Dietary Artemisia arborescens Supplementation Effects on Growth, Oxidative Status, and Immunity of Gilthead Seabream (Sparus aurata L.).

Fish infectious diseases are one of the main constraints of the aquaculture sector. The use of medicinal plants provides a sustainable way of protection using safe, eco-friendly compounds in a more cost-effective way of treatment, compared to antibiotics. The aim of the present study is the assessment of Artemisia arborescens (AA) feed-supplementation effects on gilthead seabream (Sparus aurata). Fish with an average initial body weight of 109.43 ± 3.81 g, were divided into two groups based on AA feed composition (A25 and A50). Following two months of ad libitum feeding, the effect of diets on fish weight and length were measured. Fish serum and mucus were analyzed for non-specific immune parameters (nitric oxide, lysozyme, myeloperoxidase, protease-/anti-protease activity, and complement), antibody responses, oxidative stress (cytochrome P450 1A1, metallothionein), and metabolism markers (total protein, alkaline phosphatase, and glucose). Expression levels of antioxidants (sod1, gpx1), cytokines (il-1b, il-10, tfgb1, and tnfa), hepcidin, and heat shock protein grp75 genes were measured in spleen samples. A results analysis indicated that A. arborescens use as a feed supplement has a compromised positive effect on the growth performance, immune response, and blood parameters of gilthead seabream. Overall, the suitability of A. arborescens as an efficient food supplement for gilthead seabream health improvement was investigated, setting the basis for its application assessment in Mediterranean aquaculture.

Nervous Necrosis Virus Modulation of European Sea Bass (Dicentrarchus labrax, L.) Immune Genes and Transcriptome towards Establishment of Virus Carrier State.

Viral infections of teleost fish have great environmental and economic implications in aquaculture. Nervous necrosis virus (NNV) is a pathogen affecting more than 120 different species, causing high mortality and morbidity. Herein, we studied the course of NNV experimental infection of D. labrax, focusing on survivors which indicated viral carrier state. To determine the carrier state of D. labrax head kidney, we performed a gene expression analysis of selected immune-related genes and we profiled its transcriptome 14 days post infection (dpi). All tested genes showed clear differentiations in expression levels while most of them were up-regulated 14 dpi suggesting that their role is not limited in early antiviral responses, but they are also implicated in disease persistence. To gain a better understanding of the fish that survived the acute infection but still maintained a high viral load, we studied the differential expression of 124 up-regulated and 48 down-regulated genes in D. labrax head kidney, at 14 dpi. Concluding, the NNV virus persistent profile was assessed in D. labrax, where immune-related gene modification was intense (14 dpi) and the head kidney transcriptome profile at this time point offered a glimpse into host attempts to control the infection in asymptomatic carriers.

Immune response of European sea bass (Dicentrarchus labrax L.) against combination of antigens from three different pathogens.

Three of the most important diseases of Mediterranean intensive European sea bass farming are, viral nervous necrosis (VNN) caused by the red grouper nervous necrosis virus (RGNNV) genotype of b-nodavirus, photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp) and vibriosis caused mainly by the O1 serotype of Vibrio anguillarum (VaO1). Prevention against these diseases is performed through vaccination with a monovalent vaccine against the viral disease and, usually, with bivalent vaccines against the bacterial diseases. However, it is very difficult to program two vaccinations during the same season for the same fish stock and producers are forced to either vaccinate for the viral or the bacterial diseases or to perform double vaccination with both vaccines, without any prior knowledge on any interactions that may occur due to the plethora of antigens (Ag) injected. Ideally, therefore, a trivalent vaccine should be developed against all three diseases. The objective of this work was to analyse the immune response of sea bass against combinations of Ags from all three pathogens, namely viral particles, Phdp whole cells (WC), lipopolysaccharide (LPS), capsular polysaccharide (CPS) and extracellular products (ECPs) and VaO1 WC and ECPs in respect to the identification of any phenomena of immunodominance/immunosuppression between Ags with a view to select candidate Ags for inclusion in a trivalent vaccine formulation. Eight triplicate groups of fish were immunized with different combinations of the aforementioned Ags and another triplicate group served as negative control. Blood serum was isolated at various time-points post-immunization for the measurement of specific antibodies against each Ag and, in addition, leucocytes were isolated at day 29 post-immunization for analysis of various cellular activities. Results indicated that best levels of specific a-NNV virus antibodies (Abs) were produced when VaO1 ECPs were not included in the Ag combinations, in contrast to the leucocytes proliferation assay where best stimulation against NNV Ags was measured when VaO1 ECPs were present in Ag combinations. VaO1 ECPs apparently is a strong immunogen for both humoral and cellular responses but suppresses immunological reactions against the other Ags.VaO1 WC, Phdp LPS and ECPs raised good humoral immune responses in the groups with best responses against VNN Ags, but only VaO1 WC and Phdp ECPs provided good stimulation of leucocytes, with Phdp WC and CPS effecting either similar stimulation with untrained leucocytes (control groups) or down-stimulation. Results are discussed with a view to select Ags from all three pathogens for inclusion in trivalent vaccine against all three pathogens.

The effect of temperature and challenge route on in vitro hemocyte phagocytosis activation after experimental challenge of common octopus, Octopus vulgaris (Cuvier, 1797) with either Photobacterium damselae subsp. damselae or Vibrio anguillarum O1.

Infectious diseases in aquaculture could be associated with high mortalities and morbidity rates, resulting in negative impacts to fish farming industry, consumers, and the environment. Octopods are reared near marine fish farming areas, and this may represent a major risk since fish pathogens may cause pathologies to octopods. Up to date cephalopods immune defense and pathologies, are incompletely understood. Therefore, the aim of this study was to determine the effect of water temperature and challenge route on hemocyte phagocytosis in vitro after experimental challenge of common octopus with Photobacterium damselae subsp. damselae or Vibrio anguillarum O1. Hemolymph was withdrawn at various time-points post-challenge and the number of circulating hemocytes, and phagocytosis ability were determined. No mortalities were recorded irrespective of pathogen, route of challenge and temperature employed. Great variation was observed in the number of circulating hemocytes of both control and challenged specimens in both experiments (1.04 × 105 to 22.33 × 105 hemocytes/ml for the Photobacterium damselae subsp. damselae challenge and 1.35 × 105 to 24.63 × 105 hemocytes/ml for the Vibrio anguillarum O1 and at both studied temperatures). No correlation was found between circulating hemocytes and baseline control specimens body weight. Probably, the number of circulating hemocytes is affected by many extrinsic, and intrinsic factors such as size, age, maturity stage, natural fluctuations and temperature, as indicated in the literature. The hemocyte foreign particles binding ability observed in Photobacterium damselae subsp. damselae experiments, at 21 ± 0.5 °C and 24 ± 0.5 °C, was (mean ± SD) 2.26 ± 2.96 and 11.72 ± 12.36 yeast cells/hemocyte for baseline specimens and 7.84 ± 8.88 and 8.56 ± 9.89 yeast cells/hemocyte for control and challenged specimens, respectively. The corresponding values for Vibrio anguillarum O1 experiments were (mean ± SD) 6.68 ± 9.26 and 7.00 ± 8.11 yeast cells/hemocyte for baseline specimens and 8.82 ± 9.75 and 6.04 ± 7.64 yeast cells/hemocyte for control and challenged specimens, respectively. Hemocytes of the Photobacterium damselae subsp. damselae and Vibrio anguillarum O1 challenged specimens, were more activated at lower temperature. Apparently, temperature is an important factor in hemocyte activation. In addition, our results indicated that time post challenge, route of challenge and pathogen may influence phagocytosis ability.

Genetic Basis for Resistance Against Viral Nervous Necrosis: GWAS and Potential of Genomic Prediction Explored in Farmed European Sea Bass (Dicentrarchus labrax).

Viral nervous necrosis (VNN) is an infectious disease caused by the red-spotted grouper nervous necrosis virus (RGNNV) in European sea bass and is considered a serious concern for the aquaculture industry with fry and juveniles being highly susceptible. To understand the genetic basis for resistance against VNN, a survival phenotype through the challenge test against the RGNNV was recorded in populations from multiple year classes (YC2016 and YC2017). A total of 4,851 individuals from 181 families were tested, and a subset (n∼1,535) belonging to 122 families was genotyped using a ∼57K Affymetrix Axiom array. The survival against the RGNNV showed low to moderate heritability with observed scale estimates of 0.18 and 0.25 obtained using pedigree vs. genomic information, respectively. The genome-wide association analysis showed a strong signal of quantitative trait loci (QTL) at LG12 which explained ∼33% of the genetic variance. The QTL region contained multiple genes (ITPK1, PLK4, HSPA4L, REEP1, CHMP2, MRPL35, and SCUBE) with HSPA4L and/or REEP1 genes being highly relevant with a likely effect on host response in managing disease-associated symptoms. The results on the accuracy of predicting breeding values presented 20-43% advantage in accuracy using genomic over pedigree-based information which varied across model types and applied validation schemes.

In vitro hemocyte phagocytosis activation after experimental infection of common octopus, Octopus vulgaris (Cuvier, 1797) with Photobacterium damselae subsp. piscicida or Vibrio alginolyticus at different temperatures and infection routes.

Due to the fast growth rate, the short life cycle, the high market price and the high food conversion efficiency, O. vulgaris is considered as a good candidate for aquaculture. One of the prerequisites for the successful integration of new species, such as octopi, into industrial-scale production, is the knowledge of the pathological conditions that may arise, with emphasis on infectious diseases caused by microorganisms and para-sites transmitted through wild populations, especially for the farmed organisms cul-tured in cages in proximity to teleost fish. The main objectives of this study were to investigate the sensitivity of common octopus to experimental infection with pathogenic bacteria, to assess the activation of hemocytes and more specifically their phagocytic activity after infection and to associate sensitivity of the species and phagocytic activity of hemocytes to temperature changes, route of infection and pathogen. Common octopus individuals were intramuscularly and intravenously infected with either Photobacterium damselae subsp. piscicida or Vibrio alginolyticus. The hemocyte phagocytosis activation in vitro at two temperatures (21 ± 0.5 °C and 24 ± 0.5 °C) was studied, in an effort to relate these aspects to climate change. Hemolymph was withdrawn on days 0, 3 and 7 post infections/injections. Number of circulating hemocytes/ml hemolymph, phagocytosis ability and Phagocytosis Particle Binding Intensity index were determined. Correlations between hemocytes and bodyweight and between hemocytes and phagocytosis ability were also determined. No mortalities were recorded irrespective of pathogen, route of infection and temperature employed. Circulating hemocytes in control specimens ranged between 1.60x105 hemocytes ml-1 hemolymph to 20.02x105 hemocytes/ml hemolymph at both experiments and temperatures. The interrelation between octopi weight and circulating hemocytes showed that natural fluctuations, age, maturity stage and temperature may affect this relationship. Rise of temperature influenced phagocytosis which seemed to be route of infection, time-point and pathogen related. Specimens infected with Photobacterium damselae subsp. piscicida showed decreased phagocytosis with rise of temperature while when Vibrio alginolyticus was used, phagocytosis activity increased, in most cases. Temperature also played an important role in the correlation between the circulating hemocytes and phagocytosis activity, as at lower temperatures a negative strong correlation was observed. The results prompted us to calculate the activation index. This index showed that temperature is an important factor in hemocyte activation since for Photobacterium damselae subsp. piscicida infected specimens, hemocytes were more activated at 21 ± 0.5℃ instead of 24 ± 0.5℃, and the opposite observed for Vibrio alginolyticus samples and only later post-infection. Comparing the phagocytosis ability results with those obtained from Particle Binding Intensity index important differences concerned mainly confidence levels. The use of phagocytosis ability instead of PBI index provides more accurate results.

Experimental infection of octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses.

Adult common octopus individuals were intramuscularly infected with Photobacterium damsela subsp. piscicida in order to investigate if this species is sensitive to this common and important fish pathogen. The fate of the bacterial antigens and the tissue responses of Octopus vulgaris were studied employing immunohistochemical techniques. Strong reaction at the site of injection was evident from day 2 post-infection that continued until day 14. Great numbers of hemocytes that were attracted at the site of infection were involved in phagocytosis of bacteria. Very early in the infection, a transition of cells to fibroblasts and an effort to isolate the infection was observed. During the course of the study, very large necrotic cells were seen at the site of infection, whereas during the later stages hemocytes with phagocytosed bacteria were observed in well-defined pockets inside the muscle tissue. None of the internal organs tested for the presence of the bacterium were positive with the exception of the digestive gland where antigen staining was observed which was not associated with hemocyte infiltration. The high doses of bacterial cells used in this experimental infection and the lack of disease signs from Octopus vulgaris suggest that, under normal conditions, octopus is resistant to Photobacterium damsela subsp. piscicida.