Nanoplastics are bioaccumulated in fish liver and muscle and cause DNA damage after a chronic exposure.

The extent of the widespread, planetary contamination by plastic waste is difficult to fully capture. Nanoplastics (NPs) are currently in the center of research concerning plastic litter, both for the analytical challenges they pose and for their potential to provoke hazardous effects in organisms. However, there are still many unanswered questions in this multidisciplinary field, with a crucial missing piece being the quantification of NPs in fish tissues after in vivo exposures. Another relevant question that is still greatly unexplored is how a chronic exposure to NPs will affect fish health. This study aims to provide answers to both of these relevant knowledge gaps. To this end, goldfish (Carassius auratus) were exposed to 44 nm polystyrene (PS)-NPs via water for 30 days. Following the exposure, gastrointestinal tract, liver and muscle were sampled for PS-NPs analysis by means of size exclusion chromatography coupled to high resolution mass spectrometry. PS-NPs were detected in all liver and muscle samples of exposed fish, with higher concentrations in liver than in muscle, whereas no PS-NPs were detected in the gastrointestinal tract. Nevertheless, exposure to PS-NPs did not induce changes in hematology parameters nor in cortisol and glucose levels in plasma. On the other hand, even a relatively low concentration of PS-NPs was able to cause DNA damage, measured by an increase in erythrocyte nuclear abnormalities, suggesting that PS-NPs can reach the cell nucleus and cause genotoxicity. These results show for the first time that PS-NPs find their way to fish muscle after chronic exposure, where they bioaccumulate, but do not alter fish survival nor hematological or physiological stress indicators. The accumulation of PS-NPs in fish muscle can represent a threat to human health as a possible route of exposure to small-sized plastics. The present results in a model fish species open windows for future studies in edible fish species.

Metabolic control of T cell immune response through glycans in inflammatory bowel disease.

Mucosal T lymphocytes from patients with ulcerative colitis (UC) were previously shown to display a deficiency in branched N-glycosylation associated with disease severity. However, whether this glycosylation pathway shapes the course of the T cell response constituting a targeted-specific mechanism in UC remains largely unknown. In this study, we demonstrated that metabolic supplementation of ex vivo mucosal T cells from patients with active UC with N-acetylglucosamine (GlcNAc) resulted in enhancement of branched N-glycosylation in the T cell receptor (TCR), leading to suppression of T cell growth, inhibition of the T helper 1 (Th1)/Th17 immune response, and controlled T cell activity. We further demonstrated that mouse models displaying a deficiency in the branched N-glycosylation pathway (MGAT5-/-, MGAT5+/-) exhibited increased susceptibility to severe forms of colitis and early-onset disease. Importantly, the treatment of these mice with GlcNAc reduced disease severity and suppressed disease progression due to a controlled T cell-mediated immune response at the intestinal mucosa. In conclusion, our human ex vivo and preclinical results demonstrate the targeted-specific immunomodulatory properties of this simple glycan, proposing a therapeutic approach for patients with UC.

Development of New Canned Chub Mackerel Products Incorporating Edible Seaweeds-Influence on the Minerals and Trace Elements Composition.

This study aimed to develop new canned chub mackerel products incorporating edible seaweeds (Ascophyllum nodosum, Fucus spiralis, Saccorhiza polyschides, Chondrus crispus, Porphyra sp. and Ulva sp.) harvested in the Portuguese North-Central coast, with simultaneous sensory improvement and minerals enrichment. Two processes were compared, namely the addition of seaweeds in i) the canning step and ii) in the brining step (as the replacement for salt). The concentrations of four macrominerals (Na, K, Ca and Mg), chloride, and twelve trace elements (Co, Cu, Fe, I, Li, Mn, Mo, Rb, Se, Sr, V and Zn) were determined by high-resolution continuum source flame atomic absorption spectrometry (HR-CS-FAAS) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. Results showed that canned chub mackerel incorporating C. crispus and F. spiralis was found to be the preferred sensory option, also exhibiting contents enriched with Cl, Co, Cu, Fe, I, Li, Mg, Mn, Mo, Na, Rb, Se, and Sr. This effect was more pronounced when both seaweed species were added to replace the salt added in the brining step.

Effects of polymethylmethacrylate nanoplastics on Dicentrarchus labrax.

The present study aimed to evaluate the effects of ~45 nm nanoplastics (NPs) on the marine fish Dicentrarchus labrax after a short-term exposure. Animals were exposed to a concentration range of NPs for 96 h and liver, plasma and skin mucus were sampled. Assessed endpoints included biochemical biomarkers and expression of genes related to lipid metabolism, immune system and general cell stress. Abundance of mRNA transcripts related to lipid metabolism, pparα and pparγ, were significantly increased after exposure to NPs. Biochemical endpoints revealed decreased esterase activity levels in plasma, suggesting that the immune system of fish might be compromised by exposure to NPs. Moreover, significantly lower levels of alkaline phosphatase were found in the skin mucus of animals exposed to NPs. The present results suggest that NPs may represent a hazard to this marine fish, potentially interfering with the metabolism of lipids and the correct function of the immune response.

PDX1 -MODY and dorsal pancreatic agenesis: New phenotype of a rare disease.

Maturity-Onset Diabetes of the Young (MODY) type 4 or PDX1 -MODY is a rare form of monogenic diabetes caused by heterozygous variants in PDX1 . Pancreatic developmental anomalies related to PDX1 are reported only in neonatal diabetes cases. Here, we describe dorsal pancreatic agenesis in 2 patients with PDX1 -MODY. The proband presented with diabetes since 14 years of age and maintained regular glycemic control with low doses of basal insulin and detectable C-peptide levels after 38 years with diabetes. A diagnosis of MODY was suspected. Targeted next-generation sequencing identified a heterozygous variant in PDX1 : c.188delC/p.Pro63Argfs*60. Computed tomography revealed caudal pancreatic agenesis. Low fecal elastase indicated exocrine insufficiency. His son had impaired glucose tolerance, presented similar pancreatic agenesis, and harbored the same allelic variant. The unusual presentation in this Brazilian family enabled expansion upon a rare disease phenotype, demonstrating the possibility of detecting pancreatic malformation even in cases of PDX1 -related diabetes diagnosed after the first year of life. This finding can improve the management of MODY4 patients, leading to precocious investigation of pancreatic dysgenesis and exocrine dysfunction.

Chlorogenic acid improves the quality of boar semen subjected to cooled storage at 15°C.

The aim of this study was to investigate the effect of chlorogenic acid (ChA) in boar semen stored at 15°C. Twelve ejaculates were processed into insemination doses at different concentrations of ChA (0.0, 1.5, 3.0, 4.0 and 6.0 mg/ml) or vitamin E (200 µl/ml) as positive control. Semen was analysed after 0, 24 and 72 hr of storage. ChA improved (p < .05) sperm motility, acrosome integrity and mitochondrial activity in all periods of storage. Furthermore, after 24 hr of storage, ChA above 1.5 mg/ml supported the sperm viability until 120 min after reheating (p < .05). Both ChA and vitamin E were similarly efficient in increasing the antioxidant capacity of semen, reducing the malondialdehyde levels before and after 72 hr of storage (p < .05). However, with 72 hr of storage, ChA at 3.0 mg/ml improved the mitochondrial activity over vitamin E (p < .05). In conclusion, results suggest that the concentration of 3.2 mg/ml of ChA is the best for semen stored for up to 24 hr. However, for semen stored for a longer period, 6.0 mg/ml or more should be used.

Clinical application of ACMG-AMP guidelines in HNF1A and GCK variants in a cohort of MODY families.

Maturity-onset diabetes of the young (MODY) is a form of monogenic diabetes with autosomal dominant inheritance. GCK -MODY and HNF1A -MODY are the prevalent subtypes. Currently, there is growing concern regarding the correct interpretation of molecular genetic findings. The American College of Medical Genetics and Genomics (ACMG) updated guidelines to interpret and classify molecular variants. This study aimed to determine the prevalence of MODY ( GCK / HNF1A ) in a large cohort of Brazilian families, to report variants related to phenotype, and to classify them according to ACMG guidelines. One hundred and nine probands were investigated, 45% with clinical suspicion of GCK -MODY and 55% with suspicion of HNF1A -MODY. Twenty-five different variants were identified in GCK gene (30 probands-61% of positivity), and 7 variants in HNF1A (10 probands-17% of positivity). Fourteen of them were novel (12- GCK /2- HNF1A ). ACMG guidelines were able to classify a large portion of variants as pathogenic (36%- GCK /86%- HNF1A ) and likely pathogenic (44%- GCK /14%- HNF1A ), with 16% (5/32) as uncertain significance. This allows us to determine the pathogenicity classification more efficiently, and also reinforces the suspected associations with the phenotype among novel variants.

Lipopolysaccharides isolated from Aeromonas salmonicida and Vibrio anguillarum show quantitative but not qualitative differences in inflammatory outcome in Sparus aurata (Gilthead seabream).

In fish, the defence system recognises pathogenic microorganisms via pathogen recognition receptors (PRRs) that sense particular structures of the pathogens; the so-called pathogen associated molecular patterns (PAMPs) such as bacterial lipopolysaccharides (LPSs). The result of the PAMP-PRR interactions leads to complex and orchestrated immune responses. In this study, Sparus aurata (Gilthead seabream) were intraperitoneally injected with purified lipopolysaccharide (LPS) from Aeromonas salmonicida (As)- and Vibrio anguillarum (Va) (1 mg*Kgfish(-1)), both Gram negative bacteria responsible for vibriosis and furunculosis respectively, therefore causing an impact upon marine fish cultures. Head-kidney, intestine, spleen, liver and blood samples were collected at 3, 6, 12 and 24 h post-injection. Plasma levels of cortisol, prostaglandins and lactate were measured and were significantly increased after As-LPS and Va-LPS treatment. Furthermore, tissue-specific differences of the gene regulatory patterns were evident for each LPS. When monocyte/macrophage cell cultures were challenged with As-LPS and Va-LPS, the pro-inflammatory cytokine mRNA abundances present a similar pattern of response. However, As-LPS always triggered a stronger response concerning TNFα, IL1ß and cyclooxygenase-2 (COX2) mRNA abundance as well as PGE2 levels in the supernatant. Overall, the results indicate that specific LPSs do not activate different pro-inflammatory responses and that the observed gene expression pattern is tissue and concentration dependent.

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.

Gene expression and TNF-alpha secretion profile in rainbow trout macrophages following exposures to copper and bacterial lipopolysaccharide.

Fish macrophage function can be altered after exposure to pathogens as well as to xenobiotics. Considering that wild and farmed fish can be exposed in their habitats simultaneously to different types of stressors, including chemical contaminants (e.g. heavy metals) and pathogens (e.g. bacteria), it is fundamental to study their impact either isolated or in combination. Therefore, the present study aimed to evaluate the effects of copper and bacterial lipopolysaccharide (LPS), alone and in combination, on the transcription of target genes related with immune system, respiratory burst activity and cell death, using rainbow trout macrophages as in vitro model. A cell viability experiment was performed to determine the sub-lethal concentrations of copper for rainbow trout macrophages and the LC50-24 h was estimated at 60 µM. The expression of proinflammatory cytokines, such as interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumour necrosis factor-α (TNFα) increased after copper and copper plus LPS exposure. Copper and LPS interact positively inducing an increase in cytokine expression, which may be indicative of an increased inflammatory response. However, the increase in TNFα mRNA expression induced by 50 µM copper was not accompanied by protein secretion indicating that mRNA abundance does not always reflect the level of protein and that the translation of the TNFα mRNA is somehow inhibited. Serum amyloid A (SAA) and trout C-polysaccharide binding protein (TCPBP) mRNA expression also increased after copper, LPS or LPS plus copper exposure, indicating a role of acute phase proteins in the local response to inflammation. NADPH oxidase and glutathione peroxidase gene expression increased in macrophages after 24 h exposure to copper, LPS or LPS plus copper. The results from the present study improve the understanding of mechanisms involved in copper toxicity, as well as the interaction with a simulated-inflammatory process.

A baseline study on the impact of nanoplastics on the portals of entry of xenobiotics in fish.

Mediterranean waters are particularly vulnerable to plastic pollution, with plastic particles concentrations comparable to those found in oceanic gyres. This work aimed to assess the impact of polymethylmethacrylate nanoplastics (PMMA-NPs) on the most important mucosal barriers of the gilthead seabream (Sparus aurata), a highly consumed fish species in the Mediterranean area. Fish were waterborne exposed to NPs (0.001-10 mg/L) for 24 and 96 h, and biochemical parameters associated with oxidative status (total oxidative status and total antioxidant capacity) and immune function (adenosine deaminase, ADA, acetylcholinesterase activity, AChE, and esterase activity, EA) were assessed in gills, intestine, and skin. In intestine, PMMA-NPs led to oxidative status alterations and decreased ADA and EA. In gills, PMMA-NPs induced EA decrease and AChE activity increase. Total protein values were significantly increased in skin. Overall, more alterations were observed in intestine, suggesting it may be one of the most affected tissues by exposure to NPs.

Short-term exposure to polymethylmethacrylate nanoplastics alters muscle antioxidant response, development and growth in Sparus aurata.

Polymethylmethacrylate (PMMA) plastic fragments have been found abundant in the environment, but the knowledge regarding its effects on the physiology of aquatic animals is still poorly studied. Here the short-term (96 h) effects of waterborne exposure to PMMA nanoplastics (PMMA-NPs) on the muscle of gilthead sea bream (Sparus aurata) fingerlings was evaluated at a concentration range that includes 0.001 up to 10 mg/L. The expression of key transcripts related to cell stress, tissue repair, immune response, antioxidant status and muscle development, together with several biochemical endpoints and metabolic parameters. Results indicate that exposure to PMMA-NPs elicit mildly antioxidant responses, enhanced the acetylcholinesterase (AChE) activity, and inhibited key regulators of muscle development (growth hormone receptors ghr-1/ghr-2 and myostatin, mstn-1 transcripts). However, no effects on pro-inflammatory cytokines (interleukin 1ß, il1ß and tumor necrosis factor α, tnfα) expression nor on the levels of energetic substrates (glucose, triglycerides and cholesterol) were found.

Waterborne exposure of gilthead seabream (Sparus aurata) to polymethylmethacrylate nanoplastics causes effects at cellular and molecular levels.

This study evaluated the effect of a short-term exposure to 45 nm polymethylmethacrylate nanoplastics (PMMA-NPs) on the gilthead seabream (Sparus aurata), by assessing biomarkers at different levels of biological organization in liver and plasma. Fish were exposed via water to PMMA-NPs (0, 0.001, 0.01, 0.1, 1 and 10 mg L-1) and sampled after 24 and 96 h. Results showed a general up-regulation of mRNA levels of key genes associated with lipid metabolism (e.g. apolipoprotein A1 and retinoid X receptor). Together with the modulation of the lipid pathway genes we also found a global increase in cholesterol and triglycerides in plasma. Antioxidant-related genes (e.g. glutathione peroxidase 1) were also up-regulated after 24 h of exposure, but their expression levels returned to control afterwards. Total antioxidant capacity (TAC) was increased throughout the experiment, however at 96 h the antioxidant capacity became less efficient, reflected by an increase in the total oxidative status (TOS). Concomitantly, we found an increase in the erythrocytic nuclear abnormalities (ENAs) throughout the trial. Altogether, PMMA-NPs activated the organism's antioxidant defenses and induced alterations in lipid metabolism pathways and genotoxicity in the blood cells of gilthead seabream.

Immuno-modulatory effects of nanoplastics and humic acids in the European seabass (Dicentrarchus labrax).

Pernicious effects of plastic particles, emergent contaminants worldwide, have been described in different species. In teleost species, alterations of immune function after exposure to nanoplastics (NPs) have been reported, but the interaction with cortisol - hypothalamic-pituitary-adrenal (HPI) axis has not yet been explored. Furthermore, the role of dissolved organic matter on the effects of NPs is poorly known. Thus, the aims of this research were to assess if polystyrene NPs (PSNPs) acted as a stressor on juvenile European seabass (Dicentrarchus labrax), interfering with the immune response, as well as to elucidate if humic acids (HA) modulated the potential effects of PSNPs. A short-term exposure to PSNPs and HA elicited an immuno-modulatory response, with an activation of steroidogenic stress-related pathways. An upregulation of anti-inflammatory cytokine (il10, tgfb) and stress-related (mc2r, gr1) transcripts were observed after exposure to HA and PSNPs both individually and in co-exposure. No notable alteration of inflammatory markers was consistently found, which may reflect a protective anti-inflammatory effect of HA in the presence of PSNPs. Nevertheless, there seems to be a more complex interaction between both components. Overall, data show that understanding the interaction of NPs with dissolved organic substances is key to deciphering their environmental risks.

Golden grey mullet and sea bass oxidative DNA damage and clastogenic/aneugenic responses in a contaminated coastal lagoon.

Several xenobiotics or their metabolites have redox-cycling properties and potential to induce oxidative stress and DNA damage. The current work aimed to study, under environmental conditions, oxidative DNA damage (8-hydroxy-2'-deoxyguanosine (8-OHdG)) and its association with chromosomal damage measured as erythrocytic nuclear abnormalities (ENAs), in Liza aurata and Dicentrarchus labrax, caught at a costal lagoon (Ria de Aveiro, Portugal) having sites with different contamination profiles. The quantified parameters were also used to assess the lagoon's environmental status. Five critical sites were assessed comparing to a reference site. L. aurata displayed higher 8-OHdG levels and ENAs frequency, respectively, at Laranjo and Vagos. D. labrax 8-OHdG levels were higher at the sites where quantification was possible whereas no differences were found in terms of ENAs. No correlation was found in both species between 8-OHdG and ENAs. Despite no direct linkage between the two biomarkers was found, this study demonstrates species and site dependent genotoxic responses.

Oilseeds from A Brazilian Semi-Arid Region: Edible Potential Regarding the Mineral Composition.

Oilseeds from five native plant species with edible potential from the Brazilian Caatinga semi-arid region (Diplopterys pubipetala, Barnebya harleyi, Croton adamantinus, Hippocratea volubilis, and Couroupita guianensis) were investigated regarding their mineral contents. The minerals, Na, K, Ca, Mg, Fe, Cu, Cr, Al, were analyzed by high-resolution continuum source atomic absorption spectrometry (HR-CS AAS) and P by the vanadomolybdophosphoric acid colorimetric method. K, Mg, and P were the main elements found (1.62-3.7 mg/g, 362-586 µg/g, and 224-499 µg/g dry weight (dw), respectively). B. harley seeds contained the highest amounts of K and P, while C. guianensis seeds were the richest in Mg. Fe was the most abundant oligoelement (2.3-25.6 µg/g dw). Cr contents were below the limit of quantification for all samples and Al amounts were low: 0.04-1.80 µg/g dw. A linear discriminant analysis clearly differentiated B. harleyi and C. guianensis samples from the remaining ones. In sum, these oilseeds from the Brazilian Caatinga semi-arid region seem to have the potential to be used as natural sources of minerals, mainly K.

Divergent personalities influence the myogenic regulatory genes myostatin, myogenin and ghr2 transcript responses to Vibrio anguillarum vaccination in fish fingerlings (Sparus aurata).

Myogenic regulators of muscle development, metabolism and growth differ between fish species in a context-specific manner. Commonly, the analysis of environmental influences on the expression of muscle-related gene regulators in teleosts is based on differences in swimming performance, feeding behaviour and stress-resistance, but the evaluation of behavioural phenotyping of immune and stress-related responsiveness in skeletal muscle is still scarce. Here we challenge proactive and reactive fingerlings of gilthead sea bream (Sparus aurata), one of the most commonly cultured species in the Mediterranean area, with highly pathogenic O1, O2α and O2ß serotypes of Vibrio anguillarum, a widespread opportunistic pathogen of marine animals, to analyse skeletal muscle responses to bath vaccination. Transcripts related to inflammation (interleukin 1ß, il1ß; tumour necrosis factor-α, tnfα; and immunoglobulin M, igm), and muscle metabolism and growth (lipoprotein, lpl; myostatin, mstn-1; myogenin; and growth hormone receptors type I and II, ghr1 and ghr2, respectively) were analysed. Biochemical indicators of muscle metabolism and function (creatine kinase, CK, aspartate aminotransferase, AST; esterase activity, EA; total antioxidant status, TAC and glucose) were also determined. Our results indicate that proactive, but not reactive, fish respond to Vibrio vaccination by increasing the expression levels of mstn-1, myogenin and ghr2 transcripts at short-/medium- term (1 to 3 days' post vaccination). No effect of vaccination was observed in immune indicators or biochemical parameters in either phenotypes, except for elevated levels of EA in reactive fish one-week post vaccination. This suggests that behavioural divergence should be taken into account to evaluate the crosstalk between immune, metabolic and growth processes in muscle of immune-challenged fish.

Brain and Pituitary Response to Vaccination in Gilthead Seabream (Sparus aurata L.).

Vaccination is a widely used therapeutical strategy in aquaculture, but whether vaccination elicits stress responses in the central neuroendocrine system and enhances the crosstalk between the immune and endocrine systems in the brain or pituitary after vaccination is unclear. To answer this question two experiments using two different vaccine exposure routes, i.e., bath or intraperitoneal (i.p.) injection, were carried out on gilthead seabream (Sparus aurata L.). In the first one, the stress responses of fish subjected to waterborne Vibrio anguillarum bacterin were compared with responses after air exposure or their combination. In the second experiment, fish were subjected to an intraperitoneal injection of Lactococcus garvieae bacterin and we assessed the central stress response and also whether or not a significant immune response was induced in brain and pituitary. In both experiments, blood, brain and pituitary tissues were collected at 1, 6, and 24 h post stress for plasma hormone determination and gene expression analysis, respectively. Results indicated that bath vaccination induced a decreased central stress response compared to air exposure which stimulated both brain and pituitary stress genes. In the second experiment, injection vaccination kept unchanged plasma stress hormones except cortisol that raised at 6 and 24 h. In agreement, non-significant or slight changes on the transcription of stress-related genes were recorded, including the hormone genes of the hypothalamic pituitary interrenal (HPI) axis and other stress markers such as hsp70, hsp90, and mt genes in either brain or pituitary. Significant changes were observed, however, in crhbp and gr. In this second experiment the immune genes il1ß, cox2, and lys, showed a strong expression in both brain and pituitary after vaccination, notably il1ß which showed more than 10 fold raise. Overall, vaccination procedures, although showing a cortisol response, did not induce other major stress response in brain or pituitary, regardless the administration route. Other than main changes, the alteration of crhbp and gr suggests that these genes could play a relevant role in the feedback regulation of HPI axis after vaccination. In addition, from the results obtained in this work, it is also demonstrated that the immune system maintains a high activity in both brain and pituitary after vaccine injection.

Variations in the immune and metabolic response of proactive and reactive Sparus aurata under stimulation with Vibrio anguillarum vaccine.

Environmental insults, such as exposure to pathogens, modulate the behavioural coping style of animals to stressors, and repeated exposure to stressful environments may lead to species-specific infection phenotypes. To analyse the influence of stress behavioural phenotypes on immune and metabolic performance, gilthead sea bream (Sparus aurata L.) were first screened for proactive and reactive coping styles. Once characterized, both behavioural phenotypes fish groups were bath vaccinated with bacterin from Vibrio anguillarum, an opportunistic widespread pathogen of fish. Gills and liver were sampled at 0 (control group), 1, 3 and 7 days post-vaccination. Immune-, oxidative stress- and metabolic-related transcripts (il1ß, tnfα, igm, gpx1, sod, cat, lpl, ghr1 and ghr2), metabolic endpoints (glucose, cholesterol and triglycerides), hepatic health indicators (aspartate aminotransferase, alanine transaminase and alkaline phosphatase), oxidative stress status (esterase activity, total antioxidant capacity and total oxidative status) and stress biomarkers (cortisol) were determined. Present results indicate that screening for coping styles in the gilthead sea bream segregated the two distinct phenotypes as expected: proactive and reactive. Results also indicate that under bath vaccination proactive fish show high immune response and lower metabolism, whereas reactive fish show low immune and higher metabolic responses.

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, il1ß, tnfα, il6, il10 and tgfß) and intestine (il1ß, 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.