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.

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.

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.

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.

Molecular cloning and characterization of European seabass (Dicentrarchus labrax) and Gilthead seabream (Sparus aurata) complement component C3.

We present the complete C3 cDNA sequence of Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) and its molecular characterization with a descriptive analysis of their structural elements. We obtained one sequence for Gilthead seabream (gsbC3) which encodes a predicted protein of 1656 amino acids, and two sequences for European seabass (esbC3_1 and esbC3_2) which encode two predicted proteins of 1654 and 1587 amino acids respectively. All sequences present the characteristic structural features of C3 but interestingly esbC3_2 lacks the anaphylotoxin domain and the cysteine residue responsible for thiolester bond formation. Moreover, we have detected and quantified (by real-time PCR-based absolute quantification) specific isoform expression in European seabass depending on pathogen and density conditions in vivo. In addition, we have analyzed the tissue distribution pattern of European seabass and Gilthead seabream C3 genes under crowding stress and under pathological challenges in vivo, and we have observed that crowding and infection status provoke changes in expression levels, tissue expression pattern and C3 isoform expression balance.

Cloning of the glucocorticoid receptor (GR) in gilthead seabream (Sparus aurata). Differential expression of GR and immune genes in gilthead seabream after an immune challenge.

In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1beta, TNF-alpha, Mx protein, cathepsin D and PPAR-gamma). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.

CD83 expression in sea bream macrophages is a marker for the LPS-induced inflammatory response.

CD83, a cell surface membrane glycoprotein member of the Ig superfamily which is commonly used as standard surface marker for dendritic cells, was cloned from gilthead sea bream macrophages using degenerate primers against conserved motifs of known CD83 sequences. The obtained cDNA contains an open reading frame of 669 nucleotides that translate into a 222 amino acid putative peptide. The deduced protein sequence shows conservation of features shared by vertebrate CD83 and multiple alignment with fish CD83 sequences reveals high homology. In cultured sea bream macrophages CD83 mRNA expression was significantly enhanced in a dose- and time-dependent fashion after stimulation with Escherichia coli LPS. These results indicate that in fish, macrophages express high levels of CD83 mRNA after LPS exposure and CD83 is therefore a good marker for activated mature myeloid cells in fish.