Live Yeast (Saccharomyces cerevisiae var. boulardii) Supplementation in a European Sea Bass (Dicentrarchus labrax) Diet: Effects on the Growth and Immune Response Parameters.

The present study has been aimed at evaluating the effects of the dietary inclusion of the live yeasts, Saccharomyces cerevisiae var. boulardii (LSB) administered at increasing concentrations (0, 100, and 300 mg kg-1 of feed, here referred to as LSB 0, 100, 300) for 90 days, on the health conditions of European sea bass. The main zootechnical parameters, histological and morphological analyses, innate immunity response parameters (intestinal cytokine expression, lysozyme content, spontaneous hemolytic and hemagglutinating activities, antibacterial activities, and peroxidase activity) were measured as fish welfare parameters. LSB did not impair either growth parameters or the morphometric indexes. LSB down-regulated interleukin-1ß transcription in the distal gut of fish treated with 5.4 × 105 CFU g-1 (LSB100) for 21 days. The interleukin-6 mRNA level decreased significantly in the proximal gut for both doses of yeast, after 21 days of feeding; the gene expression of interleukin-6 was significantly lower in the sea bass fed 10.81 × 105 CFU g-1 (LSB300) probiotic. The levels of TNF-α mRNA were not influenced by probiotic supplementation. Increases, although not significant, in the hematological and immunological parameters were also recorded. The data collected in the present study suggests that an LSB-supplemented diet acts on the gut immune system of sea bass by modulating the expression of the key inflammatory genes.

Influence of Hermetia illucens Larvae Meal Dietary Inclusion on Growth Performance, Gut Histological Traits and Stress Parameters in Sparus aurata.

This study provided new data and knowledge on the potential use of Hermetia illucens meal (HIM) as a new sustainable ingredient for Sparus aurata diet. The effect of HIM dietary inclusion on fish growth performance, stress indicators and gut histology was studied. For 131 days, 312 fish were fed a basal diet containing fishmeal as animal protein source, and three diets containing 25%, 35% and 50% HIM as a partial replacement for fishmeal. The main findings indicated that fishmeal can be replaced by HIM up to 110 g/kg of substitution (35% of inclusion in diet) without negative effects on growth performance, stress parameters or histological traits of the posterior gut tract, and with positive effects (p < 0.05) on the histological and morphometric characteristics of the anterior gut tract. At the same time, the results showed that the effect of Hermetia illucens meal at 50% inclusion level caused morphometric and histopathological alterations in the anterior gut tract of seabream. In conclusion, this preliminary study suggested that the dietary inclusion level of HIM35 was the most tolerated by fish showing the best gut morphometric parameters and histological conditions, with fewer signs of inflammation, as well as good nutritional and health status.

Host-associated and Environmental Microbiomes in an Open-Sea Mediterranean Gilthead Sea Bream Fish Farm.

Gilthead seabream is among the most important farmed fish species in the Mediterranean Sea. Several approaches are currently applied to assure a lower impact of diseases and higher productivity, including the exploration of the fish microbiome and its manipulation as a sustainable alternative to improve aquaculture practices. Here, using 16S rRNA gene high-throughput sequencing, we explored the microbiome of farmed seabream to assess similarities and differences among microbial assemblages associated to different tissues and compare them with those in the surrounding environment. Seabream had distinct associated microbiomes according to the tissue and compared to the marine environment. The gut hosted the most diverse microbiome; different sets of dominant ASVs characterized the environmental and fish samples. The similarity between fish and environmental microbiomes was higher in seawater than sediment (up to 7.8 times), and the highest similarity (3.9%) was observed between gill and seawater, suggesting that gills are more closely interacting with the environment. We finally analyzed the potential connections occurring among microbiomes. These connections were relatively low among the host's tissues and, in particular, between the gut and the others fish-related microbiomes; other tissues, including skin and gills, were found to be the most connected microbiomes. Our results suggest that, in mariculture, seabream microbiomes reflect only partially those in their surrounding environment and that the host is the primary driver shaping the seabream microbiome. These data provide a step forward to understand the role of the microbiome in farmed fish and farming environments, useful to enhance disease control, fish health, and environmental sustainability.

Integrated biomarker responses in European seabass Dicentrarchus labrax (Linnaeus, 1758) chronically exposed to PVC microplastics.

Few studies evaluated long-term effects of polyvinyl chloride (PVC) microplastics (MPs) ingestion in fish. The present study aimed to investigate the integrated biomarker responses in the liver and blood of 162 European seabass, Dicentrarchus labrax, exposed for 90 days to control, virgin and marine incubated PVC enriched diets (0.1 % w/w) under controlled laboratory condition. Enzymatic and tissue alterations, oxidative stress, gene expression alterations and genotoxicity were examined. Additives and environmental contaminants levels in PVC-MPs, control feed matrices and in seabass muscles were also detected. The results showed that the chronic exposure at environmentally realistic PVC-MPs concentrations in seabass, cause early warning signs of toxicological harm in liver by induction of oxidative stress, the histopathological alterations and also by the modulation of the Peroxisome proliferator-activated receptors (PPARs) and Estrogen receptor alpha (ER-α) genes expression. A trend of increase of DNA alterations and the observation of some neoformations attributable to lipomas suggest also genotoxic and cancerogenic effects of PVC. This investigation provides important data to understand the regulatory biological processes affected by PVC-MPs ingestion in marine organisms and may also support the interpretation of results provided by studies on wild species.

Rice protein concentrate as a fish meal substitute in Oreochromis niloticus: Effects on immune response, intestinal cytokines, Aeromonas veronii resistance, and gut microbiota composition.

The potential of rice protein concentrate (RPC) to substitute fishmeal (FM) protein in the diet of Oreochromis niloticus was assessed in a five-month-long feeding trial. Fishmeal protein was replaced by RPC at rates of 0% (control), 25%, 50%, and 75% (RPC0, RPC25, RPC50, and RPC75, respectively). RPC25 had no significant effect on antioxidant capacity (total antioxidant capacity; superoxide dismutase, catalase, and glutathione peroxidase activities) and immune indices (lysozyme, nitric oxide, antiprotease, and bactericidal activities) after one, two, and five months of feeding, while the values for these parameters were significantly lower in the RPC75 group compared to those in the RPC0 group. The RPC25 group showed higher mRNA levels of the intestinal cytokines IL-1ß, IL-10ß, TGF-ß, and TNF-α than the control group. In fish affected by Aeromonas veronii, the highest significant cumulative mortality was recorded in the RPC75 group, followed by the RPC50, RPC25, and control groups. Gut microbiome analyses showed a reduction in microbial diversity in response to the addition of RPC, regardless of the RPC content, and the composition of the community of the RPC samples differed from that of the control. RPC-enriched diets resulted in higher relative abundances of Bacteroidetes and Fusobacteria in the gut compared to that in the gut of the control fish. In summary, RPC can be used to replace up to 25% of the FM protein in the diet of O. niloticus, while improving the antioxidant capacity, immunocompetence, and disease resistance of the fish.

Black Soldier Fly Larvae Meal in the Diet of Gilthead Sea Bream: Effect on Chemical and Microbiological Quality of Filets.

The chemical and microbiological characteristics of filets of Spaurus aurata L. specimens fed with diets containing a Hermetia illucens meal (HIM) at the 25, 35, and 50%, as a partial replacement for fish meal (FM) were evaluated. The diets, formulated to satisfy the nutritional needs of fish, were isoenergetic (22 MJ/kg gross energy), isonitrogenous (43 g/100 g, a.f.), and isolipidic (19 g/100 g, a.f.). Seventy-two specimens were randomly killed after 186 days of growing trials. Then, the filets were analyzed for chemical profile, fatty acids, amino acids, minerals, and microbial flora. Data were subjected to statistical analysis. No significant differences were observed in chemical composition. The sum of polyunsaturated fatty acids (PUFAs) showed a similar content in the filets; eicosapentaenoic acid was similar in the filets of HIM0, HIM35%, and HIM50%, whereas docosahexaenoic acid was higher in filets of the HIM0 group. n3/n6 PUFA ratio and the sum of EPA + DHA showed a high value (p < 0.001) in filets of the group fed with FM. No significant difference was observed in thrombogenic index and hypocholesterolaemic/hypercholesterolaemic ratio in the groups; the atherogenic index showed a higher value (p = 0.001) in the HIM50% group. Indispensable amino acids showed some significant (p < 0.0001) differences in the groups; arginine and phenylalanine content was higher in the filets of fish fed with FM; isoleucine and valine content was higher in the filets of HIM50%; leucine, lysine and methionine content was lower in the filets of HIM35%; histidine content was lower in the filets of HIM25%; tryptophan content was lower in filets of the HIM50% group. EAA/NEAA ratio showed highest value in the filets of the group that received FM. The presence of HIM in the three diets kept chromium, manganese, iron, copper, zinc, and nickel levels lower than those recommended by various authorities. Ca/P ratio showed a higher level (p < 0.0001) in the group fed with FM than those fed with diets containing HIM. The insect meal in the diets did not influence the microbiological profile of fish. Use of HIM as an unconventional feed ingredient in Sparus aurata diet looks promising, although the quality of filets may be affected.

Effects of microplastics on trophic parameters, abundance and metabolic activities of seawater and fish gut bacteria in mesocosm conditions.

Plastic pollution is an emerging threat with severe implications on animals' and environmental health. Nevertheless, interactions of plastic particles with both microbial structure and metabolism are a new research challenge that needs to be elucidated yet. To improve knowledge on the effects played by microplastics on free-living and fish gut-associated microbial community in aquatic environments, a 90-day study was performed in three replicated mesocosms (control-CTRL, native polyvinyl chloride-MPV and weathered polyvinyl chloride-MPI), where sea bass specimens were hosted. In CTRL mesocosm, fish was fed with no-plastic-added food, whilst in MPV and MPI food was supplemented with native or exposed to polluted waters polyvinylchloride pellets, respectively. Particulate organic carbon (POC) and nitrogen, total and culturable bacteria, extracellular enzymatic activities, and microbial community substrate utilization profiles were analyzed. POC values were lower in MPI than MPV and CRTL mesocosms. Microplastics did not affect severely bacterial metabolism, although enzymatic activities decreased and microbes utilized a lower number of carbon substrates in MPI than MPV and CTRL. No shifts in the bacterial community composition of fish gut microflora were observed by denaturing gradient gel electrophoresis fingerprinting analysis.

Renoprotective effect of erythropoietin in zebrafish after administration of gentamicin: an immunohistochemical study for ß-catenin and c-kit expression.

BACKGROUND: Gentamicin is an aminoglycoside antibiotic widely used in the treatment of infections caused by Gram-negative bacteria. The main limitation to its therapeutic effectiveness is the potential nephrotoxicity. Erythropoietin has a tissue protective effect widely demonstrated in the kidney. The aim of the present study was to evaluate the renoprotective effects of erythropoietin in a model of zebrafish (Danio rerio) after administration of gentamicin. METHODS: Sixty adult zebrafish were subdivided into three groups: group A was treated with gentamicin; group B received gentamicin and, 24 h later, epoetin alpha; group C received drug diluent only. In order to analyze the renoprotective activity of erythropoietin, the expression of c-kit and ß-catenin was evaluated by immunohistochemistry. RESULTS: Generally, the zebrafish renal tubule regenerates 15 days after an injury. Conversely, 7 days after gentamicin administration, animals treated with erythropoietin (group B) showed a better renal injury repair as documented by: increased expression of ß-catenin, less degenerated tubules, greater number of centers of regeneration, positivity for c-kit only in immature-looking tubules and lymphohematopoietic cells. CONCLUSION: The expression of c-kit and ß-catenin suggests that erythropoietin may exert a role in regeneration reducing the extent of tubular damage from the outset after gentamicin administration.

Are mussels able to distinguish underwater sounds? Assessment of the reactions of Mytilus galloprovincialis after exposure to lab-generated acoustic signals.

This study examined the effects of lab-generated acoustic signals on the behaviour and biochemistry of Mediterranean mussels (Mytilus galloprovincialis). The experiment was carried out in a tank equipped with a video-recording system using six groups of five mussels exposed to five acoustic treatments (each treatment was replicated three times) for 30min. The acoustic signals, with a maximum sound pressure level of 150dB rms re 1µPa, differed in frequency range as follows: low (0.1-5kHz), mid-low (5-10kHz), mid (10-20kHz), mid-high (20-40kHz) and high (40-60kHz). The exposure to sweeps did not produce any significant changes in the mussels' behaviour. Conversely, the specimens exposed to the low frequency band treatment showed significantly higher values of the following biochemical stress parameters measured in their plasma and tissues: glucose, total proteins, total haemocyte number (THC), heat shock protein 70 (Hsp70) expression, and Acetylcholinesterase (AChE) activity. The responses observed in the mussels exposed to low frequency sweeps enable us to suppose a biological and ecological role for this sound, which contains the main frequencies produced by both shipping traffic and the acoustic emissions of fish.

Intestinal alterations in European sea bass Dicentrarchus labrax (Linnaeus, 1758) exposed to microplastics: Preliminary results.

This study investigates, for the first time, the intestinal responses of European sea bass Dicentrarchus labrax chronically exposed to microplastics through ingestion. Fish (n = 162) were fed with 3 different treatment diets for 90 days: control, native polyvinyl chloride (PVC) and polluted polyvinyl chloride (PVC) pellets. Intestines were fixed and processed for histological analysis using standard techniques. Histopathological alterations were examined using a score value (from 0 to 4). The distal part of intestine in all samples proved to be the most affected by pathological alterations, showing a gradual change varying from moderate to severe related to exposure times. The histological picture that characterizes both groups especially after 90 days of exposure, suggests that the intestinal functions can be in some cases totally compromised. The worst condition is increasingly evident in the distal intestine of fish fed with polluted PVC pellets respect to control groups (p < 0.05) to different exposure times. These first results underline the need to assess the impact of increasing microplastics pollution on the marine trophic web.

Vessel noise pollution as a human threat to fish: assessment of the stress response in gilthead sea bream (Sparus aurata, Linnaeus 1758).

This study examined the effects of boat noise pollution on the stress indices of gilthead sea bream (Sparus aurata, Linnaeus 1758). To assess the stress response in these fish, biometric values and plasma parameters such as ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, total protein, cholesterol, triglycerides and osmolarity were analysed. After acclimatization of the animals, the experiment was carried out in a tank fitted with underwater speakers where the fish were exposed to sound treatments (in duplicate) consisting of: 10 days of no sound (control treatment; the animals were only exposed to the experimental tank's background noise) and 10 days of noise derived from original recordings of motor boats, including recreational boats, hydrofoil, fishing boat and ferry boat (vessel noise treatment). The exposure to noise produced significant variations in almost all the plasma parameters assessed, but no differences were observed in weights and fork lengths. A PERMANOVA analysis highlighted significantly increased values (p < 0.05) of ACTH, cortisol, glucose, lactate, haematocrit, Hsp70, cholesterol, triglycerides and osmolarity in the fish exposed to vessel noise for 10 days. This study clearly highlights that anthropogenic noise negatively affects fish, and they are valuable targets for detailed investigations into the effects of this global pollutant. Finally, these experimental studies could represent part of the science that is able to improve the quality of the policies related to management plans for maritime spaces (Marine Strategy Framework Directive 56/2008 CE) that are aimed at stemming this pollutant phenomenon.

Short fasting and refeeding in red porgy (Pagrus pagrus, Linnaeus 1758): response of some haematological, biochemical and non specific immune parameters.

A short fasting-refeeding experience was applied to specimens of red porgy, Pagrus pagrus (Teleostei, Sparidae) to assess its effects on some physiological parameters. Haematological (haematocrit), biochemical (serum cortisol and glucose) and immunological (lysozyme, haemolytic and haemagglutinating activities) parameters were measured. For this study, two fish groups were considered: one was fasted for 14 days and then refed to satiation during further 7 and 15 days (indicated as fasted/refed group), the other was fed throughout the study and was taken as a control group. Significantly lower values were recorded for the condition index, the hepato-somatic index and viscero-somatic index in the fasted/refed group compared to the fed one. Fasting did not affect significantly the examined parameters, except for cortisol; refeeding for 7 days induced a significant increase in the haemoagglutinating titre and the spontaneous haemolytic activity, but when refeeding was extended to 14 days haemagglutinating and haemolytic values remained lower than those measured in fed fish.

Response to short term starvation of growth, haematological, biochemical and non-specific immune parameters in European sea bass (Dicentrarchus labrax) and blackspot sea bream (Pagellus bogaraveo).

Growth, haematological (haematocrit), biochemical (serum cortisol and glucose), and non-specific immune (lysozyme, serum haemolytic and haemagglutinating activities, extracellular respiratory burst activity) parameters, were monitored in European sea bass Dicentrarchus labrax and blackspot sea bream Pagellus bogaraveo subjected to a 31 days starvation compared to fed fish, to assess the responses to feed deprivation of these health status indicators. While haematocrit, serum cortisol, glucose and haemolytic activity of both species did not undergo significant variation following starvation, probably due to the short period applied, some non-specific immune parameters were affected significantly. In the starved sea bass, mucus lysozyme content doubled (1.8 U/mL) compared to the initial value. Haemagglutinating activity was significantly lower in starved sea bass than in fed fish after 31 days. In blackspot sea bream, a slight, not significant, reduction in haemagglutinating activity occurred 11 days after starvation. Respiratory burst activity decreased significantly in the starved fish. In spite of the limited number of examined parameters, the opportunity to use a panel of several indicators to obtain a more complete picture of health status in fish was underlined.

Regenerative medicine: does Erythropoietin have a role?

Regenerative Medicine, a recent new medical domain, aims to develop new therapies through the stimulation of natural regenerative processes also in human beings. In this field, Erythropoietin (EPO) represents a significant subject of research. Several studies allow the assertion that EPO, in different concentrations, has protective effects mainly on the central nervous system, cardiovascular system and renal tissue. This action is carried out through one of few regenerative activities of human beings: angiogenesis. This mechanism, which involves endothelial stem cells and VEGF (Vascular Endothelial Growth Factor), has been experimentally demonstrated with Recombinant human erythropoietin (rHuEPO) and Darbepoetin, a long-acting EPO derivate. Furthermore, the demonstration of a cardiac production of EPO in Fugu rubripes and in Zebrafish has led cardiologists to "discover" Erythropoietin, postulating a hypothetical role in treatment of cardiovascular disease for this hormone. This is some of the experimental evidence which demonstrates that EPO can be in reason considered an important element of research of Regenerative Medicine and put in the network of drugs able to regenerate tissues and organs.

The ultrastructure of amberjack (Seriola dumerilii) sperm.

Scanning and transmission electron microscopy were used to investigate the fine structure of sperm of the Mediterranean amberjack Seriola dumerilii. Each spermatozoon has an ovoid head which lacks an acrosome, a short, irregularly-shaped midpiece and a long flagellar tail. The midpiece houses eight spherical mitochondria, which are separated from the axoneme by the cytoplasmic canal. The centrioles are arranged approximately at right angles to each other. The proximal centriole lies inside, and the distal centriole outside, the nuclear fossa. The flagellum is inserted eccentrically into the head and is tangential to the nucleus, so that the spermatozoon is asymmetrical. It contains the conventional 9 + 2 axoneme, shows intratubular differentiations in the A microtubules of doublets 1, 2, 5 and 6, and possesses one pair of lateral fins. On the basis of its ultrastructural organization, the amberjack sperm resembles type II sperm as defined previously, except for the presence of the proximal centriole inside the nuclear fossa. This could result from a partial rotation of the nucleus during spermiogenesis.