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Resumen Hoy en dia existen numerosas estrategias desde un punto de vista cientifico que ayudan a esclarecer los casos forenses, entre ellas la datacion cadaverica. La ausencia de metodos fiables cuantitativos para estimar el intervalo post mortem explica el incremento de nuevas lineas de investigacion prometedoras con dicha finalidad. Tras la aparicion de las nuevas tecnicas de secuenciacion masiva y bioinformaticas, surge tambien el estudio del necrobioma como un area novedosa y poco estudiada dentro de las ciencias forenses, que se ha llegado a denominar «microbiologia forense». En esta revision se realiza un breve recorrido por las tecnicas y procedimientos existentes de datacion cadaverica, centrandose en la utilidad del tanatomicrobioma, o conjunto de microorganismos presentes en el momento de la muerte, que podria ser un metodo prometedor para la estimacion del intervalo post mortem en el futuro.

Nowadays there are numerous scientific strategies thathelping to clarify forensic cases, including time since death. The absence of reliable quantitative methods to estimate the post-mortem interval explains the increase in promising new lines of research for this purpose. After the appearance of the new techniques of massive sequencing and bioinformatics, also arises the study of the necrobiome through a new and little studied area within the forensic sciences, Forensic Microbiology. In this review, a tour of the existing techniques and procedures of cadaveric dating is made, which includes new cutting-edge techniques in different areas of knowledge and also mentions the utilities of Forensic Microbiology, where the thanatomicrobiome, present from the moment of death, according to recent studies, points to be a promising method for estimating the post-mortem interval in the future.

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

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

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.

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.

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.

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 potential benefits of probiotics when administering to fish could improve aquaculture production. The objective of this study was to examine the modulation of immune status and gut microbiota of gilthead seabream ( Sparus aurata L.) specimens by a probiotic when administered encapsulated. Commercial diet was enriched with Shewanella putrefaciens Pdp11 (SpPdp11, at a concentration of 10 8 cfu g −1 ) before being encapsulated in calcium alginate beads. Fish were fed non-supplemented (control) or supplemented diet for 4 weeks. After 1, 2 and 4 weeks the main humoral and cellular immune parameters were determined. Furthermore, gene expression profile of five immune relevant genes ( il1β , bd , mhcIIα , ighm and tcrβ ) was studied by qPCR in head kidney. On the other hand, intestinal microbiota of fish was analysed at 7 and 30 days by DGGE. Results demonstrated that administration of alginate encapsulated SpPdp11 has immunostimulant properties on humoral parameters (IgM level and serum peroxidase activity). Although no immunostimulant effects were detected on leucocyte activities, significant increases were detected in the level of mRNA of head-kidney leucocytes for mhcIIα and tcrβ after 4 weeks of feeding the encapsulated-probiotic diet. The administration of SpPdp11 encapsulated in alginate beads produced important changes in the DGGE patterns corresponding to the intestinal microbiota. Predominant bands related to lactic acid bacteria, such as Lactococcus and Lactobacillus strains, were sequenced from the DGGE patterns of fish fed the probiotic diet, whereas they were not sequenced from fish receiving the control diet. The convenience or not of probiotic encapsulation is discussed.

López-Goñi, Ignacio et al., Social networks are already being exploited for searching, storing, and sharing knowledge, demonstrating that they are an efficient vehicle for social learning. Consequently, they could be implemented as a competent tool for formal learning. Twitter is among the 10 most popular online social networks, integrating a community of over 500 million users around the world. Twitter has already been used in several educational programs and evaluated as a positive teaching experience with an outstanding potential in academic and educational environments (1–6). However, there are scarce examples available in the literature about its use in science teaching and communication. In this work, we present and analyze the application of Twitter to create an online space for communication and learning of basic microbiology. With this aim, a team of professionals in the field has imparted, to our knowledge, the first worldwide open access microbiology course via Twitter. Here we assess the results of our experience of using this social network as a tool for teaching, promoting, and communicating scientific knowledge to a wide audience.

Urban wastewater treatment is one of the most important challenges in villages of southern Spain. This is especially outstanding in arid and semiarid regions in which wastewater are discharged to temporary streams or wetlands. The treatment plant of the Fuente de Piedra village discharges its wastewater, passing previously through four semi-natural wetlands, into Fuente de Piedra Lake, a RAMSAR wetland. In summer 2016, a very dry year, water affluent to Fuente de Piedra was limited to wastewater plant effluents without dilution. In order to study the natural assimilation capacity of the wetland system, four key points were sampled. Physico-chemical and biological indicators were analyzed (temperature, pH, conductivity, total phosphorous, total nitrogen, bacteria, phytoplankton and zooplankton). The results show very high chlorophyll a concentration (>500 mg/l) at the water inlet, which decreased to concentration lower than

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.

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.

Aim: To characterize 16 Vibrio harveyi strains isolated from different epizootic outbreaks affecting farmed Senegalese sole. Materials and Results: The Vibrio harveyi strains tested have broad phenotypic diversity based on their biochemical and exoenzymatic patterns, outer membrane proteins (OMP), extracellular product (ECP) patterns and presence of prophages. Lethal dose 50 (LD50) of the strains and in vitro antagonism tests with two probiotic strains were also determined. The OMP analysis revealed three different patterns (A, M and V). The electrophoretic analysis of the ECP showed two different groups. All strains considered virulent based on their LD50 exhibited the same protein pattern in their ECP (pattern I), while all nonvirulent strains showed a different profile (pattern II). About 32% of the tested strains were positive for prophages, although a clear relationship between virulence and the presence of prophages has not been established. Conclusions: The results obtained have shown differences between virulent and avirulent strains isolated from diseased farmed Senegalese sole based on the protein patterns of their ECP. However, a clear relationship between virulence and presence of prophages has not been established. Significance and Impact of the Study: The differences observed between virulent and nonvirulent strains could be used to design prophylactic strategies against diseases caused by V. harveyi in farmed Senegalese sole.

Few antimicrobials are currently authorised in the aquaculture industry to treat infectious diseases. Among them, oxytetracycline (OTC) is one of the first-choice drugs for nearly all bacterial diseases. The objective of this study was to evaluate the effect of the dietary administration of OTC both alone and jointly with the probiotic Shewanella putrefaciens Pdp11 (SpPdp11) on the intestinal microbiota and hepatic expression of genes related to immunity in Senegalese sole (Solea senegalensis) juveniles. The results demonstrated that the richness and diversity of the intestinal microbiota of fish treated with OTC decreased compared with those of the control group but that these effects were lessened by the simultaneous administration of SpPdp11. In addition, specimens that received OTC and SpPdp11 jointly showed a decreased intensity of the Denaturing Gradient Gel Electrophoresis (DGGE) bands related to Vibrio genus and the presence of DGGE bands related to Lactobacillus and Shewanella genera. The relationship among the intestinal microbiota of fish fed with control and OTC diets and the expression of the NADPH oxidase and CASPASE-6 genes was demonstrated by a Principal Components Analysis (PCA) carried out in this study. In contrast, a close relationship between the transcription of genes, such as NKEF, IGF-β, HSP70 and GP96, and the DGGE bands of fish treated jointly with OTC and SpPdp11 was observed in the PCA study. In summary, the results obtained in this study demonstrate that the administration of OTC results in the up-regulation of genes related to apoptosis but that the joint administration of OTC and S.putrefaciens Pdp11 increases the transcription of genes related to antiapoptotic effects and oxidative stress regulation. Further, a clear relationship between these changes and those detected in the intestinal microbiota is established., SI

This work concerns the case study of a Mediterranean Ramsar salt lake (Fuente de Piedra, southern Spain) that receives the treated wastewater of the local village treatment plant. The wastewater goes through a system of canals, water dams, and three semi-natural ponds that were built in 2005. This work aims to investigate the capacity of the system to assimilate the impact of wastewater effluents on Lake Fuente de Piedra. For this, four points were sampled on 27–29 April 2016, at the inlet and the outlet points of the first and the third semi-natural ponds, with three replicates each. Temperature, pH, and conductivity at the inlet were 19.62 °C, 7.99, and 3262.67 μS/cm, respectively, and increased through the pond system by 7.59%, 8.04%, and 37.34%, respectively. Phytoplankton concentration indicators decreased from the inlet point to the outlet point (chlorophyll a from >500 to 5 × 1010 to 4.3 × 109 μm3/mL). Zooplankton biovolume, in contrast, increased three orders of magnitude from the inlet (3.5 × 107 μm3/mL) to the outlet point (1.6 × 109 μm3/mL). Heterotrophic bacteria (1.29 × 105 cfu/mL) and faecal enterococci (1033 ± 351 cfu/100 mL) were high at the inlet point, but decreased at the outlet point by almost three orders of magnitude. Total phosphorous and total nitrogen decreased 40.3% and 23.1% through the pond system. The results showed an improvement in water quality in its passage through the built system. Additionally, as permanent wetlands with acceptable water quality, the water system attracts wild fauna during the dry summer, leading to the conclusion that these semi-natural or artificial wetlands should be extrapolated to other aquatic ecosystems (Mediterranean wetlands) that receive contributions of residual waters. Better functioning of the treatment plant is desirable to improve the conservation of the Ramsar and adjacent wetlands systems.

Suitable nutrition at first feeding in marine fish larvae is an important factor for successful larval and juvenile rearing. In this way probiotic supplementation may contribute to better face the habitual stress of metamorphosis and weaning at early sole stages. The aimof this study was to optimize Shewanella putrefaciens Pdp11 administration to Senegalese sole (Solea senegalensis) at early stages of development. For this purpose, we studied the influence of a short pulse of S. putrefaciens Pdp11 (10–30 dah) on growth performance, gut microbiota modulation, digestive enzymes activities and body composition. These probiotic bacteria were incorporated using Artemia as live vector (2.5 × 10 7 cfu mL −1 ) and supplied to sole specimens in a co-feeding regime (Gemma, Skretting) by triplicate. S. putrefaciens Pdp11 colonized larval gut being present from23 to 119 dah. Probiotic supply significantly modulated larval and fry gut microbiota. A PCA analysis including all the parameters analyzed strongly associated S. putrefaciens Pdp11 presence with a higher fish growth, a higher digestive proteolytic activity level and a fish body composition modulation along S. senegalensis rearing. In addition less size variability was obtained frommetamorphosis until the end of weaning. In conclusion a short pulse of S. putrefaciens Pdp11 (10– 30 dah) seems sufficient to obtain a suitable microbial modulation involved in a better growth performance and body composition that suggests a great potential for sole aquaculture production., SI

Ingestion of bacteria at early stages results in establishment of a primary intestinal microbiota which likely undergoes several stages along fish life. The role of this intestinal microbiota regulating body functions is crucial for larval development. Probiotics have been proved to modulate this microbiota and exert antagonistic effects against fish pathogens. In the present study, we aimed to determine bacterial diversity along different developmental stages of farmed Senegalese sole (Solea senegalensis) after feeding probiotic (Shewanella putrefaciens Pdp11) supplemented diet for a short period (10–30 days after hatching, DAH). Intestinal lumen contents of sole larvae fed control and probiotic diets were collected at 23, 56, 87, and 119 DAH and DNA was amplified using 16S rDNA bacterial domain-specific primers. Amplicons obtained were separated by denaturing gradient gel electrophoresis (DGGE), cloned, and resulting sequences compared to sequences in GenBank. Results suggest that Shewanella putrefaciens Pdp11 induces a modulation of the dominant bacterial taxa of the intestinal microbiota from 23 DAH. DGGE patterns of larvae fed the probiotic diet showed a core of bands related to Lactobacillus helveticus, Pseudomonas acephalitica, Vibrio parahaemolyticus,and Shewanella genus, together with increased Vibri o genus presence. In addition, decreased number of clones related to Photobacterium damselae subsp piscicida at 23 and 56 DAH was observed in probiotic-fed larvae. A band corresponding to Shewanella putrefaciens Pdp11 was sequenced as predominant from 23 to 119 DAH samples, confirming the colonization by the probiotics. Microbiota modulation obtained via probiotics addition emerges as an effective tool to improve Solea senegalensis larviculture., SI

Aquaculture industry exposes fish to acute stress events, such as high stocking density, and a link between stress and higher susceptibility to diseases has been concluded. Several studies have demonstrated increased stress tolerance of fish treated with probiotics, but the mechanisms involved have not been elucidated. Shewanella putrefaciens Pdp11 is a strain isolated from healthy gilthead seabream (Sparus aurata L.) and it is considered as probiotics. The aim of this study was to evaluate the effect of the dietary administration of this probiotics on the stress tolerance of Solea senegalensis specimens farmed under high stocking density (PHD) compared to a group fed a commercial diet and farmed under the same conditions (CHD). In addition, during the experiment, a natural infectious outbreak due to Vibrio species affected fish farmed under crowding conditions. Changes in the microbiota and histology of intestine and in the transcription of immune response genes were evaluated at 19 and 30 days of the experiment. Mortality was observed after 9 days of the beginning of the experiment in CHD and PHD groups, it being higher in the CHD group. Fish farmed under crowding stress showed reduced expression of genes at 19 day probiotic feeding. On the contrary, a significant increase in immune related gene expression was detected in CHD fish at 30 day, whereas the gene expression in fish from PHD group was very similar to that showed in specimens fed and farmed with the conventional conditions. In addition, the dietary administration of S. putrefaciens Pdp11 produced an important modulation of the intestinal microbiota, which was significantly correlated with the high number of goblet cells detected in fish fed the probiotic diet., SI

Probiotic supplementation in fish aquaculture has significantly increased in the last decade due to its beneficial effect on fish performance. Probiotic use at early stages of fish development may contribute to better face metamorphosis and weaning stress. In the present work, we studied the influence of Shewanella putrefaciensPdp11 supplementation on growth, body composition and gut microbiota in Senegalese sole (Solea senegalensis) during larval and weaning development.S. putrefaciensPdp11 was incorporated usingArtemiaas live vector (2.5910 7 cfu mL -1 ) and supplied to sole specimens in a co-feeding regime (10–86 DAH) by triplicate. Probiotic addition promoted early metamorphosis and a significantly higher growth in length at 24 DAH larvae.S. putrefaciens Pdp11 also modulated gut microbiota and significantly increased protein content and DHA/EPA ratios in sole fry (90 DAH). This nutritional enhancement is considered especially important after weaning, where significantly higher growth in length and weight was observed in probiotic fish. Moreover, a less heterogeneous fish size in length was detected since metamorphosis till the end of weaning, being of interest for sole aquaculture production. After weaning, fish showed significantly higher growth (length and weight) and less variable lengths in fish when supplemented with probiotics. Both the enhancement of nutritional condition and the decrease in size variability associated with probiotic addition are highly interesting for sole aquaculture production. Versión de editor 2

Changes produced in gilthead sea bream (Sparus aurata L.) intestinal morphology and microbiota caused by dietary administration of inulin and Bacillus subtilis have been studied. Gilthead sea bream specimens were fed diets containing 0 (control), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)), or B. subtilis + inulin (10(7) cfu g(-1) + 10 g kg(-1)) for four weeks. Curiously, fish fed the experimental diets (inulin, B. subtilis, or B. subtilis + inulin) showed the same morphological alterations when studied by light and electron microscopy, while significant differences in the signs of intestinal damage were detected by the morphometric study. All of the observed alterations were present only in the gut mucosa, and intestinal morphometric study revealed no effect of inulin or B. subtilis on the intestinal absorptive area. Furthermore, experimental diets cause important alterations in the intestinal microbiota by significantly decreasing bacterial diversity, as demonstrated by the specific richness, Shannon, and range-weighted richness indices. The observed alterations demonstrate that fish fed experimental diets had different signs of gut oedema and inflammation that could compromise their body homeostasis, which is mainly maintained by the epithelial lining of the gastrointestinal tract. To our knowledge, this is the first in vivo study regarding the implications of the use of synbiotics (conjunction of probiotics and prebiotics) on fish gut morphology and microbiota.

The application of probiotics on aquatic animals is increasing for a better fish welfare status as well as an environment-friendly activity which are actual demands of modern aquaculture industry. A bacterium from skin mucus of healthy gilthead seabream (Sparus aurata L.) has been isolated and identified as Shewanella putrefaciens Pdp11. Different studies have been done to know its application as probiotic in the Senegalese sole and gilthead seabream farming. This article reviews the studies carried out with this probiotic microorganism focusing on the current knowledge of its in vitro and in vivo mechanisms of action. The results suggested that the probiotic S. putrefaciens Pdp11, due to its beneficial effects, could be used in the aquaculture activity of both species.

Fish gastrointestinal tract is one of the most important sites of interaction with the external world. Interactions between the intestinal microbiota and the host modulate the functionality of the intestinal mucosa and gene expression. Application of probiotics, prebiotics and synbiotics for aquatic animals is increasing to improve welfare and promote growth. However, data about the use of probiotics in conjunction with prebiotics, in farmed marine organisms are scarce. The objective of this work is to study the modulation ability of the intestinal microbiota and the immune system of gilthead seabream exerted by the probiotic yeast Debaromycess hansonii L2 in conjunction with the prebiotic inulin. Fish were fed a commercial diet (control diet I), and with the same diet supplemented with 1.1% D. hansenii strain L2 (106 CFU g-1) plus inulin (3%) (diet II) for 4 weeks. The whole intestines of healthy fish from each group were aseptically removed at 2 and 4 weeks after starting the experiment and they were analysed by PCR-Denaturing Gradient Gel Electrophoresis (DGGE). Samples of blood and head kidney were obtained for humoral and cellular immune parameters determination. The expression of 12 selected genes related to the immune response (IgM, MHCI?, MHCII?, C3, IL-1?,TLR, TNF?, CSF-1R, NCCRP-1, Hep, TCR? y CD8) were analyzed by real-time PCR from skin, intestine, liver and head-kidney tissue. In this study, relevant changes in the intestinal microbiota of gilthead seabream specimens fed the diets assayed has been demonstrated. An important effect on the intestinal microbiota by the dietary administration of a synbiotic mixture has also been detected, especially in fish receiving the supplemented diet for 4 weeks. These changes coincided in the same time with an up-regulation of the expression of immunological genes in skin and head kidney of gilthead seabream at 2 and 4 weeks, respectively.

Pleuronectiforms are an important group of fish, and one of their species, Solea senegalensis (Kaup 1858), has been extensively studied at different levels, although information about its intestinal microbiota and the effects of different factors on it is very scarce. Modern aquaculture industry demands strategies which help to maintain a microbiologically healthy environment and an environmentally friendly aquaculture. In this context, probiotics seem to offer an attractive alternative. The intake of probiotics could modify the composition of the intestinal microbiota, which is a key component in excluding potential invaders and maintaining health. The aim of this study was to evaluate by 16S rRNA gene analysis using polymerase chain reaction-denaturing gradient gel electrophoresis the effect of administering fresh or lyophilized cells of Pdp11 on the intestinal microbiota of farmed Senegalese sole, using sodium alginate to facilitate the incorporation of bacterial cells to the feed. The results obtained showed that the composition of fish intestinal microbiota was affected when fish received a diet supplemented with sodium alginate and fresh or lyophilized probiotic cells. In all cases, the dominant bacterial groups belonged to γ-Proteobacteria and mainly the Vibrio species. The use of sodium alginate reduced the incidence of populations with, Sí