Feed pellets containing chitosan nanoparticles as plasmid DNA oral delivery system for fish: In vivo assessment in gilthead sea bream (Sparus aurata) juveniles.

The aim of this study was the assessment of preloaded feed pellets as a delivery system for plasmid DNA (pDNA), with the purpose of evaluating the potential administration of DNA vaccines orally in aquacultured fish. Pellets were made up by usual feed ingredients, which were mixed with chitosan nanoparticles entrapping a model plasmid (pCMVß) expressible in eukaryotic cells before being elaborated. The plasmid is characterized by the insertion of the reporter gene lacZ, encoding for the bacterial enzyme ß-galactosidase (ß-gal). The possible in vivo expression of the exogenous gene was measured in different fish tissues of gilthead sea bream (Sparus aurata) juveniles by two different procedures. On the one hand, the activity of the enzyme ß-gal was detected and quantified in muscle, liver and intestine; on the other, specific IgM against ß-gal antigen was titrated in blood samples. Intramuscular (i.m.) injection of equal amounts of plasmid was also carried out for the purpose of comparison with oral administration. The expression of the reporter gene was detected in fish tissues following both oral and i. m. administration of pDNA up to 60 days. However, organ distribution of the gene expression was more evident after oral (ß-gal activity measured in gut, liver and muscle) than after parenteral administration (restricted to adjacent muscle tissues). In agreement, specific IgM titration indicated that humoral immune response was more intense and sustained throughout the experimental period after oral than after i. m. delivery of equal amounts of pDNA. These results suggest that feed pellets containing chitosan nanoparticles might enable efficient oral delivery of pDNA, a fact that might imply valuable applications in terms of on-farm mass immunization purposes, especially with regard to DNA-based vaccines and small size fish, in which i. m. administration remains unfeasible.

Assessment of the correlation among antibiotic resistance, adherence to abiotic and biotic surfaces, invasion and cytotoxicity of Pseudomonas aeruginosa isolated from diseased gilthead sea bream.

Improper uses of antibiotics to treat fish disease pose an increase of multidrug resistance in Pseudomonas aeruginosa. In order to escape host antimicrobial agents and induce cytotoxicity, different virulence properties are needed by these bacteria such as, biofilm formation, adhesion and invasion ability. This study was conducted to isolate Pseudomonas aeruginosa from diseased cultured gilthead sea bream. Seventeen isolates of Pseudomonas aeruginosa were identified by PCR. All of the isolates tested were susceptible to Gentamicin and Ciprofloxacin. Highest level of resistance was observed against Erythromycin, Ampicillin and Tetracycline. Among the 17 isolates, 11 showed multi-drug resistance. The isolates were screened for biofilm formation in abiotic surfaces, adherence, invasion and cytotoxicity against Hep-2 cells. We found that some strains were able to adhere to abiotic and biotic surface and to enter inside Hep-2 cells. Using cytochalasin D inhibitor, we observed a significant decrease in invasion of epithelial cells. The 17 washed bacterial cells induce variable degree of cytotoxicity. However, no cytotoxic effects on Hep-2 cells were obtained among the totality of cell free filtrate of Pseudomonas strains. By studying the relationship between different virulence properties, a significant positive correlation was obtained between both biofilm formation and adherence, and between adherence and invasion to epithelial cells. Subsequently, we found that the mean values of adhesion and invasion in the MDR group were significantly higher than those observed in the non-MDR group. Likewise, a significant positive correlation was found among adhesive and invasive capacities of Pseudomonas strains and their antibiotic resistance phenotypes.

Assessment of immune status of yellowfin seabream (Acanthopagrus latus) during short term exposure to phenanthrene.

The aim of the present investigation was to assess the immune status in yellowfin seabream (Acanthopagrus latus) exposed to different concentrations of phenanthrene (Phe) for 14days. In addition, the Phe accumulation in the fish muscle was measured during the experiment. Fish were injected with different concentrations (0, 2, 20 and 40mg/kg) of Phe and samples were taken from tissue and blood of fish 1, 4, 7 and 14days after injection. Exposure of fish to Phe caused a significant decrease in white blood cells, C3 and C4 levels, lysosomal membrane stability, lysozyme activity after 4days and antibacterial activity after 7days of the experiment. In contrast, cortisol level significantly increased after 4days. The concentration of Phe in fish muscle increased rapidly after 4days. The main tissue changes observed in the head kidney including increase in melanomacrophage centers (MMCs), empty spaces between cells and hemorrhage. The degree of tissue changes ranged from normal to moderate in Phe-treated fish. The size and number of MMCs in treated fish were significantly higher than control. In conclusion, Phe toxicity in yellowfin seabream can induce increased cortisol level, tissue changes and immune suppression.

Assessment of gold nanoparticle effects in a marine teleost (Sparus aurata) using molecular and biochemical biomarkers.

Gold nanoparticles (AuNP) are increasingly employed in a variety of applications and are likely to be increasing in the environment, posing a potential emerging environmental threat. Information on possible hazardous effects of engineered nanoparticles is urgently required to ensure human and environmental safety and promote the safe use of novel nanotechnologies. Nevertheless, there is a lack of comprehensive knowledge on AuNP effects in marine species. The present study aimed to assess AuNP effects in a marine teleost, Sparus aurata, by combining endpoints at different biological levels (molecular and biochemical). For that purpose, fish were exposed via water for 96h to 4, 80 and 1600µgL(-1) of AuNP (∼40nm) coated with citrate or polyvinylpyrrolidone (PVP). Results revealed a significant impact of AuNP-PVP in the hepatic expression of antioxidant, immune and apoptosis related genes. Total oxidative status was increased in plasma after exposure to the lowest concentration of AuNP-PVP, although without altering the total antioxidant capacity. Furthermore, AuNP did not induce significant damage in the liver since the activity of neither hepatic indicator (aspartate aminotransferase and alkaline phosphatase) increased. Overall, the present study demonstrated that AuNP, even with a biocompatible coating is able to alter oxidative status and expression of relevant target genes in marine fish. Another important finding is that effects are mainly induced by the lowest and intermediate concentrations of the PVP coated AuNP revealing the importance of different coatings.

Are combined AOPs effective for toxicity reduction in receiving marine environment? Suitability of battery of bioassays for wastewater treatment plant (WWTP) effluent as an ecotoxicological assessment.

Ecotoxicological assessment of three different wastewater treatment plant (WWTP) effluents D1, D2 and D3 was performed before and after tertiary treatment using combination of advanced oxidation processes (AOPs). A multibarrier treatment (MBT) consisting of microfiltration (MF), hydrogen peroxide photolysis (H2O2/UVC) and catalytic wet peroxide oxidation (CWPO) was applied for all effluents. Sparus aurata, Paracentrotus lividus, Isochrysis galbana and Vibrio fischeri, representing different trophic levels, constituted the battery of bioassays. Different acute toxicity effects were observed in each WWTP effluents tested. The percentage of sea urchin larval development and mortality fish larvae were the most sensitive endpoints. Significant reduction (p < 0.05) of effluent's toxicity was observed using a classification pT-method after MBT process. Base on obtained results, tested battery of bioassays in pT-method framework can be recommended for acute toxicity preliminary evaluation of WWTP effluents for the marine environment.

Comprehensive biometric, biochemical and histopathological assessment of nutrient deficiencies in gilthead sea bream fed semi-purified diets.

Seven isoproteic and isolipidic semi-purified diets were formulated to assess specific nutrient deficiencies in sulphur amino acids (SAA), n-3 long-chain PUFA (n-3 LC-PUFA), phospholipids (PL), P, minerals (Min) and vitamins (Vit). The control diet (CTRL) contained these essential nutrients in adequate amounts. Each diet was allocated to triplicate groups of juvenile gilthead sea bream fed to satiety over an 11-week feeding trial period. Weight gain of n-3 LC-PUFA, P-Vit and PL-Min-SAA groups was 50, 60-75 and 80-85 % of the CTRL group, respectively. Fat retention was decreased by all nutrient deficiencies except by the Min diet. Strong effects on N retention were found in n-3 LC-PUFA and P fish. Combined anaemia and increased blood respiratory burst were observed in n-3 LC-PUFA fish. Hypoproteinaemia was found in SAA, n-3 LC-PUFA, PL and Vit fish. Derangements of lipid metabolism were also a common disorder, but the lipodystrophic phenotype of P fish was different from that of other groups. Changes in plasma levels of electrolytes (Ca, phosphate), metabolites (creatinine, choline) and enzyme activities (alkaline phosphatase) were related to specific nutrient deficiencies in PL, P, Min or Vit fish, whereas changes in circulating levels of growth hormone and insulin-like growth factor I primarily reflected the intensity of the nutritional stressor. Histopathological scoring of the liver and intestine segments showed specific nutrient-mediated changes in lipid cell vacuolisation, inflammation of intestinal submucosa, as well as the distribution and number of intestinal goblet and rodlet cells. These results contribute to define the normal range of variation for selected biometric, biochemical, haematological and histochemical markers.

Dietary differences are reflected on the gut prokaryotic community structure of wild and commercially reared sea bream (Sparus aurata).

We compared the gut prokaryotic communities in wild, organically-, and conventionally reared sea bream (Sparus aurata) individuals. Gut microbial communities were identified using tag pyrosequencing of the 16S rRNA genes. There were distinct prokaryotic communities in the three different fish nutritional treatments, with the bacteria dominating over the Archaea. Most of the Bacteria belonged to the Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. The number of bacterial operational taxonomic units (OTUs) was reduced from the wild to the conventionally reared fish, implying a response of the gut microorganisms to the supplied food and possibly alterations in food assimilation. The dominant bacterial OTU in all examined fish was closely related to the genus Diaphorobacter. This is the first time that a member of the ß-Proteobacteria, which dominate in freshwaters, are so important in a marine fish gut. In total the majority of the few Archaea OTUs found, were related to methane metabolism. The inferred physiological roles of the dominant prokaryotes are related to the metabolism of carbohydrates and nitrogenous compounds. This study showed the responsive feature of the sea bream gut prokaryotic communities to their diets and also the differences of the conventional in comparison to the organic and wild sea bream gut microbiota.

Feed restriction up-regulates uncoupling protein 3 (UCP3) gene expression in heart and red muscle tissues of gilthead sea bream (Sparus aurata L.) New insights in substrate oxidation and energy expenditure.

The physiological regulation of the mitochondrial uncoupling protein 3 (UCP3) remains practically unexplored in fish and the aim of this study was to examine the effects of ration size on the regulation of UCP3 in heart, red skeletal muscle and white skeletal muscle of gilthead sea bream (Sparus aurata L.). Juvenile fish were fed at three different levels for 11 weeks: i) full ration until visual satiety (R(100) group), ii) 70% of satiation (R(70) group) and iii) 70% of satiation with two finishing weeks at the maintenance ration (20% of the satiation level) (R(70-20) group). The thirty percent feed restriction improved fish performance, increasing feed conversion efficiency and circulating levels of insulin-like growth factor-I (IGF-I). Fish of the R(70-20) group showed reduced growth and low circulating levels of IGF-I in combination with increased circulating concentrations of growth hormone and free fatty acids. Feed restriction did not alter UCP3 transcript levels in white skeletal muscle, but improved this tissue's oxidative capacity as assessed by changes in glycolytic and oxidative mitochondrial enzyme activities. In contrast, in cardiac and red skeletal muscle tissues, this dietary treatment primarily increased UCP3 mRNA expression. The respiratory control ratio of freshly isolated heart mitochondria was slightly lower in R(70-20) fish than in R(100) fish, which suggests that there was an increase in mitochondrial uncoupling concomitant with the enhanced UCP3 mRNA expression. Altogether, these findings highlight the different adaptive mechanism of glycolytic and highly oxidative muscle tissues for their rapid adjustment to varying feed intake.

Metabolomics as a powerful tool for molecular quality assessment of the fish Sparus aurata.

The molecular profiles of perchloric acid solutions extracted from the flesh of Sparus aurata fish specimens, produced according to different aquaculture systems, have been investigated. The (1)H-NMR spectra of aqueous extracts are indicative of differences in the metabolite content of fish reared under different conditions that are already distinguishable at their capture, and substantially maintain the same differences in their molecular profiles after sixteen days of storage under ice. The fish metabolic profiles are studied by top-down chemometric analysis. The results of this exploratory investigation show that the fish metabolome accurately reflects the rearing conditions. The level of many metabolites co-vary with the rearing conditions and a few metabolites are quantified including glycogen (stress indicator), histidine, alanine and glycine which all display significant changes dependent on the aquaculture system and on the storage times.

Free amino acids as indicators of nutritional status of silver bream (Vimba vimba), when using commercial and purified diets.

Studies on larval rearing of silver bream (Vimba vimba), a migratory cyprinid fish have addressed on limited scale larval and juvenile rearing using commercial and semipurified diets along with live feeds, such as brine shrimp Artemia nauplii. The objectives of the present study were (1) to determine whether experimental, protein-, peptide-, free amino acid-based diets are adequate for larval silver bream, a stomachless fish, (2) to evaluate whether commercial and purified diets are comparable as the first/exclusive feed for growth and survival of silver bream, and (3) to examine whether free amino acid concentrations in fish body are potential indicators of availability of amino acid sources. We report here the differences in diets acceptance, fish growth and diet utilization in silver bream in comparison to other cyprinid fishes. We specifically address the response in free amino acids in the body to dietary treatments. Experimental diets included: a commercial Aglo Norse feed, casein-gelatin based diet (CG), free amino acid mixture diet (FAA), dipeptide (PP), dipeptide-protein (PP50) based diet, and dipeptide diet without arginine (NoArg). In addition, live Artemia were offered to 3 groups and "fasting" control treatment was included during 3 week long trial. Fish offered Artemia overperformed those offered formulated diets both in terms of mass (80.7+/-26.3 mg) and survival (97.2%). We also indicate that commercial and purified diets are comparable as the first/exclusive feed for growth and survival of silver bream. Our experiment also showed that the whole body free amino acid concentrations of 9 indispensable amino acids (IDAA) out of 10 (His, Thr, Arg, Val, Met, Ile, Leu, Trp, Lys) in the PP50 group was the highest among 7 diet treatments and the totalfree amino acid concentration, total dispensable amino acids (DAA) and total IDAA of the PP50 diet fed fish showed the same trend. This may indicate that diets based on 50% of dipeptides and 50% of protein are adequate for silver bream larvae as they result in similar growth, survival and whole body IDAA concentrations as in fish fed CG (protein-based) diet fed fish.

Assessment of lipid and essential fatty acids requirements of black seabream (Spondyliosoma cantharus) by comparison of lipid composition in muscle and liver of wild and captive adult fish.

The primary aim of the present study was to compare the contents of total lipid, lipid classes and their associated fatty acids in muscle and liver of wild and one-year captive black seabream (Spondyliosoma cantharus) adults, in order to elucidate the lipid and fatty acids requirements of this fish species of potential interest for aquaculture. The total lipid contents (TL) of muscle and liver of the captive fish were 2.5-fold greater than those of the wild fish. In consequence, contents of triacylglycerols were much higher in tissues of the captive fish. Distribution of fatty acids in total lipids and lipid classes of muscle and liver was also different between both groups of fish. For instance, percentages of 20:4n-6, 20:5n-6 and 22:6n-3 were considerably higher in the wild fish, whereas 18:1, 20:1, and 22:1n-9 as well as 18:2n-6 and 20:5n-3 were more abundant in the captive fish. These results suggest that the lipid composition of the commercial diet supplied to the captive black seabream differed greatly from that of the diet consumed by the fish in the wild, which hypothetically contains the desirable composition for the lipid nutrition of this fish species. Despite the good growth and survival achieved by the black seabream after one year in captivity, the significant accumulation of lipids and the imbalance of essential fatty acids in their muscle and livers, together with the absence of spawning, suggest that future research on the lipid requirements of this omnivorous species is necessary in order to establish whether the administration of currently available aquaculture formulated feeds is adequate for good black seabream performance and reproduction.

Nutritional assessment of somatolactin function in gilthead sea bream (Sparus aurata): concurrent changes in somatotropic axis and pancreatic hormones.

The role of somatolactin (SL) in the regulation of energy homeostasis in gilthead sea bream (Sparus aurata) has been analysed. First, a down-regulation of plasma SL levels in response to gross shifts in dietary amino acid profile and the graded replacement of fish meal by plant protein sources (50%, 75% and 100%) has been observed. Thus, the impaired growth performance with changes in dietary amino acid profile and dietary protein source was accompanied by a decrease in plasma SL levels, which also decreased over the course of the post-prandial period irrespective of dietary nitrogen source. Secondly, we examined the effect of SL and growth hormone (GH) administration on voluntary feed intake. A single intraperitoneal injection of recombinant gilthead sea bream SL (0.1 microg/g fish) evoked a short-term inhibition of feed intake, whereas the same dose of GH exerted a marked enhancement of feed intake that still persisted 1 week later. Further, we addressed the effect of arginine (Arg) injection upon SL and related metabolic hormones (GH, insulin-like growth factor-I (IGF-I), insulin and glucagon) in fish fed diets with different nitrogen sources. A consistent effect of Arg injection (6.6 micromol/g fish) on plasma GH and IGF-I levels was not found regardless of dietary treatment. In contrast, the insulinotropic effect of Arg was found irrespective of dietary treatment, although the up-regulation of plasma glucagon and glucose levels was more persistent in fish fed a fish meal based diet (diet FM) than in those fed a plant protein diet with a 75% replacement (diet PP75). In the same way, a persistent and two-fold increase in plasma SL levels was observed in fish fed diet FM, whereas no effect was found in fish fed diet PP75. Taken together, these findings provide additional evidence for a role of SL as a marker of energy status, which may be perceived by fish as a daily and seasonal signal of abundant energy at a precise calendar time.