Fabio Brambilla

In vivo regulation of GLUT2 mRNA in sea bass (Dicentrarchus labrax) in response to acute and chronic hypoxia.

The expression and regulation of sodium-independent glucose transporter (GLUT)-2, in relation to hypoxia has not yet been explored in fish or other vertebrates. In this study, the complete open-reading frame for sea bass GLUT2 was isolated and deposited in the GenBank. The predicted 12 transmembrane domains of the protein (508 amino acids) are presented. A phylogenetic tree was constructed on GLUT2 sequences of sea bass and those of other teleost, amphibian, avian, and mammalian species. We also analyzed acute and chronic hypoxia-induced changes in the expression of hepatic GLUT2 mRNA, using one-tube, two-temperature, real-time RT-PCR with which gene expression can be absolutely quantified by the standard curve method. The number of GLUT2 mRNA copies was significantly increased in response to both acute (1.9 mg/L, dissolved oxygen for 4 h) and chronic (4.3 mg/L, DO for 15 days) hypoxia conditions. The hypoxia-related changes in GLUT2 mRNA copy number support the view that GLUT2 is involved in the adaptation response to hypoxia in sea bass, a marine hypoxia-sensitive species. We realize that the GLUT2 mRNA levels in our study do not measure the physiological effects produced by the protein. Thus, we can only speculate that, under hypoxic conditions, GLUT2 probably functions to allow the glucose produced from liver glycogen to leave the hepatocytes.

Bio-Mos®: An effective inducer of dicentracin gene expression in European sea bass (Dicentrarchus labrax)

Concern over the use of dietary antibiotics in aquaculture has encouraged the industry to search for alternatives that both enhance performance and afford protection from disease. Bio-Mos, derived from the outer cell wall of a specific strain of yeast Saccharomyces cerevisiae (Alltech Inc, USA) is a product that fits these criteria. Here, we present data on the impact of a Bio-Mos supplemented diet on the mRNA copy number of the antimicrobial peptide dicentracin, whose transcript regulation has not yet been explored in fish.We analyzed Bio-Mos-induced changes in the expression of sea bass (Dicentrarchus labrax) dicentracin,using a one-tube two-temperature real-time RT-PCR with which the gene expression can be absolutely quantified using the standard curve method. Our results revealed that 30 days of feeding fish with diets containing Bio-Mos supplemented at either 3 per thousand or 5 per thousand significantly increased the dicentracin mRNA copy number in the head kidney. Furthermore, the mRNA copy number in fish fed at 3 per thousand was significantly higher than that of the group fed at 5 per thousand for the same period of feeding Bio-Mos. A longer feeding period (60 days)did not further increase the dicentracin transcript levels as compared to the values recorded after 30 days of feeding either in the group fed at 3 per thousand or in the one fed at 5 per thousand diet. However, the transcript levels in fish fed at 3 per thousand proved to be significantly higher than those of the controls after 60 days of feeding. These findings offer new information about the response of antimicrobial peptides at the transcriptional level to diets supplemented with immune response modulators, and support a role of Bio-Mos in promoting sea bass nonspecific immune system.

Dissolved oxygen regimen (PO2) may affect osmorespiratory compromise in European sea bass (Dicentrarchus labrax, L.)

Fundamentally, in land based mediterranean aquaculture, two techniques are applied to supply water with oxygen: paddling water aeration and application of pure oxygen. The two oxygenation techniques result in quite different PO2 regimens and, consequently, different fish growth performance and gill morphology. Data exist showing a reduction in total respiratory surface (RSA) and increasing gas diffusion distance (GDD) in gills of sea bass (Dicentrarchus labrax, L.) farmed under elevated PO2 regimens. That such a modification might have an effect on the ion regulation has been defined elsewhere as osmorespiratory compromise. In this study, European sea bass previously acclimatized to two PO2 regimens, mild hypoxia and mild hyperoxia (70–80% and 130–140% of the saturation value, respectively), were challenged for 1 hour with hypo-osmotic plus manipulation stress in two separate trials. During the first trial, when only Na+ loss was determined, the ion efflux during the first 5 min resulted in a rate of 163.72±31 and 112.23±87 nmol g−1 min−1 from hypoxia and hyperoxia sea bass groups, respectively, and, if sustained, would approach 15.3 and 11.2% per hour of the total body Na+, respectively. During the second trial, in which both Na+ and Cl− loss were determined, after 60 min the Na+ loss was shown to be 76.86±12 and 179.28±32 nmol g−1 min−1 for the fish previously acclimatized to hyperoxia and hypoxia regimens, respectively, whereas for Cl− this loss was 62.02±11 and 157.28±28 nmol g−1 min−1, respectively. Our data are compatible with the hypothesis of an osmotic advantage of sea bass exposed to an elevated PO2 regimen, achievable with application of pure oxygen, instead of simple water aeration.

Effect of dietary Astaxanthin sources supplementation on muscle pigmentation and lipid peroxidation in rainbow trout (Oncorhynchus mykiss)

Abstract Astaxanthin is one of the major carotenoids in aquatic animals including salmonid fishes and is the preferred pigments added to salmon feed. It’s also a powerful antioxidant compared to other carotenoids and that may confer numerous health benefits. The aim of the present experiment was to investigate the effect of Astaxanthin deposition on the lipids peroxidation by studying the Malondialdeide (MDA) level in muscle of rainbow trout (Oncorhynchus mykiss). The Astaxanthin concentrations in fish fed with a commercial sources as Lucantin®Pink (BASF Ludwigshafen, Germany) reached values to 5.76±0.18x10-3 mg/g after 50 days feeding, while the MDA concentration decreased from 1.56x103 to 0.45x103 ng/g. The correlation between MDA and Astaxanthin concentrations decreased linearly and confirmed the antioxidant properties of the pigment by reducing the lipids peroxidation.

Next generation sequencing for gut microbiome characterization in rainbow trout (Oncorhynchus mykiss) fed animal by-product meals as an alternative to fishmeal protein sources

Animal by-product meals from the rendering industry could provide a sustainable and commercially viable alternative to fishmeal (FM) in aquaculture, as they are rich in most essential amino acids and contain important amounts of water-soluble proteins that improve feed digestibility and palatability. Among them, poultry by-product meal (PBM) have given encouraging results in rainbow trout (Oncorhynchus mykiss). However, the introduction of new ingredients in the diet needs to be carefully evaluated since diet is one of the main factors affecting the gut microbiota, which is a complex community that contributes to host metabolism, nutrition, growth, and disease resistance. Accordingly, we investigated the effects of partial replacement of dietary FM with a mix of animal by-product meals and plant proteins on intestinal microbiota composition of rainbow trout in relation to growth and feeding efficiency parameters. We used 1540 trout with an initial mean body weight of 94.6 ± 14.2 g. Fish were fed for 12 weeks with 7 different feed formulations. The growth data showed that trout fed on diets rich in animal by-product meals grew as well as fish fed on control diet, which was rich in FM (37.3%) and PBM-free. High-throughput 16S rRNA gene amplicon sequencing (MiSeq platform, Illumina) was utilised to study the gut microbial community profile. After discarding Cyanobacteria (class Chloroplast) and mitochondria reads a total of 2,701,274 of reads taxonomically classified, corresponding to a mean of 96,474 ± 68,056 reads per sample, were obtained. Five thousand three hundred ninety-nine operational taxonomic units (OTUs) were identified, which predominantly mapped to the phyla of Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. The ratio between vegetable and animal proteins proved to play a central role in determining microbiome profiles and Firmicutes and Proteobacteria phyla were particularly discriminatory for diet type in trout. Plant ingredients favoured a higher Firmicutes:Proteobacteria ratio than animal proteins. Acceptable abundance of Firmicutes was guaranteed by including at least 25% of vegetable proteins in the diet regardless of animal protein source and percentage. In summary animal by-product meals, as replacements to FM, gave good results in terms of growth performances and did not induce significant changes in gut microbial richness, thus proving to be a suitable protein source for use in rainbow trout aqua feed.

Observations of Eurasian perch (Perca fluviatilis L.) post-larvae growth performances reared in an illuminated floating cage in Varese lake (N-W Italy) over a two years period

Eurasian perch (P. fluviatilis) is a very important fish species in Varese lake (N-W Italy). Since the second half of 20th century, perch catches in the lake have steadily decreased and by the end of the ‘80s the species resulted clearly endangered. The purpose of this study was to investigate growth, mortality and feeding conditions of perch post-larvae, reared in illuminated floating cage in Varese lake, to obtain fingerlings for a restocking program. In June 2006 and 2007, groups of 280 and 300 pre-weaned post-larvae (average body weight 0.64±0.09 g and 0.25±0.08 g respectively P<0.01) were held in an illuminated net cage for 90 days. The cage was illuminated inside from 20:30 p.m. to 6:30 a.m. During the trial, the nightly zooplankton accumulation inside the cage was assessed weekly. At night time the zooplankton biomass, which resulted dominated by Cladocera family, was higher inside the cage than in the lake. In 2006, 322±36 zooplankters L−1 were observed, compared to 945±600 observed in 2007 (P<0.05). In the lake, the number of zooplankters per litre was similar in both years, resulting in 63.3±50.30 and 61.10±45 zooplankters L−1, respectively on 2006 and 2007. In order to assess perch growth performances, 25 fishes were sampled from the cage every 15–20 days and length (cm) and weight (g) were assessed for each sample. At the end of September, specific growth rate (SGR) and survival rate were assessed. In 2006 the final mean body weight of the perch fry was 4.65±1.47 g and that results significantly lower (P<0.05) than of 2007 (6.3±1.69 g). The SGR was 2.04% and 3.42%, respectively. The higher growth rate observed in 2007 was influenced by a higher zooplankton accumulation in the cage due to an improved cage management. In order to assess the cage efficiency, in September 2006 and 2007, the weight of young-of-year perch (n=50) captured in the lake were compared to those of reared fish. Wild fry showed a mean body weight significantly higher (P<0.05) than reared ones (15.90±4.25 g and 17.86±4.47 g in 2006 and 2007, respectively). The survival percentages resulted 50.7% in 2006 and 60.7% in 2007. The results demonstrate that the rearing of perch in illuminated floating cages enables the possibility to produce fry for restocking programs in Varese lake.

Molecular characterization and in vivo expression of hypoxia inducible factor (HIF)-1α in sea bass (Dicentrarchus labrax) exposed to acute and chronic hypoxia

Abstract Aquatic hypoxia is a frequent event and in fish a complex set of physiological and biochemical alterations are employed to cope with this environmental stress. Many of these adjustments depend to a large extent on changes in the expression of genes that encode for physiologically relevant proteins. Genes that are induced by hypoxia appear to share a common mode of transcriptional regulation. This induction depends upon activation of a transcription factor, the hypoxia inducible factor-1 (HIF-1), which is a heterodimer composed of two subunits: α and β . In this study we report first on the molecular cloning and characterization of HIF-1α in sea bass (Dicentrarchus labrax). The full-length sea bass cDNA for HIF-1α was isolated and deposited in the GenBank with accession no. DQ171936. It consists of 3317 base pairs (bp) carrying a single open-reading frame that encompasses 2265 bp of the coding region and 1052 bp of the 3’ UTR. We then utilized the real-time PCR technology to monitor dynamic changes in levels of HIF-1α tran-scripts, in response to acute and chronic hypoxic stress. The number of HIF-1α mRNA copies were significantly increased in response to both acute (1.9 mg/L, dissolved oxygen for 4 hours) and chronic (4.3 mg/L, DO for 15 days) hypoxia in sea bass, whereas it remained unchanged in fish exposed to hyperoxic (DO 13.5±1.2 mg/L, 155 % saturation) conditions. This is the first study carried out to investigate the behaviour of HIF-1α gene transcripts during hypoxia in representative of marine hypoxia-sensitive fish species.

The application of two benthic indices to investigate the effects of land-based fish farms in coastal transitional ecosystems: two case studies in Tuscany region (Italy)

The effects on the benthic ecosystem deriving from the activities of two land-based fish farms located in Italian coastal transitional ecosystems (CTEs) were investigated. Together with chemical and physical analysis, some biological analysis was performed. For each fish farm, three stations were sampled: two stations located inside the farm (ponds exit and farm exit) and one station located outside the fish farm, considered as reference site. The AZTI’s Marine Biotic Index (AMBI) and the BENTIX index were calculated, which are being used in assessing the ecological status of benthic communities within the European Water Framework Directive (WFD). Results were compared in order to evaluate the more suitable index for this study area. Both the indices gave similar results but AMBI resulted more sensitive than BENTIX, probably due to the small size of the collected samples. This study seems to suggest that AMBI, being not dependent from sample size, could be preferred as descriptor of benthic health status in CTEs.

A Meramod® model approach for the Environmental Impact Assessment (EIA) of the off–shore aquaculture improvement in the Alghero Bay (North western Sardinia, Italy)

Abstract Marine fish farming generates particulate wastes which are dispersed in the sea environment. To deal with this problem, particulate waste dispersion models have been developed to predict the effects of fish cage culture. In this study, we evaluated the seabed deposition of a fish farming facility located in the central western Mediterranean by using the Meramod® model. The objectives where first to assess the actual scenario, and second to forecast the possible impact due to the forthcoming enlargement of the farming area with the addition of new fish cages. By computing the hydrodynamic measurements and the daily amount of feed recorded between July and December 2006, the impact seabed surfaces forecasted by the model increased from 5.6ha in the actual scenario, up to 7.3ha in the future. The model estimated a maximum level of total solid flux deposition of 3,800g/m2bed/year and a maximum level of total carbon flux deposition of 1,350g/m2bed/year for both scenarios. Furthermore, the model predicted that the installation of 4 new fish cages (with an hypothetical mean daily amount of feed of 50kg/cage) will produce a total solid and carbon flux deposition levels ranging 0-400 and 0-150g/m2bed/year respectively, under the new fish cages location.