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Fish stress is a major concern in the aquaculture industry. Many stressors coming from routine practices can predispose fish to compromised growth, immunity and overall health. This study focuses on the characterization of the skin microbiota using next generation sequencing (NGS) platform by targeting a genomic marker 16S and to determine growth performance and immune status of gilthead seabream (Sparus aurata) during an episode of chronic stress. Two groups were established: control group and chronically stressed group. Stressed fish were subjected to 1 min air exposure twice a week for 4 weeks. Results showed that stress negatively affected fish growth performance. Cellular and humoral systemic immunity remained unaffected while local immunity in skin was positively stimulated (total IgM and peroxidase). Skin mucus microbial composition showed significant differences especially after 14 days. Stressed fish showed a decrease in the abundance of the genera Acinetobacter, NS3a_marine_group and Pseudomonas, while Pseudoalteromonas and Marinagarivorans increased significantly. In conclusion, air exposure stress was associated with alterations in skin mucosal immunity and microbial composition that may have been beneficial to the host favoring adaptation to stress.

In this work the effect of water temperature (6 +/- 1 degrees C and 22 +/- 1 degrees C) on inactivation of bacteria (104 -106 CFU mL-1; Pseudomonas spp., Aeromonas spp. and Enterobacter spp.) in simulated aquaculture streams (SAS) using UVA based advanced oxidation processes (AOP) (H2O2-assisted UVA; photocatalysis; H2O2-assisted photocatalysis) and solar driven AOPs (H2O2-assisted solar disinfection, SODIS) was studied. Efficiency at 22 degrees C in terms of inactivation rate was higher using H2O2-assisted photocatalysis (H2O2/UVA-TiO2/polysiloxane) > H2O2-assisted UVA disinfection (UVA/H2O2 - 10 mg L-1) > photocatalysis (UVA-TiO2/polysiloxane) > UVA disinfection. At low temperature (6 degrees C) the inactivation rate increased with SODIS/H2O2 > SODIS > H2O2assisted UVA disinfection (UVA/H2O2 - 10 mg L-1) > H2O2-assisted photocatalysis (H2O2/UVA-TiO2/polysiloxane) > photocatalysis (UVA-TiO2/polysiloxane). The main results indicate that the inactivation rates increased when hydrogen peroxide (10 mg L-1) was used during H2O2-assisted UVA disinfection and photocatalysis. In addition, exposure of SAS to hydrogen peroxide for 24 h (in absence of light) at room temperature decreased the subsequent exposure UVA irradiation dose by almost four times. Drastic increase of inactivation rate was observed at low water temperature (6 +/- 1 degrees C) when UVA- and solarbased AOPs were employed compared to 22 +/- 1 degrees C. The treatment with SODIS proved to be more effective in Finland than in Spain. The effect of the low temperature (6 +/- 1 degrees C) was proposed as a critical factor during UVA disinfection (UVA/H2O2 and photocatalysis) that can increase the disinfection rate constant (kmax) by 1.3-5.2 times, leading to a reduction of the treatment costs ( euro m-3) by 1.3-3.3 times. The mechanism of observed enhanced disinfection at low water temperature (6 +/- 1 degrees C) when natural solar light and UVA are employed as irradiation sources for UVA/H2O2 and photocatalytic bacteria inactivation was proposed. No regrowth was observed in case of H2O2-assisted AOPs., This work was supported by a national research project (SUNRAS PROJECT, Project AGL2016-80507-R) funded by Ministerio de Economia y Competitividad (Plan Nacional de I + D + i (2013-2016). The research was also supported by a PIF contract (UCA/REC01VI/2017) funded by Vicerrectorado de Investigacion of Universidad de Cadiz. D. Sc. Juan Jose Rueda-Marquez is grateful for financial support from Academy of Finland within the project '' Combination of Advanced Oxidation Processes and Photobiotreatment for Sustainable Resource Recovery and Wastewater Reuse'' (application number 322339). T. Homola acknowledges financial support by Czech Science Foundation project 19-14770Y and project LM2018097 funded by the Ministry of Education, Youth and Sports of the Czech Republic. Document

The aim of this research was to evaluate the possible positive effects of feeding Nile tilapia (Oreochromis niloticus) with a diet containing a commercial probiotic AQUA-PHOTO® [Lactobacillus plantarum (1.51 × 106 CFU g−1) and Bacillus subtilis (1.34 × 107 CFU g−1)] on digestive enzymes’ activity and gut microbiome profile. The experimental design was completely randomized, with two treatments [control and probiotic diets (0.08 g.100 g−1 of feed)] and six replicate tanks per group (n = 8 animals per experimental unit, mean weight 8.18 g ± 1.85 g). The 96 fish were distributed into 12 aquaria and fed with the probiotic and control diets for 7 days. After this period, intestine samples (anterior, middle, and posterior portions) were collected from two individuals per tank. The samples were analyzed for assessment of DGGE profile and digestive enzymes’ activity (amylase, alkaline protease, and alkaline phosphatase). The results were subjected to analysis of variance (ANOVA) and the means compared by t test (P

Macroalgae represent valuable sources of functional ingredients for fish diets, and the influence of supplemented aquafeeds on growth performance has been studied for some fish and seaweed species. In the present work, the potential immunomodulation exerted by U. ohnoi (5%) as dietary ingredient was investigated in Senegalese sole. After feeding with the experimental diets for 90 d, fish immune response before and after challenge with Photobacterium damselae subsp. piscicida (Phdp) was assessed. In absence of infection, systemic immune response was not modified by 5% U. ohnoi dietary inclusion for 90 d. Thus, no differences in liver and head kidney immune gene transcription or serum lysozyme, peroxidase, antiprotease and complement activities were observed based on the diet received by Senegalese sole specimens. Regarding mucosal immune parameters, no changes in gene transcription were detected in the skin and gills, whilst only tnf, cd4 and cd8 were significantly up-regulated in the intestine of fish fed with U. ohnoi, compared to the values obtained with control diet. On the contrary, when S. senegalensis specimens were challenged with Phdp, modulation of the immune response consisting in increased transcription of genes encoding complement (c1q4, c3, c9), lysozyme g (lysg), tumor necrosis factor alpha (tnfα) as well as those involved in the antioxidant response (gpx, sodmn) and iron metabolism (ferrm, hamp-1) was observed in the liver of fish fed with U. ohnoi. In parallel, decreased inflammatory cytokine and complement encoding gene transcription was displayed by the spleen of fish receiving the algal diet. Though mortality rates due to Phdp challenge were not affected by the diet received, lower pathogen loads were detected in the liver of soles receiving U. ohnoi diet. Further research to investigate the effects of higher inclusion levels of this seaweed in fish diets, feeding during short periods as wells as to assess the response against other pathogens needs to be carried out.

The effect of spray‐dried porcine plasma (SDPP) on the intestinal histological organization and autochthonous microbiota composition was evaluated in Sparus aurata. Fish were fed a basal diet (51 g/kg protein, 17 g/kg fat, 20.6 MJ/kg gross energy) and a diet containing 3 g/kg SDPP for 95 days (initial body weight, BW = 9.5 ± 0.2g, mean ± SD). The inclusion of SDPP promoted growth (p < .05), being fish fed the SDPP diet 6.2% (BW = 88.2 ± 1.6 g) heavier than the control (BW = 82.7 ± 3.2 g). SDPP increased the density of intestinal goblet cells (p < .05), whereas no differences in villi height were found (p > .05) between both groups. Intestinal microbiota was dominated by Proteobacteria (>85%) and Firmicutes (5%–12%), whereas Bacteroidetes never represented more than 1.5%. γ‐Proteobacteria, and Bacilli and Clostridia were the predominant classes. The short administration of SDPP (20 days) resulted in changes in microbiota diversity and richness associated with an increase in the sequences of the genus Lactobacillus and to a decrease in the genus Vibrio, whereas these changes were reverted at 95 days. Intestinal goblet cell density was not correlated to microbiota diversity and richness changes rather than to the immunostimulatory effect of the SDPP. info:eu-repo/semantics/acceptedVersion

Hybrid TiO2/SiO2 thin films deposited by material printing technique on flexible substrates were prepared, characterized and tested for solar photocatalytic disinfection. Effect of surface hydrophilicity/hydrophobicity of printed coatings on photocatalytic disinfection was studied by means of (i) drinking water contaminated with natural consortia of fecal bacteria (gram-negative: Escherichia coli and total coliforms; gram-positive: Enterococci), and (ii) seawater containing pathogenic gram-negative bacteria (Vibrio owensii, Vibrio alfacsensis and Vibrio harveyi). Inactivation of gram-negative bacteria in drinking water with fecal contamination by solar photocatalysis was slightly more efficient than solar disinfection, while for gram-positive bacteria similar efficiency was observed. These results, in combination with observed release of titanium from coatings (detected by means of inductively coupled plasma atomic emission spectrometer), indicate that TiO2/SiO2 needs further improvements for solar photocatalytic disinfection of drinking water. Efficiency of seawater disinfection towards gram-negative Vibrio spp. (Vibrio owensii, Vibrio alfacsensis and Vibrio harveyi) was significantly enhanced when TiO2/SiO2 coatings were used under natural solar light. Moreover, hydrophobic thin films led to faster Vibrio spp. inactivation as compared to hydrophilic ones, which was attributed to higher bacteria adhesion on hydrophobic coatings. However, decrease of photocatalytic activity of hydrophobic TiO2/SiO2 coatings was observed after ten experimental cycles mainly due to deposition of salts on the surface of photocatalyst. Generally, results of this study suggest that autochthonous bacteria such as Vibrio spp. in seawater are significantly more resistant to solar disinfection in comparison with not autochthonous bacteria such as Escherichia coli, total coliforms and Enterococci in contaminated drinking water.

Karst caves are considered a natural sink of atmospheric methane (CH 4 ). Studies generally focus on measuring CH 4 within the cave environment, and little information is available about the factors influencing CH 4 abundance, such as the surrounding vadose and saturated zones of the aquifer, inputs of carbon dioxide (CO 2 ) – a potential precursor of CH 4 – and methanotrophic bacteria, which is likely the main cause of CH 4 consumption. In this paper we report the first study of CH 4 budget in Nerja, one of the main karst caves in Spain, based on seasonal monitoring of CH 4 and CO 2 concentration and stable C isotopic ratio in the cave system, integrated with analogue data from the underlying and overlying saturated/vadose zone, and methanotrophic bacteria activity (rDNA and Polymerase Chain Reaction analyses) in cave sediments and water. The results show that the cave environment consumes, via methanotrophic activity and through γ- and α–Proteobacteria, CH 4 coming from both the atmosphere and the vadose/saturated zones of the aquifer, where it is produced microbially. However, vadose zone methanogenesis may seasonally exceed the methanotrophy capacity of the cave, resulting in a net CH 4 increase in it. This competition process may be an important factor limiting the sink potential of karst caves.

The use of lysed microalgae in the diet of carnivorous fish can increase the bioavailability of proteins and bioactive compounds, such as unsaturated fatty acids or vitamins in the digestive tract. These are essential molecules for the proper physiological development of fish in aquaculture. However, some antinutritional components and other undesirable molecules can be released from an excess of microalgae supplied, compromising the integrity of the intestine. The inclusion of small amounts of hydrolized microalgae in the fish diet can be a good strategy to avoid negative effects, improving the availability of beneficial compounds. Nannochloropsis gaditana is an interesting microalgae as it contains nutraceuticals. Previous studies reported beneficial effects after its inclusion in the diet of Sparus aurata, a widely cultured species in Europe and in all Mediterranean countries. However, administration of raw microalgae can produce intestinal inflammation, increased intestinal permeability, bacterial translocation and disturbance of digestion and absorption processes. The aim of this study was to evaluate changes in the intestinal microbiota and barrier stability of S. aurata fed with low inclusion (5%) hydrolysed N. gaditana. Intestinal microbiota was analyzed using Illumina MiSeq technology and libraries were constructed using variable regions V3–V4 of 16S rDNA molecules. Analysis were based in the identification, quantification and comparison of sequences. The predictive intestinal microbial functionality was analyzed with PICRUSt software. The results determined that the intestinal microbiota bacterial composition and the predictive intestinal microbiota functionality did not change statistically after the inclusion of N. gaditana on the diet. The study of gene expression showed that genes involved in intestinal permeability and integrity were not altered in fish treated with the experimental diet. The potential functionality and bacterial taxonomic composition of the intestinal microbiota, and the expression of integrity and permeability genes in the intestine of the carnivorous fish S. aurata were not affected by the inclusion of hydrolysed 5% N. gaditana microalgae.

Funding Information: This study was financially supported by Maa- ja vesitekniikan tuki ry. (MVTT), Aalto University, Foundation for Aalto University Science and Technology, Tekniska f?reningen i Finland (TFiF) and the Liisa and Aarre Koskenalusta Foundation. We thank the laboratory staff of Aalto Water Building for all their help in enabling the research. We also want to thank the DNA Sequencing and Genomics Laboratory of the Institute of Biotechnology (BI) at the University of Helsinki for providing sequencing services. Furthermore, we thank Shivani Singhal and Emilia Awaitey for finding local prices of PE bags in India and Ghana, and Sara Saukkonen for designing the graphical abstract. Publisher Copyright: © 2022 The Authors Solar disinfection (SODIS) is an inexpensive drinking water treatment method applied in tropical and sub-tropical low-income countries. However, it has been unclear whether it functions adequately also in colder climates. To investigate this issue, SODIS experiments were performed in the humid continental climate of Finland by exposing faecally contaminated drinking water to natural solar radiation at different water temperatures (8–23 °C) and UV intensities (12–19 W/m2) in polyethylene (PE) bags. To establish an adequate benchmark, SODIS experiments with the same experimental design were additionally conducted in the Mediterranean climate of Spain in typical conditions of SODIS application (~39 °C and 42 W/m2). Out of all experiments, the highest coliform and enterococci inactivation efficiencies in terms of lowest required doses for 4-log disinfection (25 Wh/m2 and 60 Wh/m2, respectively) were obtained in humid continental climate at the lowest studied mean water temperature (8–11 °C). Despite the low mean UV irradiance (~19 Wh/m2), 4-log disinfection of coliforms and enterococci were also reached fast in these conditions (1 h 27 min and 3 h 18 min, respectively). Overall, the doses required for disinfection increased as the water temperatures and UV intensities of the experiments rose. Disinfection of 4-logs (> 99.99%) of both bacteria was reached in all SODIS experiments within 6 h, suggesting SODIS could be a sufficient household water treatment method also in colder climates, unlike previously thought. The effects of different water temperatures on bacterial inactivation were also tested in the absence of sunlight. Together the obtained results indicate that while water temperatures below or close to the optima of coliforms and enterococci (~10 °C) alone do not cause inactivation, these temperatures may enhance SODIS performance. This phenomenon is attributed to slower bacterial metabolism and hence slower photorepair induced by the low water temperature.

Aquaculture is considered one of the largest food production sectors in the world. Probiotics have long been considered as a beneficial tool in this industry since these microorganisms improve the welfare of different fish species by modulating several physiological functions, such as metabolism, digestion, immune response, stress tolerance, and disease resistance, among others. SpPdp11, a probiotic isolated from the skin of healthy gilthead seabream, has been the center of attention in a good number of studies since its discovery. The purpose of this paper is to summarize, comment, and discuss the current knowledge related to the effects of SpPdp11 in two commercially important fish species in aquaculture (gilthead seabream and Senegalese sole). Furthermore, some considerations for future studies are also indicated.

Aquaculture is considered one of the largest food production sectors in the world. Probiotics have long been considered as a beneficial tool in this industry since these microorganisms improve the welfare of different fish species by modulating several physiological functions, such as metabolism, digestion, immune response, stress tolerance, and disease resistance, among others. SpPdp11, a probiotic isolated from the skin of healthy gilthead seabream, has been the center of attention in a good number of studies since its discovery. The purpose of this paper is to summarize, comment, and discuss the current knowledge related to the effects of SpPdp11 in two commercially important fish species in aquaculture (gilthead seabream and Senegalese sole). Furthermore, some considerations for future studies are also indicated.

Senegalese sole (Solea senegalensis) has been proposed as a high-potential species for aquaculture diversification in Southern Europe. It has been demonstrated that a proper feeding regimen during the first life stages influences larval growth and survival, as well as fry and juvenile quality. The bacterial strain Shewanella putrefaciens Pdp11 (SpPdp11) has shown very good probiotic properties in Senegalese sole, but information is scarce about its effect in the earliest stages of sole development. Thus, the aim of this study was to investigate the effect of SpPdp11, bioencapsulated in live diet, administered during metamorphosis (10-21 dph) or from the first exogenous feeding of Senegalese sole (2-21 dph). To evaluate the persistence of the probiotic effect, we sampled sole specimens from metamorphosis until the end of weaning (from 23 to 73 dph). This study demonstrated that probiotic administration from the first exogenous feeding produced beneficial effects on Senegalese sole larval development, given that specimens fed this diet exhibited higher and less dispersed weight, as well as increases in both total protein concentration and alkaline phosphatase activity, and in non-specific immune response. Moreover, real-time PCR documented changes in the expression of a set of genes involved in central metabolic functions including genes related to growth, genes coding for proteases (including several digestive enzymes), and genes implicated in the response to stress and in immunity. Overall, these results support the application of SpPdp11 in the first life stages of S. senegalensis as an effective tool with the clear potential to benefit sole aquaculture.

Interest in fish skin immunity and its associated microbiota has greatly increased among immunologists. The objective of this study is to know if skin ulcers may be associated with changes in the mucus composition and microbial diversity. The abundance of terminal carbohydrates, several enzymes (protease, antiprotease, peroxidase, lysozyme) and total immunoglobulin M levels were evaluated in skin mucus of experimentally ulcered gilthead seabream (Sparus aurata L.). Furthermore, the composition of the microbiota of ulcered and non-ulcered skin has been determined using Illumina Miseq technology. Significant decreases of terminal abundance of α-D-mannose, α-D-glucose and N-acetyl-galactosamine in skin mucus of ulcered fish, compared to control fish were detected. The levels of IgM and all the tested enzymes in mucus were decreased in ulcered fish (compared to control fish) although the observed decreases were only statistically significant for proteases and antiproteases. Concomitantly, the analysis of the composition of the skin microbiota showed clear differences between ulcered and non-ulcered areas. The genus taxonomic analysis showed that Staphylococcus and Lactobacillus were more abundant in non-ulcered skin whereas in ulcered area were Streptococcus and Granulicatella. Important decreases of the number of sequences related to Alteromonas, Thalassabius and Winogradskyella were detected in ulcered skin whilst slight increases of sequences related to Flavobacterium, Chryseobacterium and Tenacibaculum genera were observed. Overall these results demonstrated that the presence of skin ulcers provide microenvironments that perturb both the mucus composition and microbial biodiversity of this important external surface which seem to be more vulnerable to diseases.

A series of clay minerals and clay-based materials have been tested to eliminate one of the most dangerous bacteria we can find in the water: Salmonella. It has been proven that the use of clays and their PCH materials can be a suitable method for removing Salmonella from water. The results of this initial study show that all the materials analyzed have great salmonella adsorption capacities ranging from the lowest value observed in the mont-PCH sample (0.29 &times, 1010 CFU g&minus, 1) to the highest value observed in the natural palygorskite sample (1.52 &times, 1). Macroporosity, accessible external surface area, and the presence of silanol groups in the external surface of the particles appears to be the controlling factors for Salmonella adsorption capacity while it seems that the structural characteristics of the clay minerals and their respective PCH does not affect the adsorption capacity.

Recently, interest in mucosal surfaces, more specifically fish skin and its secreted mucus, has greatly increased among immunologists. The abundance of terminal carbohydrates, several enzymes (proteases, lysozyme, peroxidase, alkaline phosphatase, esterases and ceruloplasmin), bactericidal activity against fish pathogenic and non-pathogenic bacteria and several physico-chemical parameters (protein concentration, pH, conductivity, redox potential, osmolarity, density and viscosity) in the skin mucus of Senegalese sole (Solea senegalensis, Kaup) have been evaluated. Present results evidence the abundance of N-acetylneuraminic acid, mannose, glucose and N-acetyl-galactosamine in skin mucus. The levels of lysozyme, proteases, esterases and alkaline phosphatase were very similar (from 20 to 30 Units mg−1 protein). However, 93 Units mg−1 protein were detected of ceruloplasmin and only 4′88 Units mg−1 protein of peroxidase. Skin mucus of S. senegalensis showed high bactericidal activity against the tested pathogen bacteria but weak activity against non-pathogenic bacteria. Finally, a clear relationship between mucus density and temperature was detected, while viscosity showed a direct shear- and temperature-dependent behaviour. These results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system, as well as, for elucidating possible relationships between biological and physico-chemical parameters and disease susceptibility.

The marine fish pathogen Photobacterium damselae subsp. piscicida (Phdp) is responsible for important disease outbreaks affecting cultured fish species including the flatfish Solea senegalensis. In the present work, transcription of iron metabolism related genes (TF, FERR-M, HP-1 and HAMP-1) as well as innate immune system components such as complement proteins (C3 and C7), lysozyme (LYS-G), TNF family (TNFα, TRAF-3), NCCRP-1 and heat shock protein encoding genes (HSP70, HSP90AA, HSP90AB and GP96) has been determined in the liver and kidney of S. senegalensis specimens after Phdp infection. Intraperitoneal injection (IP) and immersion (IM) routes have been used for infection. Fish developed specific antibodies in both cases, higher levels being detected in IP infected specimens. Both infection routes resulted in increased relative transcript levels of FERR-M, HP-1 and HAMP-1 genes and TF decreased relative transcription, conducting to lower iron availability for the pathogen. This response can be considered as a strategy to limit iron availability for Phdp, a pathogen capable to obtain iron from transferrin. Relative transcription of genes encoding lysozyme and complement factors C3 and C7 were also increased regardless the infection route; the liver was the main organ involved in the initial stages and the kidney in later stages of the infection. TNFα and TRAF-3 relative gene transcription increased 24h post-infection. TRAF-3 gene induction was detected 30 d post-infection, whilst no changes in TNFα were observed 72h or 30 d post-infection. NCCRP-1 changes were observed after IP infection in the liver and kidney; however, IM infection resulted only in slight changes in the kidney of infected fish. This different response observed maybe related to a lower number of invaded cells by the pathogen. Finally, changes in HSP90AB and GP96 have been detected after infection by both routes. Different late modulation has been observed in assayed genes depending on the route of infection. Thus, only LYS-G, TF, NCCRP-1, GP96 and HSP90AB gene transcription was modulated 30 d post-infection in the kidney of IM infected specimens; however, IP infected fish showed modulation in a higher number of genes both in liver and kidney tissues. The implications of these responses in resistance to infection by Phdp need to be elucidated.

This study evaluated the control of streptococcosis outbreaks in Brazil, isolated from diseased sorubim and identified as Lactococcus garvieae by genetic sequencing. This report determined the potential for lactococcosis control in sorubim Pseudoplatystoma sp. with two vaccines: an aqueous-based, whole-cell inactivated vaccine (bacterin) and an oil-adjuvanted bacterin. Their efficacy was evaluated at 30 days post-vaccination (d.p.v.) by challenge with L. garvieae, and the antibody production response at 15, 30 and 60 d.p.v. and the non-specific immune response were compared amongst treatments. High protection levels (P

The development of technologically advanced recirculation aquaculture systems (RAS) implies the reuse of water in a high recirculation rate (>90%). One of the most important phases for water management in RAS involves water disinfection in order to avoid proliferation of potential pathogens and related fish diseases. Accordingly, different approaches have been assessed in this study by performing a comparison of photolytic (UV-LEDs) at different wavelengths (λ= 262, 268 and 262+268 nm), photochemical (UV-LEDs/H2O2, UV-LEDs/HSO5- and UV-LEDs/S2O82-) and photocatalytic (TiO2/SiO2/UV-LEDs and ZnO/SiO2/UV-LEDs) processes for the disinfection of water in RAS streams. Different laboratory tests were performed in batch scale with real RAS stream water and naturally occurring bacteria (Aeromonas hydrophyla and Citrobacter gillenii) as target microorganisms. Regarding photolytic processes, higher inactivation rates were obtained by combining λ262+268 in front of single wavelengths. Photochemical processes showed higher efficiencies by comparison with a single UV-C process, especially at 10 mg·L-1 of initial oxidant dose. The inactivation kinetic rate constant was improved in the range of 15-38%, with major efficiency for UV/H2O2 ~ UV/HSO5- > UV/S2O82-. According to photocatalytic tests, higher efficiencies were obtained by improving the inactivation kinetic rate constant up to 55% in comparison with a single UV-C process. Preliminary cost estimation was conducted for all tested disinfection methods. Those results suggest the potential application of UV-LEDs as promoter of different photochemical and photocatalytic processes, which are able to enhance disinfection in particular cases, such as the aquaculture industry. Post-print / Final draft

Gastrointestinal (GI) microbiota has a relevant role in animal nutrition, modulation of the immune system and protection against pathogen invasion. Interest in algae as source of nutrients and functional ingredients for aquafeeds is increasing in order to substitute conventional feedstuffs by more sustainable resources. The diet is an important factor in the modulation of the microbiota composition, and functional ingredients have been proposed to shape the microbiota and contribute benefits to the host. However, fish microbiome research is still limited compared to other hosts. Solea senegalensis is a flat fish with high potential for aquaculture in South Europe. In this study, a characterization of the microbiome of S. senegalensis (GI) tract and the effects of feeding Ulva ohnoi supplemented diet has been carried out. Differences in the composition of the microbiota of anterior and posterior sections of S. senegalensis GI tract have been observed, Pseudomonas being more abundant in the anterior sections and Mycoplasmataceae the dominant taxa in the posterior GI tract sections. In addition, modulation of the GI microbiota of juvenile Senegalese sole fed for 45 days a diet containing low percentage of U. ohnoi has been observed in the present study. Microbiota of the anterior regions of the intestinal tract was mainly modulated, with higher abundance of Vibrio spp. in the GI tract of fish fed dietary U. ohnoi.

Probiotic microorganisms are of great interest in clinical, livestock and aquaculture. Knowledge of the genomic basis of probiotic characteristics can be a useful tool to understand why some strains can be pathogenic while others are probiotic in the same species. An automatized workflow called TarSynFlow (Targeted Synteny Workflow) has been then developed to compare finished or draft bacterial genomes based on a set of proteins. When used to analyze the finished genome of the probiotic strain Pdp11 ofShewanella putrefaciensand genome drafts from seven known non-probiotic strains of the same species obtained in this work, 15 genes were found exclusive of Pdp11. Their presence was confirmed by PCR using Pdp11-specific primers. Functional inspection of the 15 genes allowed us to hypothesize that Pdp11 underwent genome rearrangements spurred by plasmids and mobile elements. As a result, Pdp11 presents specific proteins for gut colonization, bile salt resistance and gut pathogen adhesion inhibition, which can explain some probiotic features of Pdp11.

The pathogenic bacteria Photobacterium damselae subsp. piscicida affects the development of Solea senegalensis culture. Vaccines made with inactivated cells have produced a relative protection against the sickness, however the administration of subcellular and purified antigens as vaccine could increase the effectiveness of the immune response. Thus, the aim of this work was the determination of antigens of P. damselae subsp. piscicida involved in the specific immune response of S. senegalensis. Fish were immunized by intraperitoneal injection (i.p.) with inactivated extracellular polymeric substances (ECP) and whole cells of P. damselae subsp. piscicida, and Freund's incomplete adjuvant. Two months later fish were boosted with the same antigens. Serum from fish was collected to determine by ELISA the title of antibodies against subcellular fractions of bacteria (ECP, capsule, outer membrane proteins, O antigen and formalized whole cells). Significant differences were found between control and immunized fish, but differences between first immunization and booster were only found for O antigen and capsule. Western blots derived from 2D-PAGE of ECP and Outer Membrane Proteins (OMP), using sole immunized serum, detected two high reactive antigens from ECP. Proteins were identified, by mass spectrometry, as ATP-dependent metalloprotease and Telurite resistance proteins. In the case of OMP, three antigenic proteins were detected and identified as Nrfa Y218f, Anti-oxidant AhpC/TSA, and a protein domain DNA binding heat shock related.

Photobacterium damselae subsp. piscicida (Phdp) is responsible for disease outbreaks in marine aquaculture worldwide. Solea senegalensis, a valuable fish species for aquaculture in the south of Europe, is frequently affected by this pathogen. It is well established that bacteria respond to environmental signals and, in the case of pathogens, this ability may determine the outcome of their interaction with the host. Determination of gene expression under in vivo conditions constitutes a valuable tool in the assessment of microbial pathogenesis. Considering that different hosts may represent different environments for the pathogen, expression of Phdp virulence and in vivo induced antigen (IVIAT) genes during S. senegalensis infection has been determined in the present work. Increased transcription of genes encoding proteins involved in iron acquisition (Irp1, Irp2, HutB and HutD), oxidative stress defence (AhpC and Sod), adhesion (PDP_0080), toxins (AIP56) and metabolism (Impdh, Shmt and AlaRS) were detected in Phdp infecting S. senegalensis head kidney or liver. The highest increases corresponded to genes involved in survival under iron limiting conditions and oxidative stress, indicating their essential role during infection of sole. Results obtained give insight into Phdp virulence strategies and contribute to the identification of promising targets for the control of photobacteriosis.

Partial substitutions of fish meal by 5, 15, or 25 % of Gracilaria cornea or Ulva rigida in experimental diets were evaluated to study their effects on biodiversity of intestinal microbiota composition in gilthead seabream (Sparus aurata) juveniles. The diets were offered to duplicate groups of 15 juvenile fish (14.0 ± 0.5 g) for 70 days, and at the end of the experiment the intestinal microbiota from four specimens of each treatment was analysed by denaturing gel gradient electrophoresis. Results showed that the substitution of fish meal by algae meal induced important modifications in the intestinal microbiota community, as a big reduction of the biodiversity when the highest percentage (25 %) of U. rigida was included. On the contrary, an increase on the number of species was detected when a 15 % of algae was included. Various Lactobacillus delbrueckii subspecies were selectively stimulated when G. cornea was included in the feed, and other bacterial species, such as those included in the Vibrio genus, were reduced.

The aim of this study was to evaluate the inclusion of two macroalgae as dietary ingredients on growth, body composition, intermediate metabolism, intestinal enzymes and gut histology of Sparus aurata juveniles. Six isocaloric and isonitrogenous diets incorporating 5%, 15% or 25% of Gracilaria cornea or Ulva rigida meal were tested against a control diet. Duplicate groups of fifteen fish (14 g) were fed the experimental diets for 70 days. Growth performance showed an inverse linear relationship with the inclusion level of Gracilaria. Feed conversion ratio increased significantly in fish fed 25% Gracilaria. Ulva presented a quadratic relationship with the lowest SGR at 15% of inclusion. Fish fed 25% Ulva exhibited significantly lower lipid content than those fed control diet. Overall, metabolites did not show differences with specimens fed control diet, except for plasmatic glucose and muscle lactate. Intestinal proteolytic activity was reduced with the macroalgae inclusion level, but all fish showed the same enzyme pattern. The histological study revealed no negative effect on liver and intestine structure by the macroalgae inclusion. The present work demonstrated that these ingredients can be used in practical diets for sea bream, but a more comprehensive research is needed to determine the optimum dietary level in a longer feeding trial.

Solea senegalensis is a flatfish with a great potential for aquaculture, but infectious diseases restrict its production, being this fish species highly susceptible to Photobacterium damselae subsp. piscicida (Phdp) infections. A better understanding of the mechanisms related to fish immune response is crucial for the development of effective approaches in disease management. In the present work, transcriptional changes of immune related genes have been evaluated in farmed S. senegalensis specimens vaccinated against Phdp by intraperitoneal injection (IP) and immersion (IM). IP fish showed higher antibody levels and increased transcription of genes encoding lysozyme C1, complement factors involved in the classical pathway and components involved in the opsonization and the limitation of free iron availability, all of them facilitating the faster elimination of the pathogen and promoting higher RPS after the infection with Phdp. The results of this study seem to support a different intensity of the specimens immune response in the head kidney. Analysis of the immune response in 15 day post-challenged fish showed up-regulation of genes involved in all stages of S. senegalensis immune response, but especially those genes encoding proteins related to the innate response such as complement, lysozyme and iron homeostasis in the head kidney. On the other hand, liver transcription was higher for genes related to inflammation, apoptosis and cell mediated cytotoxicity (CMC). Furthermore, comparison of the differential response of S. senegalensis genes in vaccinated and unvaccinated fish to Phdp infection allowed the identification of a potential biosignature, consisting in 10 genes, as a surrogate of protection and therefore, as indicator of vaccine success against fotobacteriosis after IP vaccination. These results provide important insights into the S. senegalensis protection against Phdp induced by vaccination.

The Iberian lynx (Lynx pardinus) is an endangered species restricted to several areas of Spain and Portugal. Its low genetic diversity likely provokes immune depression and high susceptibility to infectious diseases. The intestinal microbiota is closely related to host health and nutrition. In order to contribute to the knowledge of the Iberian lynx intestinal microbiota, fecal microbiota of captive specimens from two breeding centers ("La Olivilla" and "El Acebuche"), located in Southern Spain, were studied by Denaturing Gradient Gel Electrophoresis (DGGE). Results grouped microbiota in two main clusters (I and III) which included DGGE patterns of 19 out of 36 specimens, cluster I being the most frequent in "La Olivilla" (50%) and cluster III in "El Acebuche" (55.55 %) specimens. Bacteroidetes, Firmicutes and Proteobacteria phyla were identified. Segregation of clusters I and III was attributed to different microorganism presence (Pseudomonas koreensis, Pseudomonas migulae, Carnobacterium sp., Arthrobacter, Robinsoniella peorensis and Ornithinibacillus sp.) and ability to use different carbon sources. Biolog EcoPlates® results indicate high functional diversity of fecal microbiota, it being higher in cluster III. The great impact of intestinal microbiota on host health supports the importance of its microbial composition understanding. This study is the first report of captive Iberian lynx fecal microbiota composition. [Int Microbiol 20(1): 31-41 (2017)].

Photobacterium damselae subsp. piscicida ( Phdp ), the causative agent of photobacteriosis, is an important pathogen in marine aquaculture that affects many different fish species worldwide, including Solea senegalensis , an important fish species for aquaculture in the south of Europe. Bacteria express different repertoires of proteins in response to environmental conditions and when invading a host, sense in vivo environment and adapt by changing the expression of specific proteins. In the case of pathogens, identification of genes with up-regulated expression in vivo compared to in vitro conditions might give an insight into the genes relevant to the bacterial virulence. In the present work, in vivo induced antigen technology (IVIAT) has been used to search for Phdp genes only expressed or up-regulated in infected S. senegalensis . An expression library from Phdp was assayed against pooled sera from convalescent S. senegalensis specimens and 18 clones were positive, indicating that proteins encoded are expressed by Phdp during S. senegalensis infection and are immunogenic for this fish species. In addition, five proteins were reactive against adsorbed sera, indicating their in vivo induced character. Inosine-5′-monophosphate dehydrogenase, serine hydroxy methyltransferase and alanyl-tRNA synthethase, involved in aminoacid and nucleotide metabolism, the protein with antioxidant activity alkyl hydroperoxide reductase and a non-ribosomal peptide synthetase responsible for the synthesis of the siderophore piscibactin have been identified as antigens induced in Phdp during S. senegalensis infection. Proteins induced during in vivo growth of Phdp represent promising targets for the development of novel antimicrobial or prophylactic agents in the treatment and prevention of photobacteriosis.

The interaction host-intestinal microbiota is essential for the immunological homeostasis of the host. Probiotics, prebiotics and synbiotics are promising tools for the manipulation of the intestinal microbiota towards beneficial effects to the host. The objective of this study was to evaluate the modulation effect on the intestinal microbiota and the transcription of genes involved in the immune response in head kidney of Solea senegalensis after administration of diet supplemented with the prebiotic alginate and the probiotic Shewanella putrefaciens Pdp11 CECT 7627 (SpPdp11). The results showed higher adaptability to dietary changes in the intestinal microbiota of fish fed diet with alginate and SpPdp11 together compared to those fish that received an alginate-supplemented diet. The alginate-supplemented diet produced up-regulation of genes encoding proteins involved in immunological responses, such as complement, lysozyme G and transferrin, and oxidative stress, such as NADPH oxidase and glutation peroxidase. On the other hand, the administration of alginate combined with SpPdp11 resulted in a significant increase of the transcription of genes encoding for glutation peroxidase and HSP70, indicating a potential protective effect of SpPdp11 against oxidative stress. In addition, these effects were maintained after the suspension of the probiotic treatment. The relationship between the modulation of the intestinal microbiota and the expression of genes with protective effect against the oxidative stress was demonstrated by the Principal Components Analysis., SI

The effects on histology and microbial ecology in gilthead seabream (Sparus aurata) intestine caused by dietary probiotic and microalgae were studied. Fish were fed non-supplemented (C, control) or supplemented diets with Tetraselmis chuii, Phaeodactylum tricornutum and Bacillus subtilis single or combined (diets T, P, B, BT and BP) for 4 weeks. Curiously, fish fed the experimental diets showed similar morphological alterations when studied by light and electron microscopy and significant signs of intestinal damage were detected. No effect of microalgae or B. subtilis on the intestinal absorptive area was observed, whereas the number of goblet cells and IELs were significantly lower in fish fed the T, P, B and BT diets and T, BT and BP diets, respectively. Interestingly, only the diets containing B. subtilis resulted in a significant reduction of microvilli height. Alterations such as wide intercellular spaces and large vacuoles in enterocytes were observed in fish fed T, B, BT, BT and P in lesser degrees. These observations demonstrate that fish fed experimental diets presented different signs of oedema and inflammation that could compromise their body homeostasis. Moreover, the experimental diets cause important alterations in the intestinal microbiota by a significant decrease in bacterial diversity, as demonstrated by the fall in specific richness, Shannon and range-weighted richness indices. To our knowledge, this is the first in vivo study regarding the implications of the use of probiotics in combination with immunostimulants on fish intestinal morphology and microbiota. More morphofunctional studies are needed in order to correlate the nutritional and immune aspects of fish gut.

The applicationof probiotics inaquaculture representsa promising alternative for disease prevention. Thesemicroorganisms must survive adverse conditions dur-ing handling and storage to be administered in highconcentrationstothehost.Thisisespeciallyimportantwhen stressful conditions, suchas low watercontents,arepresentinanimalfeed.Bacterialencapsulationhasbeen used to maintain probiotic viability in humanfood; however, the usefulness of the encapsulationtechniquesinanimalfeedneedstobeevaluated.Inthepresent study, optimal conditions for encapsulation inalginateofa¢shprobioticShewanellaputrefaciensstrain(Pdp11) have been determined. Diierent alginate (1%,2%,3% and 4% w/v) and calcium chloride (0.5%,1%2%and3%w/v)concentrations wereusedtoelaboratealginatebeads.TheresultsobtainedindicatethatS.pu-trefaciens Pdp11canbeencapsulatedsuccessfullyincal-cium alginate beads. The percentages of encapsulatedcellswereabove80%,theloweste⁄ciencyratescorre-spondingtohighcalciumavailability(3%).Inaddition,capsules containing viable S. putrefaciens Pdp11can bestored at 41C forat least1month, survival rates beingabove 90%. Onthe contrary, storage of the capsules at22 1C resulted in 40% viabilityloss within 30 days. Fi-nally, the survival of encapsulated probiotics through¢shgastrointestinaltracthasbeendemonstrated.Keywords probiotics, alginate, encapsulation,¢sh,microbiologyIntroductionInfectious diseases in aquaculture are of major con-cern to the ¢sh farming industry and are typicallycontrolled by antibiotic treatment or vaccination.However, vaccines cannot be used as universal dis-ease control measures in aquaculture because juve-nile ¢share not fully immuno-competentanddonotalways respondtovaccination. Also, the administra-tionof vaccines sometimes needs to be performed byan injection, a route that becomes impractical whenapplied to small ¢sh or large numbers of ¢sh. Thus,diseasecontrolusingantibioticsremainsthemethodofchoiceinmanycases.Theuseofantibioticsshouldbe minimized because of the development of poten-tially transferable antibiotic resistance and thenegative impact on the environment (Miranda Z Radu, Ahmad, Ling & Reeza 2003;FAO2006;Sarter,Nguyen,Hung,LazardM Salminen, Ouwehand, Benno & Lee 1999), theuse of probiotics in aquaculture is a relatively newconcept. Several studies have reportedaprotectoref-fect against ¢sh pathogens in diierent ¢sh speciessuch as Atlantic salmon (Salmo salar, L.), turbot(Scophthalmus maximus,L.)orAtlanticcod(Gadusmorhua, L.) that were previously treated with probio

Probiotics are currently being supplemented to cultured fish due to their benefits for fish performance. Herein, we tested the health protection and nutritional effects of probiotic Shewanella putrefaciens (Pdp11 strain), both fresh and lyophilized cells, on Senegalese sole (Solea senegalensis) juveniles. Pdp11 was incorporated into fish feed at concentration of 109 cells g−1, and then provided to juvenile sole for 2 months. Growth rates were significantly higher in fish fed the fresh probiotic compared with fish fed the control diet. Growth of fish receiving lyophilized bacteria was not improved when compared with controls. Body compositions (protein, total lipids and fatty acids profile) were similar for each dietary treatment. At the end of the study, fish were challenged by intraperitoneal inoculation with a pathogenic strain of Photobacterium damselae subsp. piscicida. Relative percentages of survival (RPS) ranged between 25% and 43.8% for fish receiving both probiotic supplemented diets. Both fresh and lyophilized Pdp11 cells conferred protection against P. damselae subsp. piscicida. However, only fresh Pdp11 enhanced Senegal sole performance. These findings should be taken into account for industry purposes in which lyophilized Pdp11 might be easier to manage.

The effects of dietary administration of probiotic Pdp11, a bacterial probiotic strain from the Alteromonadaceae family, on growth and stress tolerance to high stocking density was analyzed in juvenile gilthead sea bream Sparus auratus . In the first trial, juvenile specimens were fed during 116 days with control feed or diets supplemented with Pdp11, and growth, serum immunological parameters as well as plasmatic, hepatic and muscular metabolic parameters were assessed. Growth performance improved in group receiving probiotic compared to control group. No differences were observed for the assessed immunological or metabolic parameters. In the second trial, specimens from both groups from the first experiment were submitted to the following conditions for 15 days: i) group fed with control food and either under low density (3 kg/m 3 ), or high density (30 kg/m 3 ) and ii) group fed with probiotic supplemented feed and either under low density, or high density. Our results indicated that administration of probiotic Pdp11 in the diet promoted growth and improved stress tolerance under high stocking density, suggesting its use will be beneficial for the aquaculture industry of this species.

This study aimed to evaluate the influence of dietary administration of two probiotic strains belonging to Shewanella genus (Pdp11 and Pdp13), on juvenile Senegalese sole (Solea senegalensis, Kaup 1858). Fish were fed three experimental diets for 2 months: two diets supplemented with 109 cfu g−1 feed of Pdp11 and Pdp13 probiotics and one control group. At the end of the experiment, samples were collected to determine muscle and liver body composition and to study digestive tract and liver histology. A challenge with Photobacterium damselae subsp. piscicida (ip) was performed. Results showed a protection for fish receiving probiotic supplemented diets (Relative Percentage Survival 27.5 ± 2.5% for Pdp11; 32.5 ± 2.5% for Pdp13) and a better growth for control and Pdp11 diets. Higher total protein in muscle were detected for fish fed the probiotic diets and significant differences in muscle and liver fatty acid profiles were observed for Pdp11 and Pdp13 compared to the control. Light microscopy images from sole specimens fed the Pdp11 diet showed scarce presence of lipid droplets inside of enterocytes and hepatocytes while greater numbers were found in fish fed the control and Pdp13 diets. Finally, the DGGE technique results suggest that the addition of the probiotic strains of Shewanella genus highly influenced the intestinal microbiota of Senegalese sole specimens, although this effect may depend on the probiotic strain assayed.

The effects of dietary administration of two probiotics, Shewanella putrefaciens Pdp11 and Shewanella baltica Pdp13, on growth, respiratory burst activity of phagocytes of Senegalese sole (Solea senegalensis), and survival of fish challenged with Photobacterium damselae subsp. piscicida have been studied. Fish were fed for 60 days with three different diets: one control, and two diets supplemented with 109 cfu g− 1of probiotics Pdp11 (Pdp11 diet) and Pdp13 (Pdp13 diet). Respiratory burst activity of phagocytes from fish fed with diet Pdp11 significantly increased after 60 days of feeding, whilst this significant increase was not detected in phagocytes from fish fed control and Pdp13 diets. On the other hand, the cumulative percentage of mortality after the challenges with P. damselae subsp. piscicida, was 100% in the groups fed with control diet, whereas mortality rates observed in the groups fed with diets supplemented with Pdp11 and Pdp13 ranged from 75–100% and 65–80%, respectively. These results suggest that an increased respiratory burst activity of the phagocytes is not essential to increase the protection against the P. damselae subsp. piscicida. On the other hand, the use of both probiotics improved the growth, and the survival against the pathogen, in comparison with those fish receiving the control diet.