to other nanoparticles. Genotoxic potential of AuNP was demonstrated by increased DNA strand breaks in red blood cells of fish exposed to AuNP, suggesting that AuNP represent a potential hazard to fish.

Immune-related gene expression and physiological responses in rainbow trout (Oncorhynchus mykiss) after intraperitoneal administration of Rhodomyrtus tomentosa leaf extract: A potent phytoimmunostimulant

ABSTRACT The immunostimulatory effects of Rhodomyrtus tomentosa leaf extract were evaluated in rainbow trout through changes in expression profile of genes involved in innate immune and antioxidant response, hematology and stress indicators. The concentrations of R. tomentosa at 10 and 100 &mgr;g per fish were administrated by intraperitoneal injection, alone or in combination with LPS. After 6 h of administration, the gene expression was measured in head kidney, spleen, and intestine. Results indicated that R. tomentosa exerted immunostimulatory effects by inducing the expression of il10, saa, hepcidin, and sod in head kidney and the expression of il10, tgf&bgr;, and inos in intestine. In combination with LPS, the plant suppressed the expression of pro‐inflammtory cytokine il1&bgr;, il8 and other consisting of saa and gpx1 in head kidney and il1&bgr; in spleen, pointing out its anti‐inflammatory activities. Furthermore, the plant did not exert any impact on hematological parameters, but it was able to reduce cortisol levels when co‐administered with LPS, indicating that R. tomentosa could attenuate stress response in rainbow trout. Our observations suggest that R. tomentosa induced the expression of genes involved in cytokine and innate immune response and modulated the physiological stress response as indicated by the suppressed cortisol in rainbow trout. HighlightsImmunostimulatory effects of IP injected solutions of R. tomentosa leaf extract were evaluated in rainbow trout.R. tomentosa elevated the expression of il10, saa, hepcidin, and sod in head kidney and il10, tgf&bgr;, and inos in intestine.R. tomentosa co‐administered with LPS reduces the expression of il1&bgr;, il8, saa, and gpx1 in head kidney and il1&bgr; in spleen.R. tomentosa reduced cortisol levels when co‐administered with LPS.