Response Of Seabream Research Articles

Molecular characterization of the cytotoxic and regulatory T cell coreceptor (CRTAM), and its ligand CADM1, in the European seabass and gilthead seabream

T cell activation is a multifaceted process that depends on the activation of the T cell receptor (TCR). However, other coreceptors are also strictly necessary to provide co-signals and modulate the immune response. However, to date, most of these coreceptors are unknown in fish or their information is very limited. Therefore, in this work, we have identified the cytotoxic and regulatory T cell molecule, CRTAM, and its ligand, the cell adhesion molecule 1, CADM1, in European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata); and evaluated their transcriptional levels. Both putative proteins showed the canonical architecture observed in mammals, where CRTAM exhibited two immunoglobulin domains and CADM1, both the a and b forms, exhibited three of these domains. In addition, phylogeny and synteny analyses showed their conservation throughout vertebrate evolution. We found constitutive expression of all three genes, with crtam and cadm1a being predominant in immune tissues such as spleen, thymus and head-kidney (HK), while cadm1b expression was more limited to the brain. In vitro, only the T cell mitogen phytohemagglutinin (PHA) up-regulated the transcription of crtam and cadm1a in HK leucocytes. Nodavirus (NNV) infection elicited an up-regulation of crtam and cadm1a in brain and HK, appearing earlier in seabream than in seabass, which could explain the resistance of seabream to the development of nodavirus disease. In addition, they are up-regulated during the innate cell-mediated cytotoxic response in seabream but not in seabass. Altogether, our data seem to indicate that CRTAM is more related to the innate cytotoxicity in seabream and more in the specific and T cell-mediated cytotoxicity in seabass. Our results highlight the importance of CRTAM and CADM1 as important molecules in the activation of T lymphocytes in seabass and seabream, but further studies are needed.

Transcriptome analysis reveals a complex response to the RGNNV/SJNNV reassortant Nervous Necrosis Virus strain in sea bream larvae

The gilthead sea bream (Sparus aurata) is a marine fish of great importance for Mediterranean aquaculture. This species has long been considered resistant to Nervous Necrosis Virus (NNV), an RNA virus that causes massive mortalities in several farmed fish animals. However, the recent appearance of RGNNV/SJNNV reassortant strains started to pose a serious threat to sea bream hatcheries, as it is able to infect larvae and juveniles of this species. While host response to NNV has been extensively studied in adult fish, little attention has been devoted to early life history stages, which are generally the most sensitive ones. Here we report for the first time a time-course RNA-seq analysis on 21-day old fish gilthead sea bream larvae experimentally infected with a RGNNV/SJNNV strain. NNV-infected and mock-infected samples were collected at four time points (6 h, 12 h, 24 h, and 48 h post infection). Four biological replicates, each consisting of five pooled larvae, were analysed for each time point and group. A large set of genes were found to be significantly regulated, especially at early time points (6 h and 12 h), with several heat shock protein encoding transcripts being up-regulated (e.g. hspa5, dnaj4, hspa9, hsc70), while many immune genes were down-regulated (e.g. myd88 and irf5 at T06, pik3r1, stat3, jak1, il12b and il6st at T12). A gene set enrichment analysis (GSEA) identified several altered pathways/processes. For instance, the formation of peroxisomes, which are important anti-viral components as well as essential for nervous system homeostasis, and the autophagy pathway were down-regulated at 6 h and 24 h post infection (hpi). Finally, two custom “reactomes” (i.e. significant gene sets observed in other studies) were defined and used. The first reactome integrated the transcriptomic response to NNV in different fish species, while the second one included all genes found to be stimulated either by interferon (IFN) or by IFN and Chikungunya virus in zebrafish. Genes in both reactomes showed predominant up-regulation at 6hpi and 12hpi and a general down-regulation at 24hpi. Such evidence suggest a certain degree of similarity between the response of sea bream and that of other fish species to NNV, while the observed down-regulation of IFN- and viral-stimulated pathways argues for a possible interference of NNV against the host response.

Salinity, Temperature and Ammonia Acute Stress Response in Seabream (Sparus aurata) Juveniles: A Multidisciplinary Study.

Simple SummaryGilthead seabream (Sparus aurata) is a teleost fish of great relevance in marine aquaculture, especially in the Mediterranean area and one of the most important farmed food marine species in Europe. Nevertheless, in captivity fish are usually exposed to stressful conditions, with a consequent negative impact on animal welfare and growth. The principal goal of this study was to shed light on the acute stress response of gilthead seabream juveniles exposed to different stressors using a multidisciplinary approach. For this purpose, gilthead seabream have been exposed to three different stress tests (temperature, salinity, and ammonia changes) and several laboratory techniques have been used to evaluate their growth and stress response. Results revealed that all the tested stressors had an impact on fish growth and health, particularly thermic and chemical exposure, whereas salinity seems to have a minor effect since this species can efficiently face with extreme variations in environmental salinity. The present work aimed to obtain relevant information on acute stress response of gilthead seabream to be used for improving farming condition and ensuring fish welfare.The present study aimed to investigate the acute response of gilthead seabream (Sparus aurata) juveniles exposed to temperature, salinity and ammonia stress. Radioimmunoassay was used to evaluate cortisol levels, whereas insulin-like growth factors (igf1 and igf2), myostatin (mstn), heat-shock protein 70 (hsp70) and glucocorticoid receptor (gr) gene expression was assessed trough Real-Time PCR. The presence and localization of IGF-I and HSP70 were investigated by immunohistochemistry. In all the stress conditions, a significant increase in cortisol levels was observed reaching higher values in the thermic and chemical stress groups. Regarding fish growth markers, igf1 gene expression was significantly higher only in fish subjected to heat shock stress while, at 60 min, igf2 gene expression was significantly lower in all the stressed groups. Temperature and ammonia changes resulted in a higher mstn gene expression. Molecular analyses on stress response evidenced a time dependent increase in hsp70 gene expression, that was significantly higher at 60 min in fish exposed to heat shock and chemical stress. Furthermore, the same experimental groups were characterized by a significantly higher gr gene expression respect to the control one. Immunostaining for IGF-I and HSP70 antibodies was observed in skin, gills, liver, and digestive system of gilthead seabream juveniles.

Quantitative and qualitative analysis of sea bream,Sparus aurata(L.), humoral immune response, vaccinated with commercial and experimental vaccines against vibriosis and photobacteriosis

The specific humoral immune response of sea bream,Sparus aurata(L.), againstVibrio anguillarumO1 andPhotobacterium damselaesubsp.piscicida(Phdp) after immunization with commercial and experimental bacterins was analysed quantitatively and qualitatively. Specific anti-V. anguillarumO1 and anti-Phdp levels provoked by the adjuvanted commercial vaccine reached higher levels in comparison to the aqueous commercial and experimental bacterins. Infection of vaccinated fish withV. anguillarumO1 bacterial cells acted as a boost of the humoral immune response, except for the sera of the group vaccinated with the adjuvanted vaccine. Infection with Phdp acted as a boost of the humoral immune response mainly for the group vaccinated with a monovalent Phdp bacterin and to a lesser degree for the group vaccinated with the aqueous commercial vaccine. Western blot analysis of the sera againstV. anguillarumO1 whole cell antigens revealed strong reactions to only a few antigens below 54 kD and above 15 kD and weak reactions to other antigens. Similar reactions were observed from the sera isolated from the controls. Western blot analysis of the sera against Phdp whole cell antigens revealed strong reactions to only a handful of antigens below 20.7 and below 6.4 kD. Sera from the control group, as in the case ofV. anguillarumO1, reacted with Phdp whole cell antigens. No differences regarding antigen reactions between monovalent and bivalent formulations were noted, in contrast to the adjuvanted and aqueous bacterins.

Molecular cloning, phylogenetic analysis and functional characterization of soluble Toll-like receptor 5 in gilthead seabream, Sparus aurata

Two forms of TLR5, one membrane-anchored and one soluble, have been described in some teleost fish species. However, the exact role of each form has been poorly studied. In the present study, we show that the mRNA levels of soluble gilthead seabream TLR5 (sbTLR5S) are highly induced in head kidney, spleen, liver and blood after Vibrio anguillarum infection, suggesting an important role for sbTLR5S in the innate immune response against bacteria. Comparative genomic and phylogenetic analyses revealed a co-evolution pattern of both genes across fish species and a proximal location in their genomes, further suggesting a functional link between them. To further investigate this issue, the coding sequence of the sbTLR5S was cloned and the corresponding recombinant protein was produced in HEK293 cells. The gene product was secreted to the culture medium as a soluble factor and a physical interaction between flagellin and sbTLR5S was demonstrated. Collectively, these results suggest that sbTLR5S plays an important role in modulating the flagellin-mediated immune response in seabream.

Biochemical responses in seabream (Sparus aurata) caged in-field or exposed to benzo(a)pyrene and paraquat. Characterization of glutathione S-transferases

Gilthead seabream (Sparus aurata) specimens were caged in-field at the Téboulba harbour or exposed to benzo(a)pyrene [B(a)P] or to paraquat [PQ] plus B(a)P, and several biochemical biomarker responses were investigated. Antioxidant enzymes, such as glutathione peroxidase, catalase and glutathione reductase, significantly increased in the in-field and B(a)P+PQ exposures, but were only moderately affected by B(a)P alone. Glucose-6-phosphate and 6-phosphogluconate dehydrogenases significantly diminished after in-field exposure. Different responses with biotransformation enzymes were observed: the P4501A-associated EROD activity was highly induced in response to B(a)P and B(a)P+PQ exposures, while total activity of the glutathione S-transferase (GST) was similar to control. However, after purification of the GST proteins by affinity chromatograpy and analysis by two-dimensional electrophoresis, nineteen highly reproducible isoforms were resolved. In addition, some of reproducible isoforms showed different and specific expression patterns in response to contaminants.Thus, proteomic analysis of the purified GST subunits is a reliable tool for ecotoxicological research, useful in polluted marine ecosystem as an effective biomarker of contamination.

Stress response in sea bream (Sparus aurata) held under crowded conditions and fed diets containing linseed and/or soybean oil

The physiological response to stressors in fish, including hormonal profiles and associated tissue responsiveness, is less documented. The aim of this study was to evaluate feeding gilthead sea bream (Sparus aurata) with diets containing linseed oil (LO) and soybean oil (SO) as substitutes to fish oil (FO) and their effect on fatty acid profile of head kidney and the consequent effect on stress response to a crowding challenge. Fish were fed 8 experimental diets with different levels of substitution 0% (FO), 70% (70LO, 70SO, 20LO50SO and 50LO20SO) and 100% (100LO, 100SO and 50LO50SO) over a period of 8months. At the end of the feeding trial, samples of head kidney were collected for biochemical analysis and the fish were challenged by a crowding test. During the challenge, samples of plasma for cortisol analysis were collected at 0h, 2h, 5h, 24h, 48h and 1week in order to study acute and chronic stress responses. Results showed that fish fed vegetable oils (VO) had significantly decreased ARA, EPA, DHA and n-3 HUFA, while LA, LNA and total C18 PUFAs were significantly increased. The basal cortisol levels were significantly increased in fish fed 70LO, 100LO, 50LO20SO and 50LO50SO. The physiological response to crowding was significantly affected by the diet. After 2h of crowding, all the treatments showed higher cortisol, with fish fed 100LO had significantly the highest response registering 131.38pg/ml. After 5h and 24h, plasma cortisol was reduced in all treatments except in 50LO20SO. After 48h of crowding, the plasma cortisol was increased in all treatments with the maximum value seen in fish fed 100LO (72.12pg/ml). These levels were decreased in fish fed FO, 70LO, 100LO and 50L050SO after 1week of crowding, but remained higher in fish fed 70SO, 100SO, 20LO50SO and 50LO20SO. In conclusion, fish fed LO diets showed the same response pattern as the control but with higher intensity regaining the basal levels after 1week as the control, while fish fed SO had a slow response but changed the pattern characterized by a lower response at the beginning and longer recuperation without regaining the control value even after 1week.

Effects of feeding schedule on locomotor activity rhythms and stress response in sea bream

Feeding cycles entrain biological rhythms, which enable animals to anticipate feeding times and so maximize food utilization and welfare. In this article, the effect of mealtime was investigated in two groups of sea bream ( Sparus aurata): one group received a single daily meal at random times during the light period (random daytime feeding, RDF), whereas the other group received the meal during the light period but at the same time (scheduled daytime feeding, SDF). All the fish showed diurnal behavior, although the SDF group showed a lower percentage of diurnalism (84.4% vs. 79.5% in RDF and SDF respectively) and developed food anticipatory activity some hours before the mealtime. In addition, the mean daily locomotor activity of the RDF group was significantly higher than that of the SDF group (3132 vs. 2654 counts/day, respectively). Although the mean weight differed between both groups on day 30 (115.7 g and 125.6 g in RDF and SDF respectively), these differences had disappeared by day 60. Plasma cortisol and glucose significantly differed in both groups (cortisol: 71.8 vs. 8.7 ng/ml, glucose: 53.7 vs. 43.8 mg/dl in RDF and SDF, respectively), whereas lactate did not differ significantly. The results obtained suggest that altering the feeding time (scheduled vs. random) affects the behavior and physiology of sea bream, indicating that a single daily feeding cycle (compared to random) is beneficial for fish welfare because they can prepare themselves for the forthcoming feed.

Cortisol and haematological response in sea bream and trout subjected to the anaesthetics clove oil and 2-phenoxyethanol

Clove oil has been tested for anaesthesia induction and recovery time as well as for haematology and stress indicators in the gilthead sea bream and rainbow trout. The former parameters were compared with those generated by 2-phenoxyethanol. The results showed only slight differences between both anaesthetics in terms of anaesthetic efficiency and physiological effects. In addition, clove oil does not block the cortisol response to stress, as happens with other anaesthetics.

The response of sea bream following abrupt hyposmotic exposure

The response of a marine teleost, silver sea bream Sparus sarba, to abrupt hyposmotic exposure was investigated over a 120‐h period following direct exposure from sea water (SW, 33‰) to a hyposmotic environment of 6‰. Aspects of serum chemistry, stored metabolites and gill morphology were used to gain further insight into the biochemical, physiological and morphological alterations that take place following low salinity exposure of a marine fish. Rapid (<1 h) reductions in serum [Cl−] occurred while serum [Na+] exhibited only transient perturbations during initial exposure. Serum total [Ca] declined 24 h after exposure and returned to pre‐exposure levels by 120 h. Despite a tendency for muscle moisture to increase during the early stages of low salinity exposure to significant change could be detected. The response of the branchial chloride cell (CC) was rapid, with apical and fractional exposure area increasing after 6 h. The number of CCs exposed at the branchial surface reduced after 6 h but subsequently increased to elevated levels. Serum cortisol levels had increased three fold 1 h after hyposmotic exposure and stabilised at pre‐exposure levels within 12–24 h. Serum triiodothyronine (T3) levels exhibited a biphasic response, significantly elevating and decreasing after 3 and 6 h respectively. Significant post‐hyposmotic exposure elevations in serum glucose and protein occurred after 1 h, peaking at 3 h and returning to lower levels after 6 h. Total free ninhydrin reactive substances were significantly elevated 3 h post‐hyposmotic exposure, a phenomenon attributable to elevated levels of ammonia, alanine, arginine, glycine, isoleucine, lysine, methionine, phenylalanine, serine, taurine, threonine and valine. Of these substances, glycine, lysine, serine and taurine remained elevated for up to 12 h or longer. Serum urea levels elevated 1 h after exposure to 6%‰ and returned to SW levels 3 h post‐exposure. The relevance of these results is discussed within the context of current knowledge on the effects of hyposomotic adaptation on marine fish.

The response of sea bream following abrupt hyposmotic exposure

The response of sea bream following abrupt hyposmotic exposure