Antimicrobial Peptides from Photosynthetic Marine Organisms with Potential Application in Aquaculture.

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.

Profile of Innate Immunity in Gilthead Seabream Larvae Reflects Mortality upon Betanodavirus Reassortant Infection and Replication.

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.

Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue.

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.

The Effect of 17α-Ethynilestradiol and GPER1 Activation on Body and Muscle Growth, Muscle Composition and Growth-Related Gene Expression of Gilthead Seabream, Sparus aurata L.

Endocrine-disrupting chemicals include natural and synthetic estrogens, such as 17α-ethynilestradiol (EE2), which can affect reproduction, growth and immunity. Estrogen signalling is mediated by nuclear or membrane estrogen receptors, such as the new G-protein-coupled estrogen receptor 1 (GPER1). The present work studies the effect of EE2 and G1 (an agonist of GPER1) on body and muscle parameters and growth-related genes of 54 two-year-old seabreams. The fish were fed a diet containing EE2 (EE2 group) and G1 (G1 group) for 45 days and then a diet without EE2 or G1 for 122 days. An untreated control group was also studied. At 45 days, the shortest body length was observed in the G1 group, while 79 and 122 days after the cessation of treatments, the shortest body growth was observed in the EE2 group. Hypertrophy of white fibers was higher in the EE2 and G1 groups than it was in the control group, whereas the opposite was the case with respect to hyperplasia. Textural hardness showed a negative correlation with the size of white fibers. At the end of the experiment, all fish analyzed in the EE2 group showed a predominance of the gonadal ovarian area. In addition, the highest expression of the mafbx gene (upregulated in catabolic signals) and mstn2 (myogenesis negative regulator) was found in EE2-exposed fish.

Identification of an interferon-stimulated gene, isg15, involved in host immune defense against viral infections in gilthead seabream (Sparus aurata L.).

Interferons (IFNs) play a key role in the innate immunity of vertebrates against viral infections by inducing hundreds of IFN-stimulated genes (ISGs), such as isg15. Isg15 is an ubiquitin-like protein, which can conjugate cellular and viral proteins in a process called ISGylation, although it can also act as a cytokine-like protein. Gilthead seabream (Sparus aurata L.) is an important asymptomatic carrier of viral haemorrhagic septicaemia virus (VHSV) and nodavirus, representing a threat to other co-cultivated susceptible species. In order to better understand virus-host interactions in this fish species, this study addresses the identification and molecular characterization of seabream isg15 (sb-isg15). In addition, the modulation of transcript levels of sb-isg15 was analysed in SAF-1 cells and seabream acidophilic granulocytes (AGs) stimulated in vitro with different pathogen-associated molecular patterns (PAMPs) or inoculated with VHSV and striped jack nervous necrosis virus (SJNNV). The full-length cDNA of sb-isg15 gene, encoding a predicted protein of 155 amino acids, was identified and seen to share the same characteristics as other fish and mammalian isg15 genes. Here we report the clear induction of sb-isg15 transcript levels in SAF-1 cells and AGs stimulated with toll-like receptor (TLR) ligands, such as polyinosinic:polycytidylic acid (poly I:C) or genomic DNA from Vibrio anguillarum (VaDNA), respectively. Furthermore, VHSV and SJNNV inoculation induced a significant degree of sb-isg15 transcription in SAF-1 cells and AGs. However, the relative levels of viral RNA transcription showed that SJNNV replication seems to be more efficient than VHSV in both in vitro systems. Interestingly, sb-isg15 transcript induction elicited by VaDNA was reduced in VHSV- and SJNNV-inoculated AGs, suggesting an interference prompted by the viruses against the type I IFN system. Taken together, these findings support the use of seabream AGs as a valuable experimental system to study virus-host interactions, in which sb-isg15 seems to play an important role.

Genes related to cell-mediated cytotoxicity and interferon response are induced in the retina of European sea bass upon intravitreal infection with nodavirus.

Viral diseases are responsible for high rates of mortality and subsequent economic losses in modern aquaculture. The nervous necrosis virus (NNV) produces viral encephalopathy and retinopathy (VER), which affects the central nervous system, is considered one of the most serious viral diseases in marine aquaculture. Although some studies have localized NNV in the retina cells, none has dealt with immunity in the retina. Thus, for the first time, we intravitreally infected healthy specimens of European sea bass (Dicentrarchus labrax) with NNV with the aim of characterizing the immune response in the retina. Ultrastructural analysis detected important retinal injuries and structure degradation, including pycnosis, hydropic degeneration and vacuolization in some cell layers as well as myelin sheaths in the optic nerve fibres. Immunohistochemistry demonstrated that NNV replicated in the eyes. Regarding retinal immunity, NNV infection elicited the transcription of genes encoding proteins involved in the interferon (IFN) and cell-mediated cytotoxicity (CMC) responses as well as B and T cell markers, demonstrating that viral replication influences innate and adaptive responses. Further studies are needed to understand the retina immunity and whether the main retinal function, vision, is affected by nodavirus.

Dietary carbohydrates improve oxidative status of common dentex (Dentex dentex) juveniles, a carnivorous fish species.

Common dentex (Dentex dentex) is an appreciated carnivorous fish with high growth rate and life cycle adaptable to existing farming techniques. Since the use of carbohydrates is an economic and sustainable alternative for a protein-sparing effect, the study of how this macronutrient affects the welfare of carnivorous species must be studied. The aim of the present study was to evaluate the effects of different types and levels of carbohydrates on common dentex oxidative status. Nine isonitrogenous (43%) and isoenergetic (22MJkg-1) diets were formulated combining three types (pregelatinized starch-PS, dextrin-Dx and maltodextrin-Mx) and three levels (12, 18 and 24%) of carbohydrates. The activities of catalase-CAT, superoxide dismutase-SOD, glutathione peroxidase-GPX, glutathione reductase-GR and glucose 6-phosphate dehydrogenase-G6PDH, SOD isoenzymatic profile, lipid peroxidation-LPO and protein oxidation-PO were determined in liver and white muscle. SOD and CAT were not affected. GPX in liver and white muscle and GR in liver increased at higher inclusion carbohydrates levels. The lowest levels of GR and G6PDH in both tissues and LPO in liver were observed in maltodextrin groups. No significant effects by carbohydrate source were observed in liver PO and white muscle LPO. Regarding carbohydrate level effect, 18% and 24% dietary inclusion level decreased LPO in white muscle and PO in liver. LPO in liver was also decreased at 24% inclusion level. Altogether, results indicate the use of carbohydrates as an alternative energy source does not produce negative effects on oxidative status of common dentex, on the contrary, even contribute to their oxidative protection.

Prostaglandin E2 promotes M2 polarization of macrophages via a cAMP/CREB signaling pathway and deactivates granulocytes in teleost fish.

The profile of prostaglandin (PG) production is determined by the differential expression of the enzymes involved in their production and degradation. Although the production of PGE2 by fish leukocytes has been relatively well studied in several fish species, knowledge of how its production is regulated, its biological activities and the signaling pathways activated by this PG is scant or even contradictory. In this work we show that in the teleost fish gilthead seabream (Sparus aurata L.) macrophages regulate PGE2 release mainly by inducing the expression of the genes encoding the enzymes responsible for its synthesis, while acidophilic granulocytes (AGs) not only induce these genes quickly after activation but also inhibit the expression of the genes encoding the enzymes responsible for PGE2 degradation at later time points. In addition, treatment of macrophages with PGE2 promoted their M2 polarization, which is characterized by high expression levels of interleukin-10, mannose-receptor c-type 1 and arginase 2 genes. In sharp contrast, PGE2 promoted the deactivation of AGs, since it decreased the production of reactive oxygen species and the expression of genes encoding pro-inflammatory cytokines. These differences are the result of the alternative signaling pathways used by PGE2 in macrophages and AGs, a cAMP/CREB signaling pathway operating in macrophages, but not in AGs, downstream of PGE2. Our data identify for the first time a role for professional phagocyte-derived-PGE2 in the resolution of inflammation in fish and highlight key differences in the PGE2 signaling pathway in macrophages and granulocytes.

Transcription of histones H1 and H2B is regulated by several immune stimuli in gilthead seabream and European sea bass.

Histones (H1 to H4) are the primary proteins which mediate the folding of DNA into chromatin; however, and in addition to this function, histones have been also related to antimicrobial peptides (AMPs) activity in vertebrates, in fact, mammalian H1 is mobilized as part as the anti-viral immune response. In fish, histones with AMP activity have been isolated and characterized mainly from skin and gonads. One of most threatening pathogens for wild and cultured fish species nowadays is nodavirus (NNV), which target tissues are the brain and retina, but it is also able to colonize the gonad and display vertical transmission. Taking all this into account we have identified the h1 and h2b coding sequences in European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) fish species and studied their pattern of expression under naïve conditions and NNV in vivo infection. The data obtained prompted us to study their role on the immune response of gonad and head-kidney leucocytes upon viral (NNV), bacteria (Vibrio anguillarum or Photobacterium damselae), pathogen-associated molecular patterns (PAMPs) or mitogens stimulation. The h1 and h2b genes are expressed in a wide range of tissues and their expression is modify by infection or other immune stimuli, but further studies will be needed to determine the significance of these changes. These results suggest that h1 expression is related to the immune response against NNV in the brain, while h2b transcription seems to be more important in the head-kidney. Moreover, the potential role of histones as anti-viral agents is suggested and further characterization is in progress.

Nutritional and metabolic responses in common dentex (Dentex dentex) fed on different types and levels of carbohydrates.

The present study was aimed to evaluate the capacity of common dentex (Dentex dentex) to efficiently use dietary carbohydrates. So, the effects of different type and levels of carbohydrates on growth performance, feed utilization, fish composition, plasma metabolites and key metabolic pathways in liver and white muscle of common dentex are presented. Nine isonitrogenous (43%) and isoenergetic (22 MJ kg(-1)) diets were formulated combining three types, pregelatinized starch (PS), dextrin (Dx) and maltodextrin (Mx), and three levels (12, 18 and 24%) of carbohydrates. Growth performance was not significantly influenced by treatments. The best feed utilization was observed in 18% Mx group. Higher hepatic lipid content was found in fish fed lower dietary carbohydrate levels. PS induced higher liver and white muscle hexokinase and pyruvate kinase activities compared to the lower values observed for Mx. Malic enzyme and glucose 6-phosphate dehydrogenase in liver and white muscle were lower in Mx group. The influence of dietary carbohydrates source was more noticeable than those induced by the carbohydrate level, when glycolysis and lipogenesis pathways were considered. Common dentex is able to use properly dietary carbohydrates, although optimal dietary inclusion levels are below 24%. The greater protein-sparing effect was promoted by the less complex carbohydrate (maltodextrin) and the best feed utilization indices were obtained at intermediate levels of inclusion (18%).

Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata).

Mucosal surfaces of fish, including skin, gill and gut, contain numerous immune substances poorly studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to identify and characterize for the first time different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata). To do this, the levels of total immunoglobulin M, several enzymes and proteins (peroxidase, lysozyme, alkaline phosphatase, esterases, proteases and antiproteases), as well as the bactericidal activity against opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum, Photobacterium damselae) and non-pathogenic bacteria (Escherichia coli, Bacillus subtilis) were measured in the skin mucus and compared with those found in the serum. This study demonstrates that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. In addition, skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, while human bacteria can even grow more in the presence of mucus. The results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system with potential application for the aquaculture.

The susceptibility of shi drum juveniles to betanodavirus increases with rearing densities in a process mediated by neuroactive ligand-receptor interaction.

Nervous necrosis virus (NNV) is one of the greatest threats to Mediterranean aquaculture, infecting more than 170 fish species and causing mortalities up to 100% in larvae and juveniles of susceptible species. Intensive aquaculture implies stressed conditions that affect the welfare of fish and their ability to fight against infections. In fact, a higher susceptibility to NNV has been related to poor welfare conditions. In order to analyze the physiological link between stressed conditions and increased susceptibility to NNV, as well as its possible role in the pathogenesis of this disease, we reared shi drum (Umbrina cirrosa) juveniles (30.7 ± 3.10 g body weight), which are expected to be asymptomatic upon NNV infection, at three stocking densities (2, 15, and 30 kg/m3) for 27 days and subsequently challenged them with NNV. We firstly characterized the stressed conditions of the specimens before and after infection and recorded the mortalities, demonstrating that stressed specimens reared at 30 kg/m3 suffered mortalities. However, the viral loads in different tissues were similar in all experimental groups, allowing horizontal and vertical transmission of the virus from asymptomatic specimens. All of these data suggest that shi drum tolerates wide ranges of culture densities, although high densities might be a setback for controlling NNV outbreaks in this species. In an attempt to understand the molecular pathways orchestrating this susceptibility change in stressed conditions, we performed a transcriptomic analysis of four tissues under mock- and NNV-infected conditions. In addition to the modification of the exceptive pathways such as cell adhesion, leukocyte migration, cytokine interaction, cell proliferation and survival, and autophagy, we also observed a heavy alteration of the neuroactive ligand-receptor pathway in three of the four tissues analyzed. Our data also point to some of the receptors of this pathway as potential candidates for future pharmacological treatment to avoid the exacerbated immune response that could trigger fish mortalities upon NNV infection.

Muscle development and body growth in larvae and early post-larvae of shi drum, Umbrina cirrosa L., reared under different larval photoperiod: muscle structural and ultrastructural study.

Shi drum specimens were maintained under four different photoperiod regimes: a natural photoperiod regime (16L:8D), constant light (24L), equal durations of light and dark (12L:12D) and a reduced number of daylight hours (6L:18D) from hatching until the end of larval metamorphosis. Specimens were then kept under natural photoperiod conditions until 111 days post-hatching. Muscle and body parameters were studied. During the vitelline phase, there was little muscle growth and no photoperiod effects were reported; however, a monolayer of red muscle and immature white muscle fibres were observed in the myotome. At hatching, external cells (presumptive myogenic cells) were already present on the surface of the red muscle. At the mouth opening, some presumptive myogenic cells appeared between the red and white muscles. At 20 days, new germinal areas were observed in the apical extremes of the myotome. At this stage, the 16L:8D group (followed by the 24L group) had the longest body length, the largest cross-sectional area of white muscle and the largest white muscle fibres. Conversely, white muscle hyperplasia was most pronounced in the 24L group. Metamorphosis was complete at 33 days in the 24L and 12L:12D groups. At this moment, both groups showed numerous myogenic precursors on the surface of the myotome as well as among the adult muscle fibres (mosaic hyperplastic growth). The 16L:8D group completed metamorphosis at 50 days, showing a similar degree of structural maturity in the myotome to that described in the 24L and 12L:12D groups at 33 days. When comparing muscle growth at the end of the larval period, hypertrophy was highest in the 16L:8D group, whereas hyperplasia was higher in the 24L and 16L:8D groups. At 111 days, all groups showed the adult muscle pattern typical of teleosts; however, the cross-sectional area of white muscle, white muscle fibre hyperplasia, body length and body weight were highest in the 24L group, followed by the 12L:12D group; white muscle hypertrophy was similar in all groups. Larval survival was higher under natural photoperiod conditions compared to all the other light regimes.

Priming European Sea Bass Female Broodstock Improves the Antimicrobial Immunity of Their Offspring.

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.

Long-Term Effects of a Short Juvenile Feeding Period with Diets Enriched with the Microalgae Nannochloropsis gaditana on the Subsequent Body and Muscle Growth of Gilthead Seabream, Sparus aurata L.

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.

Reservoirs of Red-Spotted Grouper Nervous Necrosis Virus (RGNNV) in Squid and Shrimp Species of Northern Alboran Sea.

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.

Peroxisome proliferator-activated receptors alpha and beta mediate the anti-inflammatory effects of the cyclopentenone prostaglandin 15-deoxy-Δ12,14-PGJ2 in fish granulocytes.

Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor γ (PPARγ) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ2, and its biotinylated analog, as well as several PPARγ ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ2 was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1ß (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPARγ agonists were able to mimic the effects of 15dPGJ2. Conversely, the PPARγ antagonist T007097 abolished the effect of 15dPGJ2 on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ2 and its biotinylated analog signaled via Pparα and Pparß, but not by Pparγ. These results were further confirmed by HPLC/MS analysis, where Pparß was identified as an interactor of biotin-15dPGJ2 in naïve and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ2 acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Pparα and Pparß in the resolution of inflammation mediated by 15dPGJ2.

Identification of a ß1 integrin isoform with restricted tissue expression in a teleost fish.

The composition and organisation of extracellular matrix (ECM)-related molecules change during development. These components interact with different cell surface receptors to modulate the transduction of signals for cell growth, differentiation, migration, proliferation and apoptosis. Previous findings in the teleost fish gilthead seabream (Sparus aurata L., Teleostei), a marine protandrous hermaphrodite fish, showed that endocrine and immune stimuli are able to modulate the expression of ECM-related molecules, as well as specific correlations between them. In the present study, quantitative reverse transcription-polymerase chain reaction was used to examine the gene expression profile of ß(1) integrin isoform b (ITGB1b) and its possible role in reproductive physiology, especially in relation to spermatogenesis. Expression profiles were analysed in the context of the reproductive cycle (RC) and in relation with other ECM-related molecules, including matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, tissue-specific inhibitor of metalloproteinase (TIMP)-2a, TIMP-2b, collagen (COL1A1) and ITGB1a. Expression of ITGB1b was found in the testis and brain and, to some extent, in endothelial cells. In contrast, ITGB1a was expressed ubiquitously. In the testis, the ITGB1b expression peaked during spermatogenesis, whereas the expression of the other ECM-related molecules is induced mainly during the post-spawning stage, both stages of marked tissue remodelling during the first and second RC in males. In addition, in fish exposed to the endocrine disruptor 17α-ethynyloestradiol (at 5 and 50 µg g(-1) food during 7, 14 and 21 days), ITGB1b expression in the testis was inhibited in a dose- and time-dependent manner and was related to reduced serum levels of testosterone. Together, these results suggest a different functionality for the two ITGB1 isoforms in the gilthead seabream, where ITGB1b is more specifically involved in reproduction. This is the first report of an ITGB1 gene isoform whose expression is restricted to endocrine-related tissues in vertebrates.

Genetic Analysis of the Fatty Acid Profile in Gilthead Seabream (Sparus aurata L.).

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.

The molecular, functional and phylogenetic characterization of PGE2 receptors reveals their different roles in the immune response of the teleost fish gilthead seabream (Sparus aurata L.).

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.