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.

Growth performance, gut microbiota composition, health and welfare of European sea bass (Dicentrarchus labrax) fed an environmentally and economically sustainable low marine protein diet in sea cages.

The large use of fish meal/fish oil in carnivorous fish feeds is the main concern regarding environmental sustainability of aquaculture. Here, we evaluated the effects of an innovative diet, designed to be (1) environmentally sustainable by lowering the marine protein content while being (2) cost effective by using sustainable alternative raw materials with acceptable cost and produced on an industrial scale, on growth performance, gut microbiota composition, health and welfare of European sea bass (Dicentrarchus labrax), a key species of the Mediterranean marine aquaculture, reared in sea cages. Results show that the specific growth rate of fish fed the low marine protein diet was significantly lower than those fed conventional diet (0.67% vs 0.69%). Fatty acid profile of fillets from fish fed a low marine protein diet presented significant lower n-6 and higher n-3 content when compared to conventional ones. Then, a significant increase in the abundance of Vibrio and reduction of Photobacterium were found in the gut of fish fed with the low marine protein diet but effects on sea bass health needs further investigation. Finally, no major health and welfare alterations for fish fed the low marine protein diet were observed, combined with a potential slight benefit related to humoral immunity. Overall, these results suggest that despite the low marine protein diet moderately affects growth performance, it nevertheless may enhance environmental and economic sustainability of the sea bass aquaculture.

Genetic Parameters and Genotype × Diet Interaction for Body Weight Performance and Fat in Gilthead Seabream.

There has been thorough research on the genotype by diet interaction and the extent of its impact on the genetic evaluation, using a partly replaced marine animal protein on the gilthead seabream. To do that, 8356 individuals were gathered from two batches and followed different diets: a high-plant-protein diet containing 85% plant proteins and a standard commercial one containing 30% marine animal protein. During the experiment, body weight, growth and fat content were recorded. High heritability estimates were detected for the body weight performance and fat content. A small effect of genotype by diet interaction was detected in all phenotypes (presented as the genetic correlations from 0.95 to 0.97) but a medium-high ranking correlation between the breeding values for each trait was estimated (0.72-0.70). A higher expected response to selection for the body weight performance was detected using the standard commercial rather than the plant-based diet. Based on the findings, the establishment of a plant-based diet breeding strategy can be achieved provided the reduction of the cost of aquafeed is attained, though a lower genetic gain is expected.

Genomic Selection and Genome-Wide Association Analysis for Stress Response, Disease Resistance and Body Weight in European Seabass.

The majority of the genetic studies in aquaculture breeding programs focus on commercial traits such as body weight, morphology, and resistance against diseases. However, studying stress response in European seabass may contribute to the understanding of the genetic component of stress and its future use to select broodstock whose offspring may potentially be less affected by handling. A total of 865 European seabass offspring were used to measure body weight and stress response. Moreover, a disease challenge experiment with Vibrio anguillarum was conducted in a subset (332) of the above fish to study disease resistance. Fish were genotyped with a 57k SNP array, and a Genome-Wide Association study (GWAS) was performed. Five SNPs were found to be statistically significant, three of which affect stress indicators and body weight (in a subgroup of the population), and a putative SNP affects growth performance, while no SNP associated with resistance to Vibrio was found. A moderate to high genomic heritability regarding stress indicators and body weight was estimated using the Restricted Maximum Likelihood (REML) process. Finally, the accuracy, along with the correlation between Estimated Breeding Values (EBVs) and Genomic Estimated Breeding Values (GEBVs), were calculated for all the traits.

Advanced multivariate techniques for the classification and pollution of marine sediments due to aquaculture.

Aquaculture production has globally increased and its environmental impact is not well understood and assessed yet. Therefore, in this work nine metals and metalloids (Cu, Cd, Pb, Hg, Ni, Fe, Mn, Zn and As) and three nutrients (P, N and C) that seem to accumulate in marine sediments, were determined under the fish cages (zero distance) and about 50 and 100 m away from them, in three aquacultures in Greece. The analysis of these data is crucial due to the negative impact of the intensive aquaculture activities on fish population, human health and marine environment. This study investigated the environmental impact associated with aquaculture cages on marine sediments, using Supervised Artificial Neural Networks (ANNs) in parallel with Classification Trees (CTs). Optimised models were constructed in order to detect the significance of each variable, predict the origin of the sediment samples and successfully visualise their results. Three popular ANN architectures, as multi-layer perceptrons (MLPs), radial basis function (RBF) and counter propagation artificial neural networks (CP-ANNs) were used to assess the impact of the intensive aquaculture activities on marine sediments. In addition, more traditional multivariate chemometric techniques like CTs were applied to the same data set for comparison purposes. The modelling study showed that P, N, Cu, Cd were the most critical (and polluting) factors of those metals studied. Moreover, single-element models achieved elevated predictive percentages. The results were justified due to the usual practices used for fish feeding or cages maintenance.

QTL for Stress and Disease Resistance in European Sea Bass, Dicentrarhus labrax L.

There is a growing interest in selective breeding in European sea bass (Dicentrarchus labrax), especially regarding family selection based on growth performance. In particular, quantitative trait loci (QTL) identification in sea bass enhances the application of marker-assisted breeding for the genetic improvement of the production traits. The aims of the study were to identify potential QTL affecting stress and immunological indicators, body weight, and mortality after vibriosis injection in sea bass as well as to estimate heritability and genetic/phenotypic correlations for the aforementioned traits. To this end, stress test was performed on 960 offspring and a sub-group of them (420) was selected to explore the mortality after vibrio injection. Selective genotyping was performed in 620 offspring for 35 microsatellite markers and distributed into 6 linkage groups. The length of the genetic linkage map was 283.6 cM and the mean distance between the markers was 8.1 cM. QTL affecting body weight in three different growth periods detected on linkage groups LG1, LG4, LG6, and LG14. A QTL associated with weight in early growth stages (290-306 days post-hatching) was also identified on LG3. QTL analysis confirmed the existence of QTL affecting cortisol levels, on LG3 and LG14. Moreover, new QTL affecting only cortisol and glucose levels were detected on LG1 and LG23. No QTL affecting hormonal or biochemical marks was found on LG4 and LG6. Heritability of cortisol, lysozyme levels, and mortality were high (0.36, 0.55, and 0.38, respectively).

Scanning of Genetic Variants and Genetic Mapping of Phenotypic Traits in Gilthead Sea Bream Through ddRAD Sequencing.

Gilthead sea bream (Sparus aurata) is a teleost of considerable economic importance in Southern European aquaculture. The aquaculture industry shows a growing interest in the application of genetic methods that can locate phenotype-genotype associations with high economic impact. Through selective breeding, the aquaculture industry can exploit this information to maximize the financial yield. Here, we present a Genome Wide Association Study (GWAS) of 112 samples belonging to seven different sea bream families collected from a Greek commercial aquaculture company. Through double digest Random Amplified DNA (ddRAD) Sequencing, we generated a per-sample genetic profile consisting of 2,258 high-quality Single Nucleotide Polymorphisms (SNPs). These profiles were tested for association with four phenotypes of major financial importance: Fat, Weight, Tag Weight, and the Length to Width ratio. We applied two methods of association analysis. The first is the typical single-SNP to phenotype test, and the second is a feature selection (FS) method through two novel algorithms that are employed for the first time in aquaculture genomics and produce groups with multiple SNPs associated to a phenotype. In total, we identified 9 single SNPs and 6 groups of SNPs associated with weight-related phenotypes (Weight and Tag Weight), 2 groups associated with Fat, and 16 groups associated with the Length to Width ratio. Six identified loci (Chr4:23265532, Chr6:12617755, Chr:8:11613979, Chr13:1098152, Chr15:3260819, and Chr22:14483563) were present in genes associated with growth in other teleosts or even mammals, such as semaphorin-3A and neurotrophin-3. These loci are strong candidates for future studies that will help us unveil the genetic mechanisms underlying growth and improve the sea bream aquaculture productivity by providing genomic anchors for selection programs.

Environmental impact of intensive aquaculture: investigation on the accumulation of metals and nutrients in marine sediments of Greece.

The impact of intensive aquaculture activities on marine sediments along three coastal areas in Greece was studied. The content of nine metals/metalloids (Cu, Cd, Pb, Hg, Ni, Fe, Mn, Zn, As), and three nutrients (P, N and C), that seem to accumulate in marine sediments, was determined under the fish cages (zero distance) and away (50 or 100 m) from them. Elevated concentrations for phosphorus, nitrogen, copper, zinc and cadmium were recorded in the areas where farming establishments are moored. In parallel, the intrinsic differences between the aquaculture facilities and their seasonal variations were investigated. The individual characteristics of each farm (local water currents, facilities' capacity, transferring mechanisms or the geological background) were the determinant factors. On the contrary, significant seasonal differences were not recorded. Statistical techniques, as the non-parametric Mann-Whitney U and Kruskal-Wallis tests and principal components analysis (PCA), factor analysis (FA) and discriminant analysis (DA), were used for the evaluation of the results. These chemometric tools succeeded to discriminate the sampling points according to their distance from the cages or the origin of the sample. Variables' significance, correlations and potential accumulation sources were also investigated.

Structural elucidation of olive pomace fed sea bass (Dicentrarchus labrax) polar lipids with cardioprotective activities.

The purpose of this study was to structurally characterise the polar lipids of sea bass (Dicentrarchus labrax), fed with an experimental diet containing olive pomace (OP), that exhibit cardioprotective activities. OP has been added to conventional fish oil (FO) feed at 4% and this was the OP diet, having been supplemented as finishing diet to fish. Sea bass was aquacultured using either FO or OP diet. At the end of the dietary experiment, lipids in both samples of fish muscle were quantified and HPLC fractionated. The in vitro cardioprotective properties of the polar lipid fractions, using washed rabbit's platelets, have been assessed and the two most biologically active fractions were further analysed by mass spectrometry. The gas-chromatrograpy-mass spectrometric data shows that these two fractions contain low levels of myristic (14:0), oleic (18:1 cis ω-9) and linoleic acids (18:2 ω-6), but high levels of palmitic (16:0) and stearic acids (18:0) as well as eicosadienoic acid (20:2 ω-6). The first fraction (MS1) also contained significant levels of arachidonic acid (20:4 ω-6) and the omega-3 fatty acids: eicosapentaenoic acid (22:5) and docosahexaenoic acid (22:6). Electrospray-mass spectrometry elucidated that the lipid composition of the two fractions contained various diacyl-glycerophospholipids species, where the majority of them have either 18:0 or 18:1 fatty acids in the sn-1 position and either 22:6 or 20:2 fatty acids in the sn-2 position for MS1 and MS2, respectively. Our research focuses on the structure/function relationship of fish muscle polar lipids and cardiovascular diseases and structural data are given for polar lipid HPLC fractions with strong cardioprotective properties.

Evaluation of the in vitro anti-atherogenic properties of lipid fractions of olive pomace, olive pomace enriched fish feed and gilthead sea bream (Sparus aurata) fed with olive pomace enriched fish feed.

Given the pivotal role of Platelet-Activating-Factor (PAF) in atherosclerosis and the cardio-protective role of PAF-inhibitors derived from olive pomace, the inclusion of olive pomace in fish feed has been studied for gilthead sea bream (Sparus aurata). The aim of the current research was to elucidate the anti-atherogenic properties of specific HPLC lipid fractions obtained from olive pomace, olive pomace enriched fish feed and fish fed with the olive pomace enriched fish feed, by evaluating their in vitro biological activity against washed rabbit platelets. This in vitro study underlines that olive pomace inclusion in fish feed improves the nutritional value of both fish feed and fish possibly by enriching the marine lipid profile of gilthead sea bream (Sparus aurata) with specific bioactive lipid compounds of plant origin.