Functional Additives in a Selected European Sea Bass (Dicentrarchus labrax) Genotype: Effects on the Stress Response and Gill Antioxidant Response to Hydrogen Peroxide (H2O2) Treatment.

Functional ingredients have profiled as suitable candidates for reinforcing the fish antioxidant response and stress tolerance. In addition, selective breeding strategies have also demonstrated a correlation between fish growth performance and susceptibility to stressful culture conditions as a key component in species domestication processes. The aim of the present study is to evaluate the ability of a selected high-growth genotype of 300 days post-hatch European sea bass (Dicentrarchus labrax) juveniles to use different functional additives as endogenous antioxidant capacity and stress resistance boosters when supplemented in low fish meal (FM) and fish oil (FO) diets. Three isoenergetic and isonitrogenous diets (10% FM/6% FO) were supplemented with 200 ppm of a blend of garlic and Labiatae plant oils (PHYTO0.02), 1000 ppm of a mixture of citrus flavonoids and Asteraceae and Labiatae plant essential oils (PHYTO0.1) or 5000 ppm of galactomannan-oligosaccharides (GMOS0.5). A reference diet was void of supplementation. The fish were fed the experimental diets for 72 days and subjected to a H2O2 exposure oxidative stress challenge. The fish stress response was evaluated through measuring the circulating plasma cortisol levels and the fish gill antioxidant response by the relative gene expression analysis of nfΚß2, il-1b, hif-1a, nd5, cyb, cox, sod, cat, gpx, tnf-1α and caspase 9. After the oxidative stress challenge, the genotype origin determined the capacity of the recovery of basal cortisol levels after an acute stress response, presenting GS fish with a better pattern of recovery. All functional diets induced a significant upregulation of cat gill gene expression levels compared to fish fed the control diet, regardless of the fish genotype. Altogether, suggesting an increased capacity of the growth selected European sea bass genotype to cope with the potential negative side-effects associated to an H2O2 bath exposure.

Fishmeal, plant protein, and fish oil substitution with single-cell ingredients in organic feeds for European sea bass (Dicentrarchus labrax).

Single-cell ingredients (SCI) are considered promising nutrient sources which are produced using environmentally friendly biotechnological processes. The aim of the current study was to evaluate the replacement of fishmeal, plant protein sources, and fish oil with SCI in organic feeds for European sea bass (Dicentrarchus labrax). Bacterial protein, yeast protein, and microalgae were used to replace fishmeal trimmings, soya bean meal, and fish oil from trimmings. Triplicate groups (30 fish per replicate) of European sea bass (14.4 ± 2.4 g) were fed the experimental diets for 71 days. The results showed that the incorporation of SCI at all levels of inclusion significantly enhanced nutrient digestibility. Additionally, growth performance parameters were not affected by SCI inclusion, exhibiting similar or improved values. Moreover, a tendency for improved anterior and posterior gut structure was observed and a significant increase of lysozyme activity at the two highest inclusion levels of SCI was determined. Overall, the results showed that the inclusion of SCI at 15% (bacterial: yeast: algae-9.4: 4.7: 1) is possible without compromising any of the parameters evaluated. According to these findings, a higher substitution of fishmeal trimmings, plant protein sources, and fish oil from trimmings with SCI in organic diets for European sea bass (D. labrax) can be further evaluated in future studies.

The Era of Antimicrobial Peptides: Use of Hepcidins to Prevent or Treat Bacterial Infections and Iron Disorders.

The current treatments applied in aquaculture to limit disease dissemination are mostly based on the use of antibiotics, either as prophylactic or therapeutic agents, with vaccines being available for a limited number of fish species and pathogens. Antimicrobial peptides are considered as promising novel substances to be used in aquaculture, due to their antimicrobial and immunomodulatory activities. Hepcidin, the major iron metabolism regulator, is found as a single gene in most mammals, but in certain fish species, including the European sea bass (Dicentrarchus labrax), two different hepcidin types are found, with specialized roles: the single type 1 hepcidin is involved in iron homeostasis trough the regulation of ferroportin, the only known iron exporter; and the various type 2 hepcidins present antimicrobial activity against a number of different pathogens. In this study, we tested the administration of sea bass derived hepcidins in models of infection and iron overload. Administration with hamp2 substantially reduced fish mortalities and bacterial loads, presenting itself as a viable alternative to the use of antibiotics. On the other hand, hamp1 seems to attenuate the effects of iron overload. Further studies are necessary to test the potential protective effects of hamp2 against other pathogens, as well as to understand how hamp2 stimulate the inflammatory responses, leading to an increased fish survival upon infection.

Antimicrobial Peptides: Identification of two Beta-Defensins in a Teleost Fish, the European Sea Bass (Dicentrarchus labrax).

Beta-defensins consist in a group of cysteine-rich antimicrobial peptides (AMPs), widely found throughout vertebrate species, including teleost fish, with antimicrobial and immunomodulatory activities. However, although the European sea bass (Dicentrarchus labrax) is one of the most commercially important farmed fish species in the Mediterranean area, the characterization of its beta-defensins and its potential applications are still missing. In this study, we characterized two members of the beta-defensin family in this species. Phylogenetic and synteny analysis places sea bass peptides in the beta-defensin subfamilies 1 and 2, sharing similar features with the other members, including the six cysteines and the tertiary structure, that consists in three antiparallel beta-sheets, with beta-defensin 1 presenting an extra alpha-helix at the N-terminal. Further studies are necessary to uncover the functions of sea bass beta-defensins, particularly their antimicrobial and immunomodulatory properties, in order to develop novel prophylactic or therapeutic compounds to be used in aquaculture production.

Alteration of the Immune Response and the Microbiota of the Skin during a Natural Infection by Vibrio harveyi in European Seabass (Dicentrarchus labrax).

Disease outbreaks continue to represent one of the main bottlenecks for the sustainable development of the aquaculture industry. In marine aquaculture, many species from the Vibrio genus are serious opportunistic pathogens responsible for significant losses to producers. In this study, the effects on the immune response and the skin microbiota of European sea bass (Dicentrarchus labrax) were studied after a natural disease outbreak caused by V. harveyi. Data obtained from infected and non-infected fish were studied and compared. Regarding the local immune response (skin mucus) a decrease in the protease activity was observed in infected fish. Meanwhile, at a systemic level, a decrease in protease and lysozyme activity was reported while peroxidase activity showed a significant increase in serum from infected fish. A clear dysbiosis was observed in the skin mucus microbiota of infected fish in comparison with non-infected fish. Moreover, V. harveyi, was identified as a biomarker for the infected group and Rubritalea for healthy fish. This study highlights the importance of characterizing the mucosal surfaces and microbial composition of the skin mucus (as a non-invasive technique) to detect potential disease outbreaks in fish farms.

First description and diagnostics of disease caused by Piscirickettsia salmonis in farmed European sea bass (Dicentrarchus labrax Linnaeus) from Croatia.

During the winter of 2013 and 2016, several Croatian fish farms experienced mortalities in the fry of European sea bass, Dicentrarchus labrax. Affected fish showed abnormal swimming behaviour and reduced appetite, and death ensued several days after the onset of clinical signs of disease. Necropsy revealed pale liver, empty digestive tract, distended gall bladder, and hyperaemia and congestion of the meninges. Routine bacteriological examination tested negative, and virological examination ruled out nodavirus infection. Histological examination revealed multifocal necrosis and extensive inflammation in the brain with abundant cellular debris in the ventricles. Inflammatory cells displayed intra-cytoplasmic basophilic vacuoles leading to suspicion of Piscirickettsia salmonis infection. Fluorescent in situ hybridization using an oligonucleotide probe targeting Domain Bacterium applied to tissue sections tested positive. The pathogen was identified by 16S rRNA gene sequencing of brain material, and the sequence showed 99% similarity with P. salmonis. This result enabled the design of an oligonucleotide probe specifically targeting P. salmonis. In 2016, P. salmonis was successfully isolated on CHAB from the brain of an affected specimen and identified using 16S rRNA gene sequencing and MALDI-TOF. This study describes the first outbreak of disease caused by P. salmonis in sea bass in Croatia, while new diagnostic tools will enable further research on its epidemiology and pathogenicity.

The Diverse Piscidin Repertoire of the European Sea Bass (Dicentrarchus labrax): Molecular Characterization and Antimicrobial Activities.

Fish rely on their innate immune responses to cope with the challenging aquatic environment, with antimicrobial peptides (AMPs) being one of the first line of defenses. Piscidins are a group of fish specific AMPs isolated in several species. However, in the European sea bass (Dicentrarchus labrax), the piscidin family remains poorly understood. We identified six different piscidins in sea bass, performed an in-depth molecular characterization and evaluated their antimicrobial activities against several bacterial and parasitic pathogens. Sea bass piscidins present variable amino acid sequences and antimicrobial activities, and can be divided in different sub groups: group 1, formed by piscidins 1 and 4; group 2, constituted by piscidins 2 and 5, and group 3, formed by piscidins 6 and 7. Additionally, we demonstrate that piscidins 1 to 5 possess a broad effect on multiple microorganisms, including mammalian parasites, while piscidins 6 and 7 have poor antibacterial and antiparasitic activities. These results raise questions on the functions of these peptides, particularly piscidins 6 and 7. Considering their limited antimicrobial activity, these piscidins might have other functional roles, but further studies are necessary to better understand what roles might those be.

Inorganic arsenic causes apoptosis cell death and immunotoxicity on European sea bass (Dicentrarchus labrax).

Inorganic arsenic (As) is one of the most toxic pollutants in the water. We have studied their effects on the marine teleost European sea bass (Dicentrarchus labrax) at 2 and 10 days of 5 µM of As2O3 (sub-lethal doses) waterborne exposure. Arsenic accumulates in liver and gill tissues. The expression profile of five genes (bax, blc2, casp3, casp8 and casp9) involved in apoptosis cell death confirmed apoptotic effects in liver, slight changes in gill and no effects in skin according with the histopathology findings. Total IgM level and peroxidase activities were increased at 2 and 10 days, respectively. The bactericidal activity was decreased at 2 days after As exposure. A general decrease of cellular immune activities with significant differences in the case of respiratory burst activity was observed after 2 and 10 days of exposure. This work describes for the first time the effects of As exposure on European sea bass.

In vitro effects of virgin microplastics on fish head-kidney leucocyte activities.

Microplastics are well-documented pollutants in the marine environment that result from production or fragmentation of larger plastic items. The knowledge about the direct effects of microplastics on immunity, including fish, is still very limited. We investigated the in vitro effects of microplastics [polyvinylchloride (PVC) and polyethylene (PE)] on gilthead seabream (Sparus aurata) and European sea bass (Dicentrarchus labrax) head-kidney leucocytes (HKLs). After 1 and 24 h of exposure of HKLs with 0 (control), 1, 10 and 100 mg mL-1 MPs in a rotatory system, cell viability, innate immune parameters (phagocytic, respiratory burst and peroxidase activities) and the expression of genes related to inflammation (il1b), oxidative stress (nrf2, prdx3), metabolism of xenobiotics (cyp1a1, mta) and cell apoptosis (casp3) were studied. Microplastics failed to affect the cell viability of HKLs. In addition, they provoke very few significant effects on the main cellular innate immune activities, as decrease on phagocytosis or increase in the respiratory burst of HKLs with the highest dose of microplastics tested. Furthermore, microplastics failed to affect the expression of the selected genes on sea bass or seabream, except the nrf2 which was up-regulated in seabream HKLs incubated with the highest doses. Present results seem to suggest that continue exposure of fish to PVC or PE microplastics could impair fish immune parameters probably due to the oxidative stress produced in the fish leucocytes.

Vitamin D3 affects innate immune status of European sea bass (Dicentrarchus labrax L.).

The effects of vitamin D3 dietary administration on certain innate immune parameters on the expression of immune-related genes in head-kidney (HK) and gut were investigated in European sea bass Dicentrarchus labrax. Vitamin D3 (vD3) was orally administered to fish in a commercial pellet food supplemented with 0 (control); 3750; 18,750; or 37,500 U kg-1. Furthermore, gut histology was considered. This study showed a modulation in the activities examined in fish fed with the addition of vD3. After just 2 weeks of administration, diet supplementation with the vitamin resulted in increased phagocytic ability, while serum peroxidase content was increased in fish fed with all experimental diets after 4 weeks, no significant differences were observed in protease, anti-protease, natural haemolytic complement activities and total IgM level. At gene level, fbl and rbl transcripts were up-regulated in HK in fish fed with the highest concentration of vD3-supplemented diets after 4 weeks, while in the gut, an up-regulation of hep gene was observed in fish fed with the different doses of vD3. These results suggest that vD3 may be of great interest for immunostimulatory purposes in fish farms.

Impact of date palm fruits extracts and probiotic enriched diet on antioxidant status, innate immune response and immune-related gene expression of European seabass (Dicentrarchus labrax).

The application of additives in the diet as plants or extracts of plants as natural and innocuous compounds has potential in aquaculture as an alternative to antibiotics and immunoprophylactics. The aim of the current study was to evaluate the potential effects of dietary supplementation of date palm fruit extracts alone or in combination with Pdp11 probiotic on serum antioxidant status, on the humoral and cellular innate immune status, as well as, on the expression levels of some immune-related genes in head-kidney and gut of European sea bass (Dicentrarchus labrax) after 2 and 4 weeks of administration. This study showed for the first time in European sea bass an immunostimulation in several of the parameters evaluated in fish fed with date palm fruits extracts enriched diet or fed with this substance in combination with Pdp 11 probiotic, mainly after 4 weeks of treatment. In the same way, dietary supplementation of mixture diet has positive effects on the expression levels of immune-related genes, chiefly in head-kidney of Dicentrarchus labrax. Therefore, the combination of both could be considered of great interest as potential additives for farmed fish.

Skin mucus proteome map of European sea bass (Dicentrarchus labrax).

Skin mucus is the first barrier of fish defence. Proteins from skin mucus of European sea bass (Dicentrarchus labrax) were identified by 2DE followed by LC-MS/MS. From all the identified proteins in the proteome map, we focus on the proteins associated with several immune pathways in fish. Furthermore, the real-time PCR transcript levels in skin are shown. Proteins found include apolipoprotein A1, calmodulin, complement C3, fucose-binding lectin, lysozyme and several caspases. To our knowledge, this is the first skin mucus proteome study and further transcriptional profiling of the identified proteins done on this bony fish species. This not only contributes knowledge on the routes involved in mucosal innate immunity, but also establishes a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.

Terminal carbohydrate composition, IgM level and enzymatic and bacteriostatic activity of European sea bass (Dicentrarchus labrax) skin epidermis extracts.

Although the skin is one of the main defense barriers of fish to date, very little is known about the immune implications and the properties of the numerous substances present in skin cells. In the present study, terminal carbohydrate composition and some components of the skin immunity (total IgM level, and several enzymatic and bacteriostatic activities) present on aqueous and organic epidermal extracts of European sea bass (Dicentrarchus labrax) were determined. Most of the parameters measured followed a protein concentration dose-response. Curiously, both skin extracts have similar levels of total IgM. However, aqueous extracts showed higher presence of some terminal carbohydrates, alkaline phosphatase and esterase activities and lower proteases and ceruloplasmin activities than epidermal organic extracts. Regarding the bacteriostatic activity, the growth of all the bacterial strains tested was reduced when cultivated in presence of organic extracts, being the observed reduction correlated to the protein concentration present in the extract sample. On the contrary, skin aqueous extracts have no significant effect on bacterial growth or even allow bacteria to overgrow, suggesting that the bacteria could use the extracts as a nutrient source. The results are discussed and compared with the same activities studied on fish skin mucus in order to understand their possible implications on mucosal immunity.