Publications by authors named "Ariadna Sitjà-Bobadilla"

61 Publications

Infection in European Sea Bass: Revealing a Long Misunderstood Relationship.

Front Immunol 2021 11;12:645607. Epub 2021 Mar 11.

Fish Pathology Group, Institute of Aquaculture Torre de la Sal - Consejo Superior de Investigaciones Científicas (IATS-CSIC), Castellón, Spain.

(Cymothoidea, Isopoda) is a generalist crustacean parasite that negatively affects the economic sustainability of European sea bass () aquaculture in the North-East Mediterranean. While mortalities are observed in fry and fingerlings, infection in juvenile and adult fish result in approximately 20% growth delay. A transcriptomic analysis (PCR array, RNA-Seq) was performed on organs (tongue, spleen, head kidney, and liver) from infected vs. -free sea bass fingerlings. Activation of local and systemic immune responses was detected, particularly in the spleen, characterized by the upregulation of cytokines (also in the tongue), a general reshaping of the immunoglobulin (Ig) response and suppression of T-cell mediated responses. Interestingly, starvation and iron transport and metabolism genes were strongly downregulated, suggesting that the parasite feeding strategy is not likely hematophagous. The regulation of genes related to growth impairment and starvation supported the growth delay observed in infected animals. Most differentially expressed (DE) transcripts were exclusive of a specific organ; however, only in the tongue, the difference between infected and uninfected fish was significant. At the attachment/feeding site, the pathways involved in muscle contraction and intercellular junction were the most upregulated, whereas the pathways involved in fibrosis (extracellular matrix organization, collagen formation, and biosynthesis) were downregulated. These results suggest that parasite-inflicted damage is successfully mitigated by the host and characterized by regenerative processes that prevail over the reparative ones.
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http://dx.doi.org/10.3389/fimmu.2021.645607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991915PMC
March 2021

Experimental Horizontal Transmission of (Microsporea: Enterocytozoonidae) in Gilthead Sea Bream ().

Animals (Basel) 2021 Feb 1;11(2). Epub 2021 Feb 1.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), 12595 Ribera de Cabanes, Spain.

is a microsporidian enteroparasite that infects mainly the intestine of gilthead sea bream (), leading to an emaciative syndrome. Thus far, the only available information about this infection comes from natural outbreaks in farmed fish. The aim of the present study was to determine whether . could be transmitted horizontally using naturally infected fish as donors, and to establish an experimental in vivo procedure to study this host-parasite model without depending on natural infections. Naïve fish were exposed to the infection by cohabitation, effluent, or intubated either orally or anally with intestinal scrapings of donor fish in four different trials. We succeeded in detecting parasite in naïve fish in all the challenges, but the infection level and the disease signs were always milder than in donor fish. The parasite was found in peripheral blood of naïve fish at 4 weeks post-challenge (wpc) in oral and effluent routes, and up to 12 wpc in the anal transmission trial. Molecular diagnosis detected in other organs besides intestine, such as gills, liver, stomach or heart, although the intensity was not as high as in the target tissue. The infection tended to disappear through time in all the challenge routes assayed, except in the anal infection route.
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http://dx.doi.org/10.3390/ani11020362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912876PMC
February 2021

The influence of 17β-oestradiol on lymphopoiesis and immune system ontogenesis in juvenile sea bass, Dicentrarchus labrax.

Dev Comp Immunol 2021 May 16;118:104011. Epub 2021 Jan 16.

UMR-I 02 Environmental Stress and Aquatic Biomonitoring (SEBIO), University of Le Havre Normandy, F-76600, Le Havre, France. Electronic address:

The female sex steroid 17β-oestradiol (E2) is involved in the regulation of numerous physiological functions, including the immune system development and performance. The role of oestrogens during ontogenesis is, however, not well studied. In rodents and fish, thymus maturation appears to be oestrogen-dependent. Nevertheless, little is known about the function of oestrogen in immune system development. To further the understanding of the role of oestrogens in fish immune system ontogenesis, fingerlings of European sea bass (Dicentrarchus labrax) were exposed for 30 days to 20 ng E2·L, at two ages tightly related to thymic maturation, i.e., 60 or 90 days post hatch (dph). The expression of nuclear and membrane oestrogen receptors was measured in the thymus and spleen, and the expression of several T cell-related gene markers was studied in both immune organs, as well as in the liver. Waterborne E2-exposure at 20.2 ± 2.1 (S.E.) ng·L was confirmed by radioimmunoassay, leading to significantly higher E2-contents in the liver of exposed fish. The majority of gene markers presented age-dependent dynamics in at least one of the organs, confirming thymus maturation, but also suggesting a critical ontogenetic window for the implementation of liver resident γδ and αβ T cells. The oestrogen receptors, however, remained unchanged over the age and treatment comparisons with the exception of esr2b, which was modulated by E2 in the younger cohort and increased its expression with age in the thymus of the older cohort, as did the membrane oestrogen receptor gpera. These results confirm that oestrogen-signalling is involved in thymus maturation in European sea bass, as it is in mammals. This suggests that esr2b and gpera play key roles during thymus ontogenesis, particularly during medulla maturation. In contrast, the spleen expressed low or non-detectable levels of oestrogen receptors. The E2-exposure decreased the expression of tcrγ in the liver in the cohort exposed from 93 to 122 dph, but not the expression of any other immune-related gene analysed. These results indicate that the proliferation/migration of these innate-like T cell populations is oestrogen-sensitive. In regard to the apparent prominent role of oestrogen-signalling in the late thymus maturation stage, the thymic differentiation of the corresponding subpopulations of T cells might be regulated by oestrogen. To the best of our knowledge, this is the first study investigating the dynamics of both nuclear and membrane oestrogen receptors in specific immune organs in a teleost fish at very early stages of immune system development as well as to examine thymic function in sea bass after an exposure to E2 during ontogenesis.
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http://dx.doi.org/10.1016/j.dci.2021.104011DOI Listing
May 2021

Genetic selection for growth drives differences in intestinal microbiota composition and parasite disease resistance in gilthead sea bream.

Microbiome 2020 11 23;8(1):168. Epub 2020 Nov 23.

Nutrigenomics and Fish Endocrinology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, Spain.

Background: The key effects of intestinal microbiota in animal health have led to an increasing interest in manipulating these bacterial populations to improve animal welfare. The aquaculture sector is no exception and in the last years, many studies have described these populations in different fish species. However, this is not an easy task, as intestinal microbiota is composed of very dynamic populations that are influenced by different factors, such as diet, environment, host age, and genetics. In the current study, we aimed to determine whether the genetic background of gilthead sea bream (Sparus aurata) influences the intestinal microbial composition, how these bacterial populations are modulated by dietary changes, and the effect of selection by growth on intestinal disease resistance. To that aim, three different groups of five families of gilthead sea bream that were selected during two generations for fast, intermediate, or slow growth (F3 generation) were kept together in the same open-flow tanks and fed a control or a well-balanced plant-based diet during 9 months. Six animals per family and dietary treatment were sacrificed and the adherent bacteria from the anterior intestinal portion were sequenced. In parallel, fish of the fast- and slow-growth groups were infected with the intestinal parasite Enteromyxum leei and the disease signs, prevalence, intensity, and parasite abundance were evaluated.

Results: No differences were detected in alpha diversity indexes among families, and the core bacterial architecture was the prototypical composition of gilthead sea bream intestinal microbiota, indicating no dysbiosis in any of the groups. The plant-based diet significantly changed the microbiota in the intermediate- and slow-growth families, with a much lower effect on the fast-growth group. Interestingly, the smaller changes detected in the fast-growth families potentially accounted for more changes at the metabolic level when compared with the other families. Upon parasitic infection, the fast-growth group showed significantly lower disease signs and parasite intensity and abundance than the slow-growth animals.

Conclusions: These results show a clear genome-metagenome interaction indicating that the fast-growth families harbor a microbiota that is more flexible upon dietary changes. These animals also showed a better ability to cope with intestinal infections. Video Abstract.
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http://dx.doi.org/10.1186/s40168-020-00922-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686744PMC
November 2020

Passive Immunization Delays Disease Outcome in Gilthead Sea Bream Infected With (Myxozoa), Despite the Moderate Changes in IgM and IgT Repertoire.

Front Immunol 2020 11;11:581361. Epub 2020 Sep 11.

Fish Pathology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain.

Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that gilthead sea bream (GSB, ) that survive . infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific IgM in serum. Thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in GSB and to study in detail the nature of these protective antibodies. Serum from a pool of resistant (SUR) or naïve (NAI) animals was intracoelomically injected 24 h prior to the . -effluent challenge and at 9 days post-challenge (dpc). Effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. In parallel, we performed an immunoglobulin repertoire analysis of the fish generating SUR and NAI sera. The results showed that, fish injected with parasite-specific antibodies (spAbs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. Repertoire analysis revealed that induced a polyclonal expansion of diverse IgM and IgT subsets that could be in part an evasion strategy of the parasite. Nonetheless, GSB was able to produce sufficient levels of parasite-spAbs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. These results highlight the crucial role of spAb responses against and set the basis for the development of effective treatment or prophylactic methods for aquaculture.
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http://dx.doi.org/10.3389/fimmu.2020.581361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516018PMC
September 2020

(Microsporidia) in Gilthead Sea Bream (): Pathological Effects and Cellular Immune Response in Natural Infections.

Vet Pathol 2020 07 12;57(4):565-576. Epub 2020 Jun 12.

Instituto de Acuicultura Torre de la Sal, Castellón, Spain.

is a microsporidian responsible for an emaciative disease in gilthead sea bream (). Its intranuclear development and the lack of and models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM and IgT cells were identified by hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of in gilthead sea bream, an important aquaculture species.
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http://dx.doi.org/10.1177/0300985820927707DOI Listing
July 2020

Water temperature, time of exposure and population density are key parameters in Enteromyxum leei fish-to-fish experimental transmission.

J Fish Dis 2020 Apr 25;43(4):491-502. Epub 2020 Feb 25.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal, Consejo Superior de Investigaciones Científicas, Ribera de Cabanes, Spain.

Enteromyxum leei is a myxozoan histozoic parasite that infects the intestine of several teleost fish species. In gilthead sea bream (Sparus aurata), it provokes a chronic disease, entailing anorexia, delayed growth, reduced marketability and mortality. Direct fish-to-fish transmission, relevant in aquaculture conditions, has been demonstrated for E. leei via effluent, cohabitation, and oral and anal routes. However, the minimum time of exposure for infection has not been established, nor the possible effect on the fish immune response. Two effluent trials were performed at different temperatures (high: average of 25.6°C; and low: constant at 18°C), different times of exposure to the effluent (1, 3, 5 and 7 weeks) and different population densities. The results showed that 1 week was enough to infect 100% of fish at high temperature and 58.3% at low temperature. High temperature not only increased the prevalence of infection in posterior intestine, but also induced a higher production of specific antibodies, limiting the progression of the infection along the intestine. Longer time of exposure to the parasite and higher fish densities facilitated E. leei infection. These results show that effective diagnosis, lowering animal density and removal of infected fish are key aspects to manage this disease in aquaculture facilities.
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http://dx.doi.org/10.1111/jfd.13147DOI Listing
April 2020

Effects of Enteromyxum spp. (Myxozoa) infection in the regulation of intestinal E-cadherin: Turbot against gilthead sea bream.

J Fish Dis 2020 Mar 26;43(3):337-346. Epub 2020 Jan 26.

Departamento de Anatomía, Producción Animal y Ciencias Clínicas Veterinarias, Universidade de Santiago de Compostela, Lugo, Spain.

Enteromyxoses are relevant diseases for turbot and gilthead sea bream aquaculture. The myxozoan parasites invade the intestinal mucosa, causing a cachectic syndrome associated with intestinal barrier alteration; nonetheless, their pathological impact is different. Turbot infected by Enteromyxum scophthalmi develop more severe intestinal lesions, reaching mortality rates of 100%, whereas in E. leei-infected gilthead sea bream, the disease progresses slowly, and mortality rates are lower. The mechanisms underlying the different pathogenesis are still unclear. We studied the distribution and expression changes of E-cadherin, a highly conserved protein of the adherens junctions, in the intestine of both species by immunohistochemistry and quantitative PCR, using the same immunohistochemical protocol and common primers. The regular immunostaining pattern observed in control fish turned into markedly irregular in parasitized turbot, showing an intense immunoreaction at the host-parasite interface. Nevertheless, E-cadherin gene expression was not significantly modulated in this species. On the contrary, no evident changes in the protein distribution were noticed in gilthead sea bream, whereas a significant gene downregulation occurred in advanced infection. The results contribute to the understanding of the different host-parasite interactions in enteromyxoses. Host and parasite cells appear to establish diverse relationships in these species, which could underlie the different pathological picture.
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http://dx.doi.org/10.1111/jfd.13130DOI Listing
March 2020

Sex, Age, and Bacteria: How the Intestinal Microbiota Is Modulated in a Protandrous Hermaphrodite Fish.

Front Microbiol 2019 31;10:2512. Epub 2019 Oct 31.

Nutrigenomics and Fish Growth Endocrinology Group, Institute of Aquaculture Torre de la Sal (CSIC), Castellón, Spain.

Intestinal microbiota is key for many host functions, such as digestion, nutrient metabolism, disease resistance, and immune function. With the growth of the aquaculture industry, there has been a growing interest in the manipulation of fish gut microbiota to improve welfare and nutrition. Intestinal microbiota varies with many factors, including host species, genetics, developmental stage, diet, environment, and sex. The aim of this study was to compare the intestinal microbiota of adult gilthead sea bream () from three groups of age and sex (1-year-old males and 2- and 4-year-old females) maintained under the same conditions and fed exactly the same diet. Microbiota diversity and richness did not differ among groups. However, bacterial composition did, highlighting the presence of and starting at 2 years of age (females) and a higher presence of and in 1-year-old males. The core microbiota was defined by 14 Operational Taxonomic Units (OTUs) and the groups that showed more OTUs in common were 2- and 4-year-old females. Discriminant analyses showed a clear separation by sex and age, with bacteria belonging to the phyla Firmicutes, Proteobacteria and Actinobacteria driving the separation. Pathway analysis performed with the inferred metagenome showed significant differences between 1-year-old males and 4-year-old females, with an increase in infection-related pathways, nitrotoluene degradation and sphingolipid metabolism, and a significant decrease in carbohydrate metabolism pathways with age. These results show, for the first time, how intestinal microbiota is modulated in adult gilthead sea bream and highlight the importance of reporting age and sex variables in these type of studies in fish.
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http://dx.doi.org/10.3389/fmicb.2019.02512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834695PMC
October 2019

Disruption of gut integrity and permeability contributes to enteritis in a fish-parasite model: a story told from serum metabolomics.

Parasit Vectors 2019 Oct 16;12(1):486. Epub 2019 Oct 16.

Associated Unit of Marine Ecotoxicology (IATS-IUPA), Castellon, Spain.

Background: In the animal production sector, enteritis is responsible for serious economic losses, and intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and animal production efficiency. The effect of enteric parasites on the gut function of teleost fish, which represent the most ancient bony vertebrates, is far from being understood. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe enteritis in gilthead sea bream (Sparus aurata), anorexia, cachexia, growth impairment, reduced marketability and increased mortality.

Methods: This study aimed to outline the gut failure in this fish-parasite model using a multifaceted approach and to find and validate non-lethal serum markers of gut barrier dysfunction. Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits.

Results: The immunodetection of Tjp1 and Cldn3 was significantly lower in the intestine of parasitized fish, while no strong differences were found in E-cadherin. Parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses. Of these compounds, serum inosine (decreased) and creatine (increased) were identified as relevant and validated with commercial kits.

Conclusions: The results demonstrate the disruption of tight junctions and the loss of gut barrier function, a metabolomic profile of absorption dysfunction and anorexia, which further outline the pathophysiological effects of E. leei.
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http://dx.doi.org/10.1186/s13071-019-3746-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796429PMC
October 2019

Acquired protective immune response in a fish-myxozoan model encompasses specific antibodies and inflammation resolution.

Fish Shellfish Immunol 2019 Jul 5;90:349-362. Epub 2019 May 5.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, Spain. Electronic address:

The myxozoan parasite Enteromyxum leei causes chronic enteritis in gilthead sea bream (GSB, Sparus aurata) leading to intestinal dysfunction. Two trials were performed in which GSB that had survived a previous infection with E. leei (SUR), and naïve GSB (NAI), were exposed to water effluent containing parasite stages. Humoral factors (total IgM and IgT, specific anti-E. leei IgM, total serum peroxidases), histopathology and gene expression were analysed. Results showed that SUR maintained high levels of specific anti-E. leei IgM (up to 16 months), expressed high levels of immunoglobulins at the intestinal mucosa, particularly the soluble forms, and were resistant to re-infection. Their acquired-type response was complemented by other immune effectors locally and systemically, like cell cytotoxicity (high granzyme A expression), complement activity (high c3 and fucolectin expression), and serum peroxidases. In contrast to NAI, SUR displayed a post-inflammatory phenotype in the intestine and head kidney, characteristic of inflammation resolution (low il1β, high il10 and low hsp90α expression).
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http://dx.doi.org/10.1016/j.fsi.2019.04.300DOI Listing
July 2019

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization.

J Fish Dis 2019 Jun 10;42(6):809-815. Epub 2019 Apr 10.

Institute of Aquaculture "Torre de la Sal" (IATS-CSIC), Castellón, Spain.

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.
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http://dx.doi.org/10.1111/jfd.12993DOI Listing
June 2019

Acting locally - affecting globally: RNA sequencing of gilthead sea bream with a mild Sparicotyle chrysophrii infection reveals effects on apoptosis, immune and hypoxia related genes.

BMC Genomics 2019 Mar 11;20(1):200. Epub 2019 Mar 11.

Fish Pathology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Ribera de Cabanes, Castellón, Spain.

Background: Monogenean flatworms are the main fish ectoparasites inflicting serious economic losses in aquaculture. The polyopisthocotylean Sparicotyle chrysophrii parasitizes the gills of gilthead sea bream (GSB, Sparus aurata) causing anaemia, lamellae fusion and sloughing of epithelial cells, with the consequent hypoxia, emaciation, lethargy and mortality. Currently no preventive or curative measures against this disease exist and therefore information on the host-parasite interaction is crucial to find mitigation solutions for sparicotylosis. The knowledge about gene regulation in monogenean-host models mostly comes from freshwater monopysthocotyleans and almost nothing is known about polyopisthocotyleans. The current study aims to decipher the host response at local (gills) and systemic (spleen, liver) levels in farmed GSB with a mild natural S. chrysophrii infection by transcriptomic analysis.

Results: Using Illumina RNA sequencing and transcriptomic analysis, a total of 2581 differentially expressed transcripts were identified in infected fish when compared to uninfected controls. Gill tissues in contact with the parasite (P gills) displayed regulation of fewer genes (700) than gill portions not in contact with the parasite (NP gills) (1235), most likely due to a local silencing effect of the parasite. The systemic reaction in the spleen was much higher than that at the parasite attachment site (local) (1240), and higher than in liver (334). NP gills displayed a strong enrichment of genes mainly related to immune response and apoptosis. Processes such as apoptosis, inflammation and cell proliferation dominated gills, whereas inhibition of apoptosis, autophagy, platelet activation, signalling and aggregation, and inflammasome were observed in spleen. Proteasome markers were increased in all tissues, whereas hypoxia-related genes were down-regulated in gills and spleen.

Conclusions: Contrasting forces seem to be acting at local and systemic levels. The splenic down-regulation could be part of a hypometabolic response, to counteract the hypoxia induced by the parasite damage to the gills and to concentrate the energy on defence and repair responses. Alternatively, it can be also interpreted as the often observed action of helminths to modify host immunity in its own interest. These results provide the first toolkit for future studies towards understanding and management of this parasitosis.
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http://dx.doi.org/10.1186/s12864-019-5581-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6416957PMC
March 2019

Gene expression analysis of Atlantic salmon gills reveals mucin 5 and interleukin 4/13 as key molecules during amoebic gill disease.

Sci Rep 2018 09 12;8(1):13689. Epub 2018 Sep 12.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, 12595, Spain.

Amoebic gill disease (AGD) is one of the main diseases affecting Atlantic salmon (Salmo salar L.) mariculture. Hallmarks of AGD are hyperplasia of the lamellar epithelium and increased production of gill mucus. This study investigated the expression of genes involved in mucus secretion, cell cycle regulation, immunity and oxidative stress in gills using a targeted 21-gene PCR array. Gill samples were obtained from experimental and natural Neoparamoeba perurans infections, and sampling points included progressive infection stages and post-freshwater treatment. Up-regulation of genes related to mucin secretion and cell proliferation, and down-regulation of pro-inflammatory and pro-apoptotic genes were associated with AGD severity, while partial restoration of the gill homeostasis was detected post-treatment. Mucins and Th2 cytokines accoun ted for most of the variability observed between groups highlighting their key role in AGD. Two mucins (muc5, muc18) showed differential regulation upon disease. Substantial up-regulation of the secreted muc5 was detected in clinical AGD, and the membrane bound muc18 showed an opposite pattern. Th2 cytokines, il4/13a and il4/13b2, were significantly up-regulated from 2 days post-infection onwards, and changes were lesion-specific. Despite the differences between experimental and natural infections, both yielded comparable results that underline the importance of the studied genes in the respiratory organs of fish, and during AGD progression.
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http://dx.doi.org/10.1038/s41598-018-32019-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6135806PMC
September 2018

Hints on T cell responses in a fish-parasite model: Enteromyxum leei induces differential expression of T cell signature molecules depending on the organ and the infection status.

Parasit Vectors 2018 Jul 31;11(1):443. Epub 2018 Jul 31.

Fish Pathology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Ribera de Cabanes, Castellón, Spain.

Backgroud: Enteromyxum leei is a myxozoan parasite that produces a slow-progressing intestinal disease. This parasite invades the paracellular space of the intestinal epithelium and progresses from the posterior to the anterior intestine. The aim of the present study was to gain insights into fish T cell responses in the gilthead sea bream-E. leei infection model using a PCR-array with 30 signature molecules for different leukocyte responses in head kidney, spleen, anterior and posterior intestine.

Results: The PCR-array results suggest that E. leei induced migration of T cells from head kidney to intestines where T, CTL and T profiles were activated and kept in balance by the upregulation of regulatory cytokines. These results were partially validated by the use of cross-reacting antibodies and BrdU immunostaining to monitor proliferation. Zap70 immunostaining supported the increased number of T cells in the anterior intestine detected by gene expression, but double staining with BrdU did not show active proliferation of this cell type at a local level, supporting the migration from lymphohaematopoietic tissues to the site of infection. Global analyses of the expression profiles revealed a clear separation between infected and exposed, but non-infected fish, more evident in the target organ. Exposed, non-infected animals showed an intermediate phenotype closer to the control fish.

Conclusions: These results evidence a clear modulation of the T cell response of gilthead sea bream upon E. leei infection. The effects occurred both at local and systemic levels, but the response was stronger and more specific at the site of infection, the intestine. Altogether, this research poses a promising basis to understand the response against this important parasite and establish effective preventive or palliative measures.
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http://dx.doi.org/10.1186/s13071-018-3007-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069777PMC
July 2018

Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets.

Microbiome 2017 12 28;5(1):164. Epub 2017 Dec 28.

Nutrigenomics and Fish Growth Endocrinology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, Spain.

Background: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested.

Results: The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome.

Conclusions: This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.
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http://dx.doi.org/10.1186/s40168-017-0390-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5745981PMC
December 2017

Sodium salt medium-chain fatty acids and -based probiotic strategies to improve growth and intestinal health of gilthead sea bream ().

PeerJ 2017 4;5:e4001. Epub 2017 Dec 4.

Nutrigenomics and Fish Growth Endocrinology Group, Institute of Aquaculture Torre de la Sal, CSIC, Castellón, Spain.

Background: The increased demand for fish protein has led to the intensification of aquaculture practices which are hampered by nutritional and health factors affecting growth performance. To solve these problems, antibiotics have been used for many years in the prevention, control and treatment against disease as well as growth promoters to improve animal performance. Nowadays, the use of antibiotics in the European Union and other countries has been completely or partially banned as a result of the existence of antibiotic cross-resistance. Therefore, a number of alternatives, including enzymes, prebiotics, probiotics, phytonutrients and organic acids used alone or in combination have been proposed for the improvement of immunological state, growth performance and production in livestock animals. The aim of the present study was to evaluate two commercially available feed additives, one based on medium-chain fatty acids (MCFAs) from coconut oil and another with a -based probiotic, in gilthead sea bream (GSB, ), a marine farmed fish of high value in the Mediterranean aquaculture

Methods: The potential benefits of adding two commercial feed additives on fish growth performance and intestinal health were assessed in a 100-days feeding trial. The experimental diets (D2 and D3) were prepared by supplementing a basal diet (D1) with MCFAs in the form of a sodium salt of coconut fatty acid distillate (DICOSAN; Norel, Madrid, Spain), rich on C-12, added at 0.3% (D2) or with the probiotic CECT 5940, added at 0.1% (D3). The study integrated data on growth performance, blood biochemistry, histology and intestinal gene expression patterns of selected markers of intestinal function and architecture.

Results: MCFAs in the form of a coconut oil increased feed intake, growth rates and the surface of nutrient absorption, promoting the anabolic action of the somatotropic axis. The probiotic (D3) induced anti-inflammatory and anti-oxidant effects with changes in circulating cortisol, immunoglobulin M, leukocyte respiratory burst, and mucosal expression levels of cytokines, lymphocyte markers and immunoglobulin T.

Discussion: MCFA supplementation showed positive effects on GSB growth and intestinal architecture acting mainly in the anterior intestine, where absorption takes place. The probiotic CECT 5940 exhibited key effects in the regulation of the immune status inducing anti-inflammatory and anti-oxidant effects which can be potentially advantageous upon infection or exposure to other stressors. The potential effects of these feed additives in GSB are very promising to improve health and disease resistance in aquaculture.
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http://dx.doi.org/10.7717/peerj.4001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719961PMC
December 2017

Skin Mucus of Gilthead Sea Bream ( L.). Protein Mapping and Regulation in Chronically Stressed Fish.

Front Physiol 2017 1;8:34. Epub 2017 Feb 1.

Fish Pathology Group Group, Biology, Culture and Pathology of Marine Species, Institute of Aquaculture Torre de la Sal (IATS-CSIC) Castellón, Spain.

The skin mucus of gilthead sea bream was mapped by one-dimensional gel electrophoresis followed by liquid chromatography coupled to high resolution mass spectrometry using a quadrupole time-of-flight mass analyzer. More than 2,000 proteins were identified with a protein score filter of 30. The identified proteins were represented in 418 canonical pathways of the Ingenuity Pathway software. After filtering by canonical pathway overlapping, the retained proteins were clustered in three groups. The mitochondrial cluster contained 59 proteins related to oxidative phosphorylation and mitochondrial dysfunction. The second cluster contained 79 proteins related to antigen presentation and protein ubiquitination pathways. The third cluster contained 257 proteins where proteins related to protein synthesis, cellular assembly, and epithelial integrity were over-represented. The latter group also included acute phase response signaling. In parallel, two-dimensional gel electrophoresis methodology identified six proteins spots of different protein abundance when comparing unstressed fish with chronically stressed fish in an experimental model that mimicked daily farming activities. The major changes were associated with a higher abundance of cytokeratin 8 in the skin mucus proteome of stressed fish, which was confirmed by immunoblotting. Thus, the increased abundance of markers of skin epithelial turnover results in a promising indicator of chronic stress in fish.
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http://dx.doi.org/10.3389/fphys.2017.00034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288811PMC
February 2017

Differential Modulation of IgT and IgM upon Parasitic, Bacterial, Viral, and Dietary Challenges in a Perciform Fish.

Front Immunol 2016 27;7:637. Epub 2016 Dec 27.

Instituto de Acuicultura Torre de la Sal, Consejo Superior de Investigaciones Científicas (IATS-CSIC) , Castellón , Spain.

Three different immunoglobulin (Ig) isotypes can be found in teleost fish, IgM, IgD, and the teleost-specific IgT. IgM is considered to have a systemic activity, and IgT is attributed a mucosal role, similar to mammalian IgA. In this study, the complete sequence of gilthead sea bream IgM and IgT in their membrane (m) and soluble (s) forms are described for the first time in a perciform fish. Their constitutive gene expression is analyzed in different tissues, and their regulation upon viral, bacterial, parasitic, mucosal vaccination and dietary challenges are studied. GCB IgM and IgT have the prototypical structure when compared to other fish Igs. The constitutive expression of was the highest overall in all tissues, whereas expression was highest in mucosal tissues, such as gills and intestine. and were differentially regulated upon infection. was highly upregulated locally upon infection with the intestinal parasite or systemically after Nodavirus infection. Long-term intestinal parasitic infections increased the serum titer of both isotypes. Mucosal vaccination against subsp. finely regulated the Ig response inducing a systemic increase of IgM titers in serum and a local IgT response in skin mucus when animals were exposed to the pathogen by bath challenge. Interestingly, plant-based diets inhibit IgT upregulation upon intestinal parasitic challenge, which was related to a worse disease outcome. All these results corroborate the mucosal role of IgT and emphasize the importance of a finely tuned regulation of Ig isotypes upon infection, which could be of special interest in vaccination studies.
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http://dx.doi.org/10.3389/fimmu.2016.00637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5186763PMC
December 2016

Dietary Butyrate Helps to Restore the Intestinal Status of a Marine Teleost (Sparus aurata) Fed Extreme Diets Low in Fish Meal and Fish Oil.

PLoS One 2016 29;11(11):e0166564. Epub 2016 Nov 29.

Nutrigenomics and Fish Endocrinology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, Spain.

There is a constant need to find feed additives that improve health and nutrition of farmed fish and lessen the intestinal inflammation induced by plant-based ingredients. The objective of this study was to evaluate the effects of adding an organic acid salt to alleviate some of the detrimental effects of extreme plant-ingredient substitution of fish meal (FM) and fish oil (FO) in gilthead sea bream diet. Three experiments were conducted. In a first trial (T1), the best dose (0.4%) of sodium butyrate (BP-70 ®NOREL) was chosen after a short (9-weeks) feeding period. In a second longer trial (T2) (8 months), four diets were used: a control diet containing 25% FM (T2-D1) and three experimental diets containing 5% FM (T2-D2, T2-D3, T2-D4). FO was the only added oil in D1, while a blend of plant oils replaced 58% and 84% of FO in T2-D2, and T2-D3 and T2-D4, respectively. The latter was supplemented with 0.4% BP-70. In a third trial (T3), two groups of fish were fed for 12 and 38 months with D1, D3 and D4 diets of T2. The effects of dietary changes were studied using histochemical, immunohistochemical, molecular and electrophysiological tools. The extreme diet (T2-D3) modified significantly the transcriptomic profile, especially at the anterior intestine, up-regulating the expression of inflammatory markers, in coincidence with a higher presence of granulocytes and lymphocytes in the submucosa, and changing genes involved in antioxidant defences, epithelial permeability and mucus production. Trans-epithelial electrical resistance (Rt) was also decreased (T3-D3). Most of these modifications were returned to control values with the addition of BP-70. None of the experimental diets modified the staining pattern of PCNA, FABP2 or ALPI. These results further confirm the potential of this additive to improve or reverse the detrimental effects of extreme fish diet formulations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0166564PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5127657PMC
June 2017

Gene Expression Profiling Reveals Functional Specialization along the Intestinal Tract of a Carnivorous Teleostean Fish (Dicentrarchus labrax).

Front Physiol 2016 25;7:359. Epub 2016 Aug 25.

Nutrigenomics and Fish Growth Endocrinology Group, Biology, Culture and Pathology of Marine Species, Institute of Aquaculture Torre de la Sal (IATS-CSIC) Castellón, Spain.

High-quality sequencing reads from the intestine of European sea bass were assembled, annotated by similarity against protein reference databases and combined with nucleotide sequences from public and private databases. After redundancy filtering, 24,906 non-redundant annotated sequences encoding 15,367 different gene descriptions were obtained. These annotated sequences were used to design a custom, high-density oligo-microarray (8 × 15 K) for the transcriptomic profiling of anterior (AI), middle (MI), and posterior (PI) intestinal segments. Similar molecular signatures were found for AI and MI segments, which were combined in a single group (AI-MI) whereas the PI outstood separately, with more than 1900 differentially expressed genes with a fold-change cutoff of 2. Functional analysis revealed that molecular and cellular functions related to feed digestion and nutrient absorption and transport were over-represented in AI-MI segments. By contrast, the initiation and establishment of immune defense mechanisms became especially relevant in PI, although the microarray expression profiling validated by qPCR indicated that these functional changes are gradual from anterior to posterior intestinal segments. This functional divergence occurred in association with spatial transcriptional changes in nutrient transporters and the mucosal chemosensing system via G protein-coupled receptors. These findings contribute to identify key indicators of gut functions and to compare different fish feeding strategies and immune defense mechanisms acquired along the evolution of teleosts.
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http://dx.doi.org/10.3389/fphys.2016.00359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997091PMC
September 2016

RNA-seq analysis of early enteromyxosis in turbot (Scophthalmus maximus): new insights into parasite invasion and immune evasion strategies.

Int J Parasitol 2016 07 21;46(8):507-17. Epub 2016 Apr 21.

Departamento de Genética, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, Spain.

Enteromyxum scophthalmi, an intestinal myxozoan parasite, is the causative agent of a threatening disease for turbot (Scophthalmus maximus, L.) aquaculture. The colonisation of the digestive tract by this parasite leads to a cachectic syndrome associated with high morbidity and mortality rates. This myxosporidiosis has a long pre-patent period and the first detectable clinical and histopathological changes are subtle. The pathogenic mechanisms acting in the early stages of infection are still far from being fully understood. Further information on the host-parasite interaction is needed to assist in finding efficient preventive and therapeutic measures. Here, a RNA-seq-based transcriptome analysis of head kidney, spleen and pyloric caeca from experimentally-infected and control turbot was performed. Only infected fish with early signs of infection, determined by histopathology and immunohistochemical detection of E. scophthalmi, were selected. The RNA-seq analysis revealed, as expected, less intense transcriptomic changes than those previously found during later stages of the disease. Several genes involved in IFN-related pathways were up-regulated in the three organs, suggesting that the IFN-mediated immune response plays a main role in this phase of the disease. Interestingly, an opposite expression pattern had been found in a previous study on severely infected turbot. In addition, possible strategies for immune system evasion were suggested by the down-regulation of different genes encoding complement components and acute phase proteins. At the site of infection (pyloric caeca), modulation of genes related to different structural proteins was detected and the expression profile indicated the inhibition of cell proliferation and differentiation. These transcriptomic changes provide indications regarding the mechanisms of parasite attachment to and invasion of the host. The current results contribute to a better knowledge of the events that characterise the early stages of turbot enteromyxosis and provide valuable information to identify molecular markers for early detection and control of this important parasitosis.
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http://dx.doi.org/10.1016/j.ijpara.2016.03.007DOI Listing
July 2016

Immunity to gastrointestinal microparasites of fish.

Dev Comp Immunol 2016 11 29;64:187-201. Epub 2016 Jan 29.

Nutrigenomics and Fish Growth Endocrinology Group, Institute of Aquaculture Torre de la Sal (IATS-CSIC), Consejo Superior de Investigaciones Científicas, Castellón, Spain.

Fish intestinal parasites cause direct mortalities and also morbidity, poor growth, higher susceptibility to opportunistic pathogens and lower resistance to stress. This review is focused on microscopic parasites (Protozoa and Metazoa) that invade the gastrointestinal tract of fish. Intracellular parasites (mainly Microsporidia and Apicomplexa) evoke almost no host immune reaction while they are concealed in the cytoplasmic and nuclear compartments, and can even use fish cells (macrophages) as Trojan horses to spread in the host. Inflammatory reaction only appears when the parasite bursts infected cells. Immunity against extracellular parasites is depicted for the myxozoans Ceratonova shasta and Enteromyxum spp. The cellular and humoral innate responses and the production of antibodies are crucial for resolving some of these myxozoonoses, but an excessive inflammatory reaction (concerted by cytokines) can become a fatal pathophysiological consequence. The local immune response plays a key role, with numerous genes more strongly regulated in the intestine than at lymphohaematopoietic organs.
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http://dx.doi.org/10.1016/j.dci.2016.01.014DOI Listing
November 2016

European Sea Bass (Dicentrarchus labrax) Immune Status and Disease Resistance Are Impaired by Arginine Dietary Supplementation.

PLoS One 2015 8;10(10):e0139967. Epub 2015 Oct 8.

Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Universidade do Porto, Rua dos Bragas 289, 4050-123, Porto, Portugal.

Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139967PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598043PMC
June 2016

Immunohistochemical detection and gene expression of TNFα in turbot (Scophthalmus maximus) enteromyxosis.

Fish Shellfish Immunol 2015 Nov 16;47(1):368-76. Epub 2015 Sep 16.

Departamento de Ciencias Clínicas Veterinarias, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002, Lugo, Spain. Electronic address:

Enteromyxum scophthalmi (Myxozoa) constitutes one of the most devastating pathogens for turbot (Scophthalmus maximus, L.) aquaculture. This parasite causes a severe intestinal parasitosis that leads to a cachectic syndrome with high morbidity and mortality rates for which no therapeutic options are available. Presence of inflammatory infiltrates, increased apoptotic rates and epithelial detaching have been described at intestinal level, as well as leukocyte depletion in lymphohaematopoietic organs. Previous investigations on enteromyxosis in turbot showed the high susceptibility of this species to the parasite and reported the existence of a dysregulated immune response against the parasite. The pleiotropic cytokine tumour necrosis factor alpha (TNFα) plays a major role in immune response and is involved in a wide range of biological activities. In teleost, the gene expression of this cytokine has been found regulated under several pathological conditions. Teleost TNFα shows some analogous functions with its mammalian counterparts, but the extent of its activities is still poorly understood. Cytokines are generally considered as a double-edge sword and TNFα has been implicated in the pathogenesis of different inflammatory diseases as well as in wasting syndromes described in mammals. The aim of this work was to analyse the expression of TNFα during enteromyxosis with molecular (Q-PCR) and morphological (immunohistochemistry) tools. Kidney, spleen and pyloric caeca from turbot with moderate and severe infections were analysed and compared to healthy naïve fish. TNFα expression was increased in both spleen and kidney in the earlier stages of the disease, whereas in severely infected fish, the expression decreased, especially in kidney. At the intestinal level, an increase in the number of TNFα-positive cells was noticed, which was proportional to the infiltration of inflammatory cells. The results demonstrate the involvement of TNFα in the immune response to E. scophthalmi in turbot, which could be related to the development of the clinic signs and lesions.
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http://dx.doi.org/10.1016/j.fsi.2015.09.032DOI Listing
November 2015

Phylogenomics Reveals Convergent Evolution of Lifestyles in Close Relatives of Animals and Fungi.

Curr Biol 2015 Sep 10;25(18):2404-10. Epub 2015 Sep 10.

Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Passeig Marítim de la Barceloneta 37-49, Barcelona 08003, Catalonia, Spain; Departament de Genètica, Universitat de Barcelona, Avinguda Diagonal 645, Barcelona 08028, Catalonia, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys 23, Barcelona 08010, Catalonia, Spain. Electronic address:

The Opisthokonta are a eukaryotic supergroup divided in two main lineages: animals and related protistan taxa, and fungi and their allies [1, 2]. There is a great diversity of lifestyles and morphologies among unicellular opisthokonts, from free-living phagotrophic flagellated bacterivores and filopodiated amoebas to cell-walled osmotrophic parasites and saprotrophs. However, these characteristics do not group into monophyletic assemblages, suggesting rampant convergent evolution within Opisthokonta. To test this hypothesis, we assembled a new phylogenomic dataset via sequencing 12 new strains of protists. Phylogenetic relationships among opisthokonts revealed independent origins of filopodiated amoebas in two lineages, one related to fungi and the other to animals. Moreover, we observed that specialized osmotrophic lifestyles evolved independently in fungi and protistan relatives of animals, indicating convergent evolution. We therefore analyzed the evolution of two key fungal characters in Opisthokonta, the flagellum and chitin synthases. Comparative analyses of the flagellar toolkit showed a previously unnoticed flagellar apparatus in two close relatives of animals, the filasterean Ministeria vibrans and Corallochytrium limacisporum. This implies that at least four different opisthokont lineages secondarily underwent flagellar simplification. Analysis of the evolutionary history of chitin synthases revealed significant expansions in both animals and fungi, and also in the Ichthyosporea and C. limacisporum, a group of cell-walled animal relatives. This indicates that the last opisthokont common ancestor had a complex toolkit of chitin synthases that was differentially retained in extant lineages. Thus, our data provide evidence for convergent evolution of specialized lifestyles in close relatives of animals and fungi from a generalist ancestor.
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http://dx.doi.org/10.1016/j.cub.2015.07.053DOI Listing
September 2015

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

Br J Nutr 2015 Sep 29;114(5):713-26. Epub 2015 Jul 29.

1Nutrigenomics and Fish Growth Endocrinology Group,Instituto de Acuicultura Torre de la Sal,IATS-CSIC,12595 Castellón,Spain.

Seven isoproteic and isolipidic semi-purified diets were formulated to assess specific nutrient deficiencies in sulphur amino acids (SAA), n-3 long-chain PUFA (n-3 LC-PUFA), phospholipids (PL), P, minerals (Min) and vitamins (Vit). The control diet (CTRL) contained these essential nutrients in adequate amounts. Each diet was allocated to triplicate groups of juvenile gilthead sea bream fed to satiety over an 11-week feeding trial period. Weight gain of n-3 LC-PUFA, P-Vit and PL-Min-SAA groups was 50, 60-75 and 80-85 % of the CTRL group, respectively. Fat retention was decreased by all nutrient deficiencies except by the Min diet. Strong effects on N retention were found in n-3 LC-PUFA and P fish. Combined anaemia and increased blood respiratory burst were observed in n-3 LC-PUFA fish. Hypoproteinaemia was found in SAA, n-3 LC-PUFA, PL and Vit fish. Derangements of lipid metabolism were also a common disorder, but the lipodystrophic phenotype of P fish was different from that of other groups. Changes in plasma levels of electrolytes (Ca, phosphate), metabolites (creatinine, choline) and enzyme activities (alkaline phosphatase) were related to specific nutrient deficiencies in PL, P, Min or Vit fish, whereas changes in circulating levels of growth hormone and insulin-like growth factor I primarily reflected the intensity of the nutritional stressor. Histopathological scoring of the liver and intestine segments showed specific nutrient-mediated changes in lipid cell vacuolisation, inflammation of intestinal submucosa, as well as the distribution and number of intestinal goblet and rodlet cells. These results contribute to define the normal range of variation for selected biometric, biochemical, haematological and histochemical markers.
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http://dx.doi.org/10.1017/S0007114515002354DOI Listing
September 2015

Effects of dietary NEXT ENHANCE®150 on growth performance and expression of immune and intestinal integrity related genes in gilthead sea bream (Sparus aurata L.).

Fish Shellfish Immunol 2015 May 11;44(1):117-28. Epub 2015 Feb 11.

Fish Pathology Group, Instituto de Acuicultura Torre de la Sal (IATS-CSIC), Castellón, Spain. Electronic address:

Gilthead sea bream juveniles were fed different doses (0, 50, 100, 200, 300 ppm) of NEXT ENHANCE®150 (NE) for 9 weeks. Feed gain ratio (FGR) was improved by a 10% with all the doses, but feed intake decreased in a dose dependent manner. The optimum inclusion level to achieve maximum growth was set at 100 ppm. The hepatosomatic index did not vary and only at the highest dose, viscerosomatic and splenosomatic indexes were significantly decreased. No significant changes were found in haematological parameters, plasma biochemistry, total antioxidant capacity and respiratory burst. In a second trial, NE was given at 100 ppm alone (D1) or in combination with the prebiotic PREVIDA® (0.5%) (PRE) (D2) for 17 weeks. There were no differences in the growth rates, and FGR was equally improved for D1 and D2. No significant changes in haematology and plasma antioxidant capacity were detected. The histological examination of the liver and the intestine showed no outstanding differences in the liver, but the number of mucosal foldings appeared to be higher in D1 and D2 vs CTRL diet and the density of enterocytes and goblet cells also appeared higher, particularly in the anterior intestine. A 87-gene PCR-array was constructed based on our transcriptomic database (www.nutrigroup-iats.org/seabreamdb) and applied to samples of anterior (AI) and posterior (PI) intestine. It included 54 new gene sequences and other sequences as markers of cell differentiation and proliferation, intestinal architecture and permeability, enterocyte mass and epithelial damage, interleukins and cytokines, pattern recognition receptors (PRR), and mitochondrial function and biogenesis. More than half of the studied genes had significantly different expression between AI and PI segments. The functional significance of this differential tissue expression is discussed. The experimental diets induced significant changes in the expression of 26 genes. The intensity of these changes and the number of genes that were significantly regulated were higher at PI than at AI. At PI, both diets invoked a clear down-regulation of genes involved in cell differentiation and proliferation, some involved in cell to cell communication, cytokines and several PRR. By contrast, up-regulation was mostly found for genes related to enterocyte mass, cell epithelial damage and mitochondrial activity at AI. The changes were of the same order for D1 and D2, except for fatty acid-binding proteins 2 and 6 and the PRR fucolectin, which were higher in D2 and D1 fed fish, respectively. Thus, NE alone or in combination with PRE seems to induce an anti-inflammatory and anti-proliferative transcriptomic profile with probable improvement in the absorptive capacity of the intestine that would explain the improved FGR.
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http://dx.doi.org/10.1016/j.fsi.2015.01.039DOI Listing
May 2015

RNA-seq analysis reveals significant transcriptome changes in turbot (Scophthalmus maximus) suffering severe enteromyxosis.

BMC Genomics 2014 Dec 19;15:1149. Epub 2014 Dec 19.

Departamento de Ciencias Clínicas Veterinarias, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo 27002, Spain.

Background: Enteromyxosis caused by the intestinal myxozoan parasite Enteromyxum scophthalmi is a serious threat for turbot (Scophthalmus maximus, L.) aquaculture, causing severe catarrhal enteritis leading to a cachectic syndrome, with no therapeutic options available. There are still many aspects of host-parasite interaction and disease pathogenesis that are yet to be elucidated, and to date, no analysis of the transcriptomic changes induced by E. scophthalmi in turbot organs has been conducted. In this study, RNA-seq technology was applied to head kidney, spleen and pyloric caeca of severely infected turbot with the aim of furthering our understanding of the pathogenetic mechanisms and turbot immune response against enteromyxosis.

Results: A huge amount of information was generated with more than 23,000 identified genes in the three organs, amongst which 4,762 were differently expressed (DE) between infected and control fish. Associate gene functions were studied based on gene ontology terms and available literature, and the most interesting DE genes were classified into five categories: 1) immune and defence response; 2) apoptosis and cell proliferation; 3) iron metabolism and erythropoiesis; 4) cytoskeleton and extracellular matrix and 5) metabolism and digestive function. The analysis of down-regulated genes of the first category revealed evidences of a connexion failure between innate and adaptive immune response, especially represented by a high number of DE interferon-related genes in the three organs. Furthermore, we found an intense activation of local immune response at intestinal level that appeared exacerbated, whereas in kidney and spleen genes involved in adaptive immune response were mainly down-regulated. The apoptotic machinery was only clearly activated in pyloric caeca, while kidney and spleen showed a marked depression of genes related to erythropoiesis, probably related to disorders in iron homeostasis. The genetic signature of the causes and consequences of cachexia was also demonstrated by the down-regulation of the genes encoding structural proteins and those involved in the digestive metabolism.

Conclusions: This transcriptomic study has enabled us to gain a better understanding of the pathogenesis of enteromyxosis and identify a large number of DE target genes that bring us closer to the development of strategies designed to effectively combat this pathogen.
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http://dx.doi.org/10.1186/1471-2164-15-1149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320470PMC
December 2014