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Aquaculture is an extremely prosperous market threatened by pathogen outbreaks, including viruses as nodavirus (NNV), which infect fish species with special interest in trading such as European sea bass. Antimicrobial peptides (AMPs) might constitute potential antiviral agents, which had been previously evaluated in fish with positive prospects, based on their properties as immunomodulators or directly killing pathogens. In this line, we aimed to evaluate this dual role by administering two European sea bass synthetic AMPs (Hamp or Dic) prior to NNV infection. Both treatments conferred partial protection against NNV though viral replication and load were not affected. Both AMPs elicited, prior to infection, AMP response and leukocyte mobilization whilst down-regulated pro-inflammatory markers. Upon infection, Hamp and Dic peptides abrogated the inflammatory response provoked by NNV as well as avoid NNV-induced disturbance of the leucocyte distribution in the brain, mainly neutrophils, macrophages and CD8+ T cells. This study points that preventive applications of synthetic Hamp and Dic peptides exert their antiviral actions through the immunomodulatory role and not by a direct action of the antimicrobial on NNV. This work opens the door to the use of AMPs as potential prophylactic tools against NNV as well as immunostimulant in fish farms.

Previous studies have shown that Nannochloropsis gaditana can partially replace fishmeal in the diet of gilthead seabream, Sparus aurata. However, its effect on muscle growth is hardly known. This experiment was carried out with gilthead seabream adults that were fed with N. gaditana at two inclusion levels (2.5 or 5%) either raw (R2.5 and R5 groups) or cellulose-hydrolyzed (H2.5 and H5 groups) for 45 days in the final fattening phase. The body length and body weight were measured in all fish at the beginning and end of the experiment. Also, the white muscle transverse area (WM), size, number and fibrillar density of the white fibers were measured in 9 fish group−1. After 45 days, the body parameters and the WM did not show significant differences among the groups. However, muscle cellularity did show significant differences, such that the hypertrophy values were higher in the H2.5 and H5 than in the R2.5 and R5 groups. On the contrary, R2.5 and R5 showed the highest fibrillar density and hyperplasia values, which are often positively correlated with the fillet firmness and therefore could improve the final quality of the fish. No significant differences attributable to the inclusion levels of N. gaditana were observed

Numerous studies evaluating the effects of the incorporation of microalgae in feeds have reported favourable impacts on different physiological aspects of aquacultured fish. Although productivity is the major goal in terms of profitability in fish farming, qualitative aspects are gaining the attention of producers, given the relevance of quality attributes related to organoleptic parameters, proximal composition, and shelf life on the commercial value of fish. Indeed, microalgae are acknowledged for their richness in substances with potential positive effects on all those quality attributes. In this context, this study assesses the effects of finishing diets enriched with the microalga Nannochloropsis gaditana, either crude or enzymatically hydrolysed, on several quality parameters of gilthead seabream (Sparus aurata) fillets. Two inclusion levels (2.5 and 5%) of raw and enzymatically hydrolysed microalgal biomass were incorporated into diets, plus a microalgae-free control diet, and a 42-day feeding trial was carried out on fish of commercial size (approx. 500 g body weight). The influence of the experimental diets on fish biometry, fillet quality parameters, and shelf life was evaluated. The results indicate, overall, that microalgae-enriched diets yielded favourable, dose-dependent effects on several objective quality parameters of fillets, namely, improved fatty acid profile, reduced microbial counts, enhanced lipid oxidative status, and improved textural and skin colour attributes. Although the enzymatic pre-treatment of the microalgal biomass was expected to impact positively its functional effects on all quality parameters, however, no general trend was observed.

[EN] Currently, microalgae are used in fish diets, but their long-term growth effect is unknown. In this experiment, juvenile seabream specimens were fed with microalgae-enriched diets for three months, and then transferred to a microalgae-free diet for 10 months to assess long-term effects up to commercial size (≈27 cm and ≈300 g). The juvenile diets contained Nannochloropsis gaditana at 2.5 or 5% inclusion levels, either raw (R2.5 and R5 groups) or cellulose-hydrolyzed (H2.5 and H5 groups). The body length and weight were measured in 75 fish group-1 at commercial stage. The size, number, and fibrillar density of white muscle fibers and the white muscle transverse area were measured in nine fish group-1 at commercial stage. The results showed the highest body weight in H5 at commercial stage. The white muscle transverse area and the white fibres hyperplasia and density also showed the highest values in H5, followed by H2.5. In contrast, the highest hypertrophy was observed in C and R2.5, being associated with the lowest muscle growth in both groups. These results showed a microalgae concentration-dependent effect in hydrolyzed diets as well as an advantageous effect of the hydrolyzed versus raw diets on the long-term growth of Sparus aurata.

[EN] Acquiring immunocompetence is essential in the development of fish embryos, as they are exposed to environmental pathogens even before they are fertilized. Despite the importance of the antimicrobial function as the first line of defense against foreign microorganisms, little knowledge is available about its role in larval development. In vertebrates, transgenerational immune priming influences the acquisition of immunocompetence of specimens, regulating the selective allocation of nongenetic resources to their progeny and modulating their development. In this work, we primed teleost European sea bass broodstock females with a viral protein expression vector in order to evaluate the innate immunity development of their offspring. Several antimicrobial functions, the pattern of expression of gene coding for different antimicrobial peptides (AMPs), and their protein levels, were evaluated in eggs and larvae during development. Our data determined the presence of antimicrobial proteins of maternal origin in eggs, and that female vaccination increases antimicrobial activities and the transcription and synthesis of AMPs during larval development.

Aquaculture production is at a record level and is estimated to increase in the coming years. However, this production can be negatively affected by infectious diseases produced by viruses, bacteria, and parasites, causing fish mortality and economic losses. Antimicrobial peptides (AMPs) are small peptides that may be promising candidates to replace antibiotics because they are the first line of defense in animals against a wide variety of pathogens and have no negative effects; they also show additional activities such as antioxidant or immunoregulatory functions, which makes them powerful alternatives for use in aquaculture. Moreover, AMPs are highly available in natural sources and have already been used in the livestock farming and food industries. Photosynthetic marine organisms can survive under all kinds of environmental conditions and under extremely competitive environments thanks to their flexible metabolism. For this reason, these organisms represent a powerful source of bioactive molecules as nutraceuticals and pharmaceuticals, including AMPs. Therefore, in this study we reviewed the present knowledge about AMPs from photosynthetic marine organism sources and analyzed whether they could be suitable for use in aquaculture.

Introduction: The diversification of species in aquaculture requires the continuous study of animal welfare parameters and the susceptibility to pathogens. An inadequate welfare state results in stress, poor health and increased severity of infectious diseases. In fact, several stress conditions have been related with increased mortalities and betanodavirus (NNV) spread. The shi drum (Umbrina cirrosa) has attracted attention due to its good growth rates and great adaptability to culture conditions together with a great flesh quality. However, this species is susceptible to the four genotypes of NNV and displays stress behavior at high rearing densities. Methodology: In order to determine the response of shi drum upon NNV infection at different stressed conditions, specimens reared at low or high density were experimentally infected. A transcriptomic study was performed comparing the gene expression pattern of several tissues of infected and non-infected specimens from the group showing mortalities. The presence of NNV in the gonad of survivors was analyzed. Results: The mortalities observed were low but only occurred at the group reared at high density. The transcriptomic study showed that the number of tissue-specific genes expressed increased in head-kidney, liver and brain, and decreased in spleen. In the four tissues, cell adhesion, leukocyte migration, cytokine interaction, cell proliferation, cell survival and autophagy related pathways were differentially expressed. NNV was detected in the gonad of surviving fish at all rearing densities analyzed. Conclusions: No differences in the viral capacity of colonizing the gonad were observed between low- and high-density conditions. At this last condition, NNV infection produced a deep systemic alteration in shi drum. The main pathways altered were related with immune response, cell cycle and autophagy, accordingly with studies performed in other fish species.

Diet plays a crucial role in causing intestinal dysbiosis, leading to disruptions in the intestinal barrier and increased permeability. This condition facilitates the entry of harmful substances and microorganisms into the bloodstream, directly and indirectly contributing to the development of bacterial pathologies and physiological imbalances in the body. Consequently, the analysis and study of the intestinal microbiota have become significantly important for designing and implementing new diets or supplements in aquaculture. By studying the diet and gut microbiota at different life stages of Sparus aurata, this research has the potential to provide valuable information on how to promote a healthy digestive tract and improve aquaculture production in the future. In this context, Nannochloropsis gaditana are microalgae rich in beneficial lipids, antioxidant compounds, and amino acids, which have shown to improve growth and product quality in Sparus aurata. The objective of this study is to investigate the changes in the microbiota during the transition from larval to juvenile S. aurata when fed a diet supplemented with microalgae in both raw and hydrolyzed forms for a period of 45 days

Introduction. Nodavirus (NNV) is the most threatening marine virus in the Mediterranean area, and it is spreading in the number and type of viral isolates and host fish species, either wild or culture. In our research group we have been studying the immune response of fish against NNV for 2 decades, focusing on gilthead seabream and European sea bass models. While European sea bass is highly susceptible to some classical NNV genotypes the gilthead seabream acts as a resistant and reservoir species. However, their potential vertical transmission, the emergence of new natural NNV recombinants, producing high mortalities to traditionally-resistant species such as gilthead seabream, the climate change and the aquaculture diversification might increase the deleterious effects of NNV infection in the aquaculture sector. Based on this, we are now increasing our knowledge about fish-NNV interactions in the following topics: 1. Generation of biotechnological tools to study NNV and the fish immune response. We have developed several fish cell lines (from seabream, sea bass, European eel or Senegalese sole) susceptible to NNV, antibodies for localization studies and transcriptomic databases, useful as research and diagnostic tools. These tools allow us to increase their applications to other host, marine viruses or, even, other fields. 2. Deepening of the antiviral immune response of fish. We are identifying and characterizing, among others, the involvement of antimicrobial peptides (AMPs) and cell-mediated cytotoxicity as two of the most important antiviral immune mechanisms. Thus, we are able to analyze levels of antibodies and AMPs, or the adaptive cellular cytotoxic activity in fish, novel techniques that are routinely applied in our laboratory. 3. Effect of culture conditions and welfare on susceptibility to NNV. We are characterizing the lethality and presence of the virus in survivors and its success on vertical transmission under different culture conditions, size, or

Autophagy is an intracellular degradation process playing an important role in homeostasis. Viral infections can modulate autophagy and, when induced, can be host-beneficial. To date evidence points to the interest of investigating strategies that upregulate autophagy as fish virus preventive treatments. Nodavirus (NNV) is a great threat for Mediterranean aquaculture as it produces severe natural outbreaks leading to great economic losses to the industry. Regarding autophagy, the NNV capsid protein is able to induce autophagic processes. Antimicrobial peptides (AMPs) are short proteins with lytic activity against a broad range of pathogens, and exert great regulatory effects on the immune response. Recently, in insects, a positive relationship between the AMP and autophagy-related genes has been successfully established. Thus, the objective of this work was to analyze whether the administration of synthetic AMPs might regulate the NNV-induced expression of autophagy-related genes. To accomplish our objective, hepcidin and dicentracin peptides were intramuscularly administered to European sea bass juveniles and a day later infected with NNV. Brain, the NNV target tissue, and muscle, the site of infection, were removed in order to ascertain NNV replication in both tissues as well as analyze the transcription levels of autophagy-related genes. Mortality rates were recorded daily. Our data show that NNV replicates in both studied tissues and AMPs administration resulted in lower mortality rates. The transcription profile observed in brain suggests that the treatment with AMPs prior to NNV infection induces autophagy in the main target site of the virus. Therefore, the autophagy induced by AMPs could be an effective mode of viral clearance and could contribute to the increase of survival during NNV infection. Funded by MCIN/AEI 10.13039/501100011033 (grants RTI2018-096625-B-C33, PID2019-105522GB-100 and PRE2020-093771 to L.C) and “Fundación Séneca” (grant 19883/GERM/15).

Aquaculture is an economic sector which is continuously growing. However, natural outbreaks of pathogens constitute an important difficulty that this sector must face. Nodavirus (NNV) is one of the most prevalent pathogens in the Mediterranean Sea, causing viral encephalopathy and retinopathy to more than 170 fish species. One of the most affected species is European sea bass (Dicentrarchus labrax L.), which is greatly important for the Spanish aquaculture sector. Antimicrobial peptides (AMPs) are short aminoacidic sequences, cationic and amphipathic with lytic activity against a broad spectrum of pathogens including viruses. Until now, scare studies have focused on AMPs antiviral activity in fish but with positive prospects. Therefore, we aimed to evaluate the potential stimulatory effects of AMPs on the European sea bass immune system, as well as their potential as a prophylactic agent against NNV. Thus, Hepcidin (Hamp) and Dicentracin (Dic) antimicrobial peptides were chemically synthesized and intramuscularly administered to European sea bass juveniles. Head-kidney (HK) and muscle were removed after one day from the treatment to evaluate the immunomodulatory capability of the AMPs. In that moment, fish were infected with NNV and sampled two days after infection, where HK, muscle and brain were removed. In all samplings, serum samples were taken in order to perform functional analysis. Mortality and clinical signs of infection (ranked according to the severity) were recorded daily. Hamp and Dic administration resulted in a local recruitment of immune cells such as macrophages, neutrophils and T and B-cells to the site of injection. Moreover, the levels of AMPs were modulated both systemic and locally. Regarding the disease pattern upon NNV infection, AMPs administration reduced the transcription levels of proinflammatory gene markers in brain, leading to the prevention of neuronal damage and ameliorating the mortality rates. In addition, Hamp is also able to prev

Spain leads the aquaculture production in the European Union; however the appearance of new competitors in the Mediterranean area resulted in lower prices for most cultured species. In this framework shi drum (Umbrina cirrosa) has attracted attention due to its good growth rates and great adaptability to culture conditions together with a great flesh quality. An inadequate welfare state results in stress, poor health and increased severity of infectious diseases, including those caused by viral pathogens. The betanodavirus (NNV) is the causative agent of viral encephalopathy and retinopathy and infects more than 170 fish species. Nowadays, NNV is a great concern due to its high prevalence in the Mediterranean basin. In addition, several kind of physiological and induced stress conditions have been related with increased mortalities and NNV spread. The diversification of species in aquaculture requires the continuous study of the physiological process of stress to determine appropriate animal welfare parameters for each species together with the study of the susceptibility to pathogens. Shi drum is susceptible to the four genotypes of NNV, although with different clinical signs and replication rates. To increase the knowledge on the biological aspects of fish welfare in general and of shi drum in particular, we have studied different rearing densities and their impact on the response to stress and to NNV infection. Our data show an increase in stress behavior in the highest density that in turn affects the physiological performance of fish. This study was financed by the project OWI TWO (Operational welfare indicators in two aquaculture species) cofunded by the European Maritime and Fisheries Fund (EMFF) and IEO-CSIC, and by MCIN/AEI 10.13039/501100011033 (grants RTI2018-096625-B-C33 and PID2019-105522GB-100) and “Fundación Séneca” (grant 19883/GERM/15).

Historically, gilthead seabream (Sparus aurata) has been considered a fish species resistant to nervous necrosis virus (NNV) disease. Nevertheless, mortality in seabream hatcheries, associated with typical clinical signs of the viral encephalopathy and retinopathy (VER) disease has been confirmed to be caused by RGNNV/SJNNV reassortants. Because of this, seabream larvae at 37 and 86 days post-hatching (dph) were infected by immersion with RGNNV/SJNNV and SJNNV/RGNNV reassortants under laboratory conditions, and mortality, viral replication and immunity were evaluated. Our results show that gilthead seabream larvae, mainly those at 37 dph, are susceptible to infection with both NNV reassortant genotypes, with the highest impact from the RGNNV/SJNNV reassortant. In addition, viral replication occurs at both ages (37 and 86 dph) but the recovery of infective particles was only confirmed in 37 dph larvae,; this value was also highest with the RGNNV/SJNNV reassortant. Larvae immunity, including the expression of antiviral, inflammatory and cell-mediated cytotoxicity genes, was affected by NNV infection. Levels of the natural killer lysin (Nkl) peptide were increased in SJNNV/RGNNV-infected larvae of 37 dph, though hepcidin was not. Our results demonstrate that the seabream larvae are susceptible to both NNV reassortants, though mainly to RGNNV/SJNNV, in an age-dependent manner.

Among the pathogens that most affect Mediterranean aquaculture, betanodavirus (NNV), a non-enveloped icosahedral RNA virus with a genome composed of two single-stranded positive-sense RNA segments known as RNA1 and RNA2, stands out. Apart from the traditional NNV genotypes, there are two reassortant genotypes called RGNNV/SJNNV and SJNNV/RGNNV, due to the origin of the RNA genome. It has been confirmed that gilthead seabream, a species resistant to traditional NNV genotypes, is susceptible to developing NNV disease in larval stages when infected by the RGNNV/SJNNV genotype, bringing a new focus to the prevention of infection in aquaculture farms. Therefore, the main objective of this research was to examine the susceptibility of 37 and 86 days post-hatching (dph) seabream larvae against RGNNV/SJNNV an SJNNV/RGNNV reassortants and the infective capacity of both viruses. Our results confirmed that both genotypes induced mortality in seabream larvae of both ages, being higher for RGNNV/SJNNV genotype and in 36 dph larvae. For both ages, the mortality rates were related to the replicative capacity of the virus. In 36 dph larvae, the high mortality can be explained by the high mRNA levels of viral genes from the beginning of the infection, increasing exponentially until the end of the infection, being always higher for RGNNV/SJNNV. In addition, we were able to recover viral particles for both genotypes, again higher for RGNNV/SJNNV. In contrast, the low, but statistically significant, mortality rate for 86 dph larvae might be due to the low replicative capacity of both viruses at this age. This issue together with a low expression level of viral genes and the failure in recovering infective particles seems to indicate that, even when the virus is initially competent to infect cells, its growth is dismissed or even blocked by different immune mechanisms and, therefore, the virus loses its pathogenic capacity. Further studies are needed to understand the host-viral interac

The production of the aquaculture industry has increased to be equal to that of the world fisheries in recent years. However, aquaculture production faces threats such as infectious diseases. Betanodaviruses induce a neurological disease that affects fish species worldwide and is caused by nervous necrosis virus (NNV). NNV has a nude capsid protecting a bipartite RNA genome that consists of molecules RNA1 and RNA2. Four NNV strains distributed worldwide are discriminated according to sequence homology of the capsid protein encoded by RNA2. Since its first description over 30 years ago, the virus has expanded and reassortant strains have appeared. Preventive treatments prioritize the RGNNV (red-spotted grouper nervous necrosis virus) strain that has the highest optimum temperature for replication and the broadest range of susceptible species. There is strong concern about the spreading of NNV in the mariculture industry through contaminated diet. To surveil natural reservoirs of NNV in the western Mediterranean Sea, we collected invertebrate species in 2015 in the Alboran Sea. We report the detection of the RGNNV strain in two species of cephalopod mollusks (Alloteuthis media and Abralia veranyi), and in one decapod crustacean (Plesionika heterocarpus). According to RNA2 sequences obtained from invertebrate species and reported to date in the Mediterranean Sea, the strain RGNNV is predominant in this semienclosed sea. Neither an ecosystem- nor host-driven distribution of RGNNV were observed in the Mediterranean basin.

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

This work evaluates the effects of the dietary inclusion of crude or hydrolysed Arthrospira platensis (Cyanobac- teria) biomass on growth, muscle composition, digestive functionality and immune activities in gilthead seab- ream (Sparus aurata) fry (20.32 mg mean body weight). A 40-day feeding trial was conducted, aimed at assessing four experimental diets that included 5 or 10% (w/w) A. platensis, either crude or hydrolysed, plus a microalgae- free diet as control batch. Overall, none of the dietary treatments caused negative impacts on fish growth, body composition, muscle fatty acid profile, or innate immune response. Thus, the dietary inclusion of both crude and hydrolysed A. platensis reduced significantly the oxidation of muscle lipids, especially when using hydrolysed biomass, regardless of the dietary inclusion level. In relation to digestive enzymes, significantly higher levels of trypsin, chymotrypsin and leucine aminopeptidase activities were measured in fish fed on A. platensis-supple- mented diets compared to control fish. In addition, within each inclusion level (5 or 10% w/w), those animals fed with diets that included the hydrolysed biomass yielded consistently higher digestive enzyme activities than those receiving the crude biomass. Microalgae dietary inclusion also induced favourable changes in fish gut morphology, according to the increase in microvilli length and diameter observed. This fact might well have contributed to reinforce the role of the intestinal mucosa as a protective barrier against microorganisms, as well as to enhance the absorptive capacity of the intestinal mucosa. Finally, 10% inclusion of microalgae hydrolysate enhanced lysozyme activity in liver, this fact suggesting improved protection against infectious diseases. In conclusion, the positive effects observed in fish fed with the diets including A. platensis up to 10% (not least the hydrolysed biomass) with regard to the different parameters assessed (digestive enzyme activit

Aquaculture is one of the most prosperous economic sectors. Nevertheless, the natural outbreaks of several infectious diseases make the sector to deal with important economic losses. One of the most important pathogens in the Mediterranean Sea is nodavirus (NNV). NNV is the agent causing viral encephalopathy and retinopathy in more than 170 fish species including some of the most impact in Spanish hatcheries as European sea bass (Dicentrarchus labrax). Antimicrobial peptides (AMPs) are short aminoacidic sequences which constitute important mediators of the innate immune response in teleost fish. AMPs can kill directly a broad range of pathogens such as bacteria or viruses and modulate the host immune response leading to a more effective clearance of pathogens. These properties along with the world emergency in antimicrobial resistance (AMR) make AMPs good candidates to replace traditional antimicrobials. Therefore, we aimed to analyze the molecular regulation of European sea bass defensin beta genes upon NNV infection and after NNV vaccination. To achieve our objective, European sea bass were infected with NNV and samples of head-kidney (HK), brain and gonad were taken. In addition, gonadal cells from healthy males were in vitro stimulated with NNV. Then, ovary samples of control females or vaccinated against NNV (pBAD vaccine) were taken, as well as fertilized eggs and larvae from the same groups. To support our data, we also analyzed in silico the potential antiviral activity of the protein encoded by the studied genes. Our results show that defensin beta 1 gene is up-regulated upon in vivo NNV infection even if in the in silico study showed the lesser predicted activity. Interestingly, the in vitro NNV challenge resulted in no variation of the defensin beta 1 gene, while defensin beta 2.1 and 2.2 genes were blocked upon this stimulus. Strikingly, females vaccinated with pBAD greatly down-regulate defensin beta 2.2 expression. Moreover, larvae from vaccinated moth

The nervous necrosis virus (NNV) leads to viral encephalopathy and retinopathy (VER) disease in more than 170 fish species, mainly from marine habitats. It replicates in the central nervous tissues, reaching up to 100% mortalities after a few days of infection, mainly in the larvae and juvenile stages. This is continuously spreading and affecting more species, both wild and cultured, posing a risk to the development of the aquaculture industry. In the Mediterranean Sea, it mainly affects European sea bass (Dicentrarchus labrax) and some grouper species (Epinephelus spp.). Interestingly, in the gilthead seabream (Sparus aurata), typically resistant to common NNV strains, great mortalities in hatcheries associated with typical clinical signs of VER have been confirmed to be caused by RGNNV/SJNNV reassortants. Thus, we have evaluated the susceptibility of seabream larvae to either RGNNV/SJNNV or SJNNV/RGNNV reassortants, as well as the larval immunity. Based on our results we can conclude that: (i) gilthead seabream larvae are susceptible to infection with both NNV reassortant genotypes, but mainly to RGNNV/SJNNV; (ii) virus replicated and infective particles were isolated; (iii) larval immunity was correlated with larval survival; and (iv) larval resistance and immunity were correlated with age of the larvae. Further investigations should be carried out to ascertain the risks of these new pathogens to Mediterranean larviculture.

The gilthead seabream is one of the most valuable species in the Mediterranean basin both for fisheries and aquaculture. Marine fish, such as gilthead seabream, are a source of n3 polyunsaturated fatty acids, highly appreciated for human food owing to their benefits on the cardiovascular and immune systems. The aim of the present study was to estimate heritability for fatty acid (FA) profile in fillet gilthead seabream to be considered as a strategy of a selective breeding program. Total of 399 fish, from a broodstock Mediterranean Sea, were analysed for growth, flesh composition and FA profile. Heritabilities for growth traits, and flesh composition (fat, protein, and moisture content) were medium. Heritability was moderate for 14:0, 16:0 and 18:1n9 and for sum of monounsaturated FA and n6/n3 ratio, and it was low for 20:1n11 and 22:6n3 and the ratio unsaturated/saturated FA. Breeding programs in gilthead seabream usually include growth as the first criterion in the selection process of the fish. However, other quality traits, such as fillet fat content and its fatty acids profile should be considered, since they are very important traits for the consumer, from a nutritional point of view and the benefits for the health.

A challenge test for Photobacterium damselae subsp. piscicida (Phdp) resistance was carried out in two juvenile populations of gilthead seabream (Sparus aurata L.): F2_ATL and F0_MED. At 250 days post-hatching (dph), a fish plasma sample was collected to measure humoral immune markers (peroxidase activity, bactericidal activity, and IgM immunoglobulin levels), and at 272 dph fish were weighed and inoculated with bacteria Phdp. From that time onwards, surviving fish were recorded for nine days, and days to death was registered. Heritabilities for body weight and Phdp survival were moderate, although for days to death the heritability was low. Regarding humoral immune markers, for peroxidase activity it was moderate, and for IgM levels and for bactericidal activity it was low. Genetic correlations for body weight with Phdp survival and days to death were high and positive, while with peroxidase activity and IgM levels they tended to be positive, although these estimates were not accurate. Regarding genetic correlations between Phdp survival and humoral immune markers, they were very high, positive with peroxidase activity, and negative with IgM levels and bactericidal activity. Some humoral immune markers, particularly peroxidase activity, along with performance traits such as body weight and absence of deformities, are proposed to be included in a selective breeding program to raise fish that are capable of coping with diseases

The quest for sustainable fisheries and procurement of food supply has increased aquaculture production up to the world fisheries capture. Viral encephalopathy and retinopathy (VER), also known as viral nervous necrosis (VNN), is caused by nervous necrosis virus (NNV) and results in high mortality of larvae and juveniles of continuous increasing amount of fish species. The disease causes vacuolation and necrosis of the central nervous system. The virus has a nude capsid protecting a bipartite RNA genome that consists of positive stranded molecules RNA1 and RNA2. Four NNV genotype strains distributed worldwide are discriminated according to sequence homology of the capsid protein encoded by RNA2. Preventive treatments prioritize the RGNNV (Redspotted grouper nervous necrosis virus) genotype that has the highest optimum temperature for replication and the broadest range of susceptible species. A flow of NNV between wild and cultured fish had been demonstrated, and reservoirs of NNV have been reported in invertebrates, raising concern on the spreading of NNV in the mariculture industry through contaminated food. The present study aimed to contribute in the surveillance of reservoirs of NNV in invertebrates of the unexplored western Mediterranean Sea. We report the detection of the RGNNV strain in two species of squid (Alloteuthis media and Abralia veranyi), and in one shrimp (Plesionika heterocarpus) collected in 2015 in the Alboran Sea. According to RNA2 sequences obtained from invertebrates and reported to date in the Mediterranean Sea, the strain RGNNV is predominant in this semi-enclosed sea. Our results suggest that RGNNV distribution is apparently independent of host species and ecosystem, and similar between invertebrates and fish species that feed on invertebrates, calling for an increase in surveillance of NNV reservoirs in the wild.

A challenge test for Photobacterium damselae subsp. piscicida (Phdp) resistance was carried out in two juvenile populations of gilthead seabream (Sparus aurata L.): F2_ATL and F0_MED. At 250 days post-hatching (dph), a fish plasma sample was collected to measure humoral immune markers (peroxidase activity, bactericidal activity, and IgM immunoglobulin levels), and at 272 dph fish were weighed and inoculated with bacteria Phdp. From that time onwards, surviving fish were recorded for nine days, and days to death was registered. Heritabilities for body weight and Phdp survival were moderate, although for days to death the heritability was low. Regarding humoral immune markers, for peroxidase activity it was moderate, and for IgM levels and for bactericidal activity it was low. Genetic correlations for body weight with Phdp survival and days to death were high and positive, while with peroxidase activity and IgM levels they tended to be positive, although these estimates were not accurate. Regarding genetic correlations between Phdp survival and humoral immune markers, they were very high, positive with peroxidase activity, and negative with IgM levels and bactericidal activity. Some humoral immune markers, particularly peroxidase activity, along with performance traits such as body weight and absence of deformities, are proposed to be included in a selective breeding program to raise fish that are capable of coping with diseases

The gilthead seabream is one of the most valuable species in the Mediterranean basin both for fisheries and aquaculture. Marine fish, such as gilthead seabream, are a source of n3 polyunsaturated fatty acids, highly appreciated for human food owing to their benefits on the cardiovascular and immune systems. The aim of the present study was to estimate heritability for fatty acid (FA) profile in fillet gilthead seabream to be considered as a strategy of a selective breeding program. Total of 399 fish, from a broodstock Mediterranean Sea, were analysed for growth, flesh composition and FA profile. Heritabilities for growth traits, and flesh composition (fat, protein, and moisture content) were medium. Heritability was moderate for 14:0, 16:0 and 18:1n9 and for sum of monounsaturated FA and n6/n3 ratio, and it was low for 20:1n11 and 22:6n3 and the ratio unsaturated/saturated FA. Breeding programs in gilthead seabream usually include growth as the first criterion in the selection process of the fish. However, other quality traits, such as fillet fat content and its fatty acids profile should be considered, since they are very important traits for the consumer, from a nutritional point of view and the benefits for the health.

Betanodavirus (NNV), the causative agent of viral retinopathy and encephalopathy (VER) disease in fish, is the greatest viral threat for aquaculture growth and diversification in the Mediterranean area, where several genotypes and reassortants have been described. The shi drum (Umbrina cirrosa) is an excellent candidate for marine aquaculture diversification in the Mediterranean area, as this species is easily adapted to culture conditions and shows high growth rates, low mortalities during larval development and high market value. Although outbreaks of RGNNV, the most common NNV genotype in the Mediterranean Sea, have been detected in wild and farmed shi drums, little is known about host–virus interactions, clinical signs, mortality rates and viral load in this species. In this framework, we have evaluated the mortality rates, the signs of the VER disease including behaviour, histopathological alterations in the brain and retina and the rescue of infective particles in shi drums after infection with the four NNV genotypes (RGNNV, SJNNV, BFNNV and TPNNV). We found that all of the genotypes produce mortalities and analogous time-lapses between the first signs of disease and mortalities. However, infective particles were only recovered from RGNNV-, BFNNV- and TPNNV-infected specimens. Interestingly, clinical signs and histopathological lesions in the brain and retina were different depending on the genotype used.

In this study, a total of 18 novel productive traits, three related to carcass [cNiT] and fifteen related to morphometric [mNiT]), were measured in gilthead seabream (Sparus aurata) using Non-invasive Technologies (NiT) as implemented in IMAFISH_ML (MatLab script). Their potential to be used in industrial breeding programs were evaluated in 2348 offspring reared under different production systems (estuarine ponds, oceanic cage, inland tank) at harvest. All animals were photographed, and digitally measured and main genetic parameters were estimated. Heritability for growth traits was medium (0.25–0.37) whereas for NiT traits medium-high (0.24–0.61). In general, genetic correlations between mNiT, cNiT and growth and traits were high and positive. Image analysis artifacts such as fin unfold or shades, that may interfere in the precision of some digital measurements, were discarded as a major bias factor since heritability of NiT traits after correcting them were no significantly different from original ones. Indirect selection of growth traits through NiT traits produced a better predicted response than directly measuring Body Weight (13–23%), demonstrating that this methodological approach is highly cost-effective in terms of accuracy and data processing time.

Nervous necrosis virus (NNV; family Nodaviridae, genus Betanodavirus) is the causative agent of viral retinopathy and encephalopathy (VER) disease, which mainly affects the larval and juvenile stages of fish. The virus has an acute lethal effect in larval stages and juveniles. According to the RNA2 sequence, NNV are mainly divided into four genotypes: RGNNV, SJNNV, BFNNV and TPNNV. The shi drum (Umbrina cirrosa) is a serious candidate for the diversification of Mediterranean aquaculture. Although aspects related to nutrition or reproduction have been elucidated others related to pathology or immunity have been poorly studied. In this regard, the shi drum is a susceptible species for betanodavirus (RGNNV) infection, as several natural outbreaks have been reported. In order to expand the actual knowledge and understand the shi drum-NNV interactions we evaluated whether this fish species is susceptible to all the NNV genotypes. Our data demonstrate that the laboratory infections with all the NNV genotypes produced clinical signs of the VER disease and mortalities in shi drum juveniles. Interestingly, clinical signs and histopathological lesions in the brain and retina were different depending on the genotype used. Finally, viral capsid protein was immunodetected in the brain and retina from all infected fish whilst infective particles were only recovered from RGNNV-, BFNNV- and TPNNV-infected specimens. In conclusion, this work demonstrates that shi drum juveniles are susceptible to all four genotypes of NNV and represent the first step in studying host–NNV interactions and immune responses in this species, which should be further characterized.

Seven-month-old shi drum (Umbrina cirrosa) specimens (mean weight 18.98 g, mean length 11.80 cm) were classified into 2 groups, a control group (C) fed a standard diet and a vegetal group (V) fed a diet with partial replacement of fish meal and fish oil by vegetable products. The body and muscle parameters were studied on days 0, 27, and 62 of the experiment. On day 27, body length was similar in both groups, but body weight was significantly lower in group V than in group C. The transverse area of the white muscle was also significantly lower in V than in C. Hypertrophy was higher in C than in V, whereas hyperplasia was higher in V than in C. On day 62, the body parameters showed similar results to those found on day 27. Muscle cellularity was different from that found on day 27, since hypertrophy was higher in V than in C and hyperplasia was higher in C than in V. The feed conversion rate and specific growth rate were similar in both groups, but the daily intake rate was lower in V than in C. Therefore, it seems that the percentage of vegetables in the vegetal feed was excessively high and this caused less acceptance by the fish. The hepatosomatic, viscerosomatic, and intestinal length indices were similar in both groups. Survival was nearly 100% in both groups.

Common name Umbrina cirrosa specimens (mean weight 9.5 g, mean length 9 cm) were categorized in three densities (4, 9 and 15 kg m-3 ) in order to study the influence of stocking density on the growth, survival rate and feed conversion rates. The body and muscle parameters were studied at 0, 43 and 84 days of the experiment. After 43 days, the highest body parameters values (body length and weight) were found in specimens reared at 9 kg m-3 , followed by the specimens reared at 4 kg m-3 , showing the specimens reared at 15 kg m-3 the lowest growth. These results showed a negative effect of high density on the growth, being the intermediate density (9 kg m-3 ) the most favorable density for the growth at this stage. Muscle cellularity was different among the groups, being the hypertrophy values higher at high density of rearing (15 kg m-3 ) than at 4 and 9 kg m-3 . In contrast, the hyperplasia was higher at 4 kg m-3 than in the rest of densities. The group reared at 9 kg m-3 showed intermediate values of hypertrophy and hyperplasia. At 84 days of the experiment, the muscle cellularity was similar at 4 and 9 kg m-3 , this indicating a compensatory hypertrophic growth at 4 kg m3 . The highest values of body weight were reached at 9 kg m-3 . Moreover, the feed conversion rates and the percentage of survival rate were better at 9 than at 4 kg m-3 .

Prostaglandin E2 (PGE2) plays an important role in immune activities in teleost fish, including seabream. However, receptors involved in PGE2 signaling, as well as the pathways activated downstream, are largely unknown. In this study, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced higher mRNA levels of ptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE2, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE2 receptors in the regulation of the immune responses.

A 90-d feeding trial was conducted in which five groups of gilthead seabream (11.96 g initial body weight) were fed with a microalgae-free diet (control group, C) or four diets containing the microalgae Nannochloropsis gaditana at two inclusion levels (2.5% or 5%), either raw (R2.5 and R5 batches) or cellulose-hydrolyzed (H2.5 and H5 batches), to study their effect on the body and muscle growth. At 40 days, the highest values of body length and weight were reached in R5 group, but at 64 and 90 days, these were reached in R2.5. However, feed conversion rate, specific growth, daily intake, and survival (100%) were similar in all the groups. The acquisition of a discoid body shape was accelerated depending on the inclusion level of N. gaditana in the diets. Moreover, H5 diet affected the fish geometric morphology compared to R5 diet. The white muscle transverse area was similar in all groups at 40 days, with the exception of H2.5 group, which showed the lowest area. At day 90, C and R2.5 displayed the highest muscle growth, attributable to increased hyperplasia in C, and higher hypertrophy in R2.5. However, the highest proportion of small and medium fibers was observed in R5 and H5.

Farmed gilthead sea bream (Sparus aurata) is able to grow efficiently with new feed formulations based on plant ingredients. Here, two experimental diets with standard and high inclusion levels of plant ingredients were formulated to assess the suited use of plant-based diets in fish with different growth genetic backgrounds. To pursue this issue, a long-term feeding trial (12-months) was conducted with fish (17 g initial body weight) of 16 families coming from the broodstock of PROGENSA project, that were grown communally in the IATS-CSIC experimental facilities. All fish in the study (2545) were PIT-tagged, and their pedigree was re-constructed with 96% success by using a SMsa1 multiplex of 11 microsatellites, which revealed the main parents contributions of 5 females and 6 males. Each diet was randomly assigned to replicate 3000 L tanks, gathering each replicate a similar family composition through all the feeding trial. Data on growth performance highlighted a strong ge- netic effect on growth trajectories, associated with enhanced growth during winter in fish selected for faster growth. No main dietary effects were found on growth rates or condition factor, and regression-correlation analyses of growth rates across families on both diets suggest that genome by diet interaction was weak, while genetic variation accounted for most of the growth phenotypic variation. Hepatosomatic index (HSI) and me- senteric fat index (MSI) of five families, covering the growth variability of the population, were regulated nu- tritionally and genetically, but without statistically significant genome by diet interactions. Fish from faster growing families showed shorter intestines after being fed the control diet, but this phenotype was masked by the enriched plant-based diet. Collectively, the results demonstrate that selection for faster growth is associated in gilthead sea bream with different growth trajectories and a high diet flexibility and intestine plasticity.

Antimicrobial peptides (AMPs) are considered to be amongst the most powerful tools for the fight against pa- thogens in fish, since they form part of the innate immune response, which is especially vital in eggs and early larval stages, when the immune system is developing. The fish responsible for a large part of the profits in Mediterranean aquaculture is European sea bass (Dicentrarchus labrax), a species greatly susceptible to nodavirus (NNV), especially in the larval and juvenile stages. In this work, polyclonal antibodies were developed and used to detect and quantify NK-lysin, dicentracin and hepcidin AMPs in European sea bass eggs and during larval development, as well as to evaluate their regulation in juvenile specimens upon NNV infection. Basal and de- tectable levels of all the AMPs studied were present in eggs, confirming the maternal transfer of peptides, which increased in one or two waves during larval development up to 69 days post-fertilization. After NNV infection, the mRNA of all the AMPs analysed was up-regulated five days after infection in most of the tissues, whilst peptide quantification of all three AMPs decreased in the brain, the target tissue for NNV, but increased in the head-kidney 5 days after infection. Further research should be carried out to ascertain the role of AMPs in fish innate immunity and to understand how NNV evades the immune response to be disseminated.

Sparus aurata specimens of 11 months of age (mean weight: 90.2 g, mean length: 18.3 cm) were classified in two diet groups: one control group was fed with conventional diet, and one experimental group was fed with an organic diet. The body and muscle parameters were studied at 0, 41 and 71 days of the experiment. The results showed the highest values of body length and weight as well as the best biological feed conversion rates in the organic group. The transverse area of white muscle, the hyperplasia and the muscle fibres density were also higher in the organic than in the conventional group (P>0.05). In contrast, hypertrophy was greater in the conventional than in the organic group (P<0.05). Survival was 100% in both groups. In conclusion, the organic feeding regime was more favourable for growth than conventional feeding.

Sparus aurata specimens of 11 months of age (mean weight: 90.2 g, mean length: 18.3 cm) were classified in two diet groups: one control group was fed with conventional diet, and one experimental group was fed with an organic diet. The body and muscle parameters were studied at 0, 41 and 71 days of the experiment. The results showed the highest values of body length and weight as well as the best biological feed conversion rates in the organic group. The transverse area of white muscle, the hyperplasia and the muscle fibres density were also higher in the organic than in the conventional group (P>0.05). In contrast, hypertrophy was greater in the conventional than in the organic group (P<0.05). Survival was 100% in both groups. In conclusion, the organic feeding regime was more favourable for growth than conventional feeding.