Enteromyxum leei (Myxozoa: Myxosporea) infection in gilthead sea bream (Sparus aurata): Immune-related studies

La presente tesis doctoral estudia diversos aspectos de la enteromixosis de la dorada, relacionados principalmente con la transmisión y la caracterización del parásito, con los factores que afectan a su establecimiento en el hospedador y a la interacción parásito-hospedador, y con la respuesta inmunitaria inducida por el parásito. En primer lugar, se desarrolló un nuevo método de transmisión de E. leei, que mejora los resultados obtenidos por los hasta ahora disponibles. El parásito se transmitió con éxito a doradas mediante intubación por vía anal de raspados intestinales de peces infectados. Con este método, se consiguió una infección más rápida y extensa que por efluente y por cohabitación, y más eficaz, y uniforme y repetitiva que por vía oral. En términos de intensidad de infección y de maduración del parásito, la progresión de la infección siguió un gradiente posterior-anterior en el intestino. El daño histopatológico causado por el parásito por esta vía fue similar a la de otras formas de infección, con la consabida alteración de la integridad de la mucosa intestinal junto con descamación epitelial. Así mismo, la reacción inflamatoria local del hospedador consistió en la infiltración de linfocitos y granulocitos eosinófilos, todo ello en tiempos más tempranos que los registrados mediante las otras vías de infección. Por lo tanto, la prevalencia y la intensidad de la infección obtenidas en dorada por vía anal son comparables a los niveles de infección de E. leei alcanzados en el sargo picudo Diplodus puntazzo, especie muy susceptible a E. leei, y a los niveles de infección de Enteromyxum scophthalmi, cuya patogenicidad en rodaballo, Psetta maxima, es mayor. La intubación anal de la dorada es, por tanto, un método rápido y eficaz para la transmisión experimental de E. leei, que aporta simultáneamente para cada individuo una dosis infectiva uniforme aplicada directamente en el principal tejido diana del parásito. En referencia a los factores que afectan a la transmisión de E. leei, se demostró inequívocamente que la temperatura del agua juega un papel importante, constatándose una evidente relación entre temperatura y prevalencia. La prevalencia de infección en las infecciones experimentales disminuyó gradualmente de las temperaturas registradas en periodos estivales (22-25 °C), a las temperaturas de otoño (19-22 °C) y a una temperatura constante de 18 °C. Se confirmó el efecto inhibidor de las bajas temperaturas sobre el desarrollo de la enteromixosis, ya que a las bajas temperaturas invernales (11-12 °C) los peces no se infectaron, pero la infección reapareció al aumentar la temperatura de nuevo en primavera. Esta reaparición de E. leei indica que el parásito es capaz de permanecer latente e indetectable durante los períodos más fríos, convirtiéndose los animales clasificados como falsos negativos en reservorios del parásito, con las consiguientes implicaciones epidemiológicas. Con el fin de desarrollar inmunoensayos que permitan la detección del parásito, su localización y la caracterización funcional de sus antígenos, se obtuvieron anticuerpos policlonales de conejo contra E. leei (aPab-Eleei) y E. scophthalmi (aPab-Escoph). Se caracterizó su sensibilidad y especificidad mediante las técnicas de ELISA e inmunohistoquímica. Tras su adsorción con raspados intestinales no infectados del hospedador correspondiente, los anticuerpos mostraron una alta especificidad de unión a los estadios proliferativos del respectivo parásito (células primarias y secundarias) y a los estadios esporogónicos correspondientes (esporoblastos, valvas de las esporas). El título de los anticuerpos fue diferente en ELISA (aPab-Eleei 1:32.000; aPab-Escoph 1:16.000) que en inmunohistoquímica (aPab-Eleei 1:8.000; aPab-Escoph 1:16.000). El immunógeno de E. leei inyectado a los conejos tenía más diversidad de estadios parasitariosy probablemente por ello aPab-Eleei presentó reacción cruzada con otros mixosporidios (Sphaerospora dicentrarchi y S. testicularis), mientras que aPab-Escoph no detectó ninguna otra especie de mixozoo. La reactividad cruzada entre mixozoos se atribuye a la existencia de epítopos antigénicos compartidos, especialmente residuos de carbohidratos. Ambos anticuerpos permiten la detección de Enteromyxum spp. en cualquier fase de su ciclo vital. Para la caracterización antigénica del parásito, se obtuvieron extractos parcialmente purificados de E. leei, que contenían grandes cantidades de esporas además de algunos esporoblastos diespóricos. Los extractos de parásito se separaron mediante SDS-PAGE y se compararon con extractos intestinales sanos. El perfil proteico de E. leei en geles teñidos con azul Coomassie brillante consistió en seis bandas antigénicas reducidas y desnaturalizadas con un peso molecular entre 10 y 49 kDa. En Western blots, aPab-Eleei y el anticuerpo policlonal heterólogo dirigido contra el filamento polar de Myxobolus pendula (PabMPPF) detectaron epítopos proteicos (de 15 a 165 kDa con aPab-Eleei; de 15 a >209 kDa con PabMPPF) en cinco bandas antigénicas y en una amplia zona inmunorreactiva. Estos antígenos se compararon con las bandas obtenidas al aplicar lectinas en blots, resultando que ambos anticuerpos detectaron bandas glicoprotéicas de 15 kDa y de 165 kDa, lo que sugiere que podría tratarse de antígenos comunes entre mixozoos. Estas dos glicoproteínas tenían residuos glicosídicos de glucosa y de manosa, un rasgo común entre los mixozoos. El antígeno de 165 kDa también presentó residuos de galactosa, de N-acetil-glucosamina y de N-acetil-galactosamina, lo que apunta a un posible origen en las valvas de quitina de las esporas. El antígeno glicoprotéico de 15 kDa podría corresponder por su peso molecular con el homólogo de minicolágeno encontrado en otros mixozoos. También se detectó un antígeno glicoprotéico de 34 kDa mediante aPab-Eleei (positivo para residuos glicosídicos de glucosa y manosa) que podría corresponder con otro antígeno común de E. leei y M. pendula. En las zimografías realizadas con los extractos de E. leei, se detectaron varias proteasas gelatinolíticas funcionales (entre 43 y 245 kDa), que podrían tener un papel importante en la patogénesis del parásito. Se estudió el efecto de algunos factores nutricionales sobre la enteromixosis de la dorada. Para ello, se alimentaron juveniles de dorada durante 9 meses con una dieta a base de proteína vegetal que contenía una mezcla de aceites vegetales como principal fuente de lípidos (al 66% de sustitución) (dieta 66VO), en oposición a doradas alimentadas con una dieta en la que la fuente de lípidos era exclusivamente de aceite de pescado (FO). Posteriormente ambos grupos se expusieron a E. leei mediante efluente. Las doradas 66VO (vs las FO) presentaron una mayor progresión de la enteromixosis (mayor prevalencia e intensidad de infección, invasión más extensa y más rápida de los distintos tramos intestinales) y signos de la enfermedad más severos (menor crecimiento, factor de condición, la tasa específica de crecimiento y hematocrito). La pérdida de masa corporal en los peces receptores se debió principalmente a la anorexia y probablemente se agravó por la deficiente absorción de nutrientes debido al daño intestinal ejercido por el parásito, la insuficiencia osmorreguladora y el coste metabólico de la respuesta inmune del propio hospedador. Los antecedentes nutricionales por sí mismos no produjeron ningún efecto perjudicial sobre los parámetros biométricos y hematológicos de las doradas en el grupo control, no expuesto 66VO. Sin embargo, este grupo presentó valores significativamente más bajos de óxido nítrico y lisozima séricos, mientras que la vía alternativa del complemento sérico (ACH50) aumentó significativamente. En cualquier caso, la intensidad de la infección se correlacionó negativamente con los parámetros de crecimiento y de hematocrito para los peces receptores de ambas dietas. Por lo tanto, la sustitución 66VO en la alimentación de la dorada puede constituir un factor que predispone para la enteromixosis. Este posible efecto de la dieta se podría deducir también de la significativa disminución de las células goblet positivas para mucinas neutras y ácidas en las secciones del intestino anterior y medio, y de las positivas para mucinas carboxílicas y para ácido siálico en el intestino medio de las doradas control 66VO, ya que estas secciones intestinales presentaron una mayor prevalencia de infección en el grupo de peces receptores de esta dieta (en comparación con el grupo FO). Este menor contenido de tales tipos de mucinas, podría provocar algún tipo de deficiencia en la barrera mucosa protectora de los peces 66VO. Sin embargo, la enteromixosis conllevó en las dos dietas una disminución significativa de la mayoría de tipos de células goblets, es decir, neutras, ácidas, carboxílicas y con ácido siálico, y su disminución fue más intensa en el intestino posterior (vs anterior y medio) y en peces infectados tempranamente, que albergaron el parásito durante más tiempo (vs tardíamente infectados). Las células goblets en las zonas intestinales infectadas no sólo fueron menos numerosas, sino también de menor tamaño. Este fenotipo de reducción de células goblet en doradas infectadas por E. leei puede estar relacionado con el daño histopatológico directamente causado por la invasión epitelial del parásito, aunque no se puede descartar la implicación de cierta inmunomodulación inducida por el parásito o mediada por el propio hospedador. Los cambios observados en el fenotipo de células goblet nos llevaron a estudiar en mayor profundidad la composición del mucus intestinal en doradas parasitadas. Las mucinas intestinales secretadas, aisladas de doradas infectadas por E. leei, tuvieron un menor contenido glicoproteíco y un menor grado de glicosilación en las secciones intestinales anterior y media, y mayores en el intestino posterior, en comparación con la secreción mucosa de doradas sanas. Independientemente del tramo intestinal y del estado parasitario de los peces, estas mucinas presentaron dos rangos distintos de tamaño: las de tamaño mayor (> 2.000 kDa), con mayor contenido de glicoproteíco y grado de glicosilación, se consideraron las mucinas maduras y las que de tamaño menor (69-670 kDa) se consideraron mucinas inmaduras. La variación de la glicosilación terminal de las mucinas intestinales secretadas apuntó a una secreción de mucinas inmaduras en el intestino posterior de los peces parasitados debido al aumento de residuos de N-acetil-galactosamina y a la reducción de fucosa y ácido neuramínico. Estos monosacáridos terminales aparentemente participan en el reconocimiento y la unión de microorganismos al observarse una reducción de las adhesión Aeromonas hydrophila y Vibrio alginolyticus a las mucinas de mayor tamaño en doradas infectadas, comparadas con las no expuestas, independientemente del tramo intestinal. Por lo tanto, se demostró la modulación bioquímica de la secreción de mucosa intestinal en dorada en respuesta a la enteromixosis, probablemente con consecuencias sobre la interacción con microorganismos tanto patógenos como comensales. Se analizó a nivel local y sistémico la respuesta inmunitaria inducida por E. leei en la dorada y el efecto de la dieta 66VO. Se detectó un aumento significativo pero tardío de la expresión génica de la IgM en el intestino posterior de los peces infectados crónicamente por efluente, (vs peces control), que se correlacionó con el aumento de células IgM inmunorreactivas, principalmente plasmacitos y linfocitos B. Esta respuesta local frente al parásito se magnificó en los peces alimentados con la dieta 66VO, lo que sugiere un efecto pro-inflamatorio a nivel local, mientras que no se detectó ningún efecto de la dieta sobre el perfil de IgM en las doradas control, sin exponer al parásito. Con respecto a los tejidos linfohematopoyéticos, únicamente se observó un aumento de plasmacitos y linfocitos B en el riñón anterior de las doradas alimentadas con la dieta FO e infectadas tempranamente (que llevaban más tiempo infectadas). Se observó una agregación de plasmacitos y linfocitos B alrededor de los centros melanomacrofágicos y de los vasos sanguíneos tanto del riñón anterior como del bazo, lo que sugiere el comienzo de una respuesta inmunitaria adaptativa, interconectando la inmunidad humoral sistémica con la respuesta local. El factor que resultó ser más determinante para el fenotipo de aumento de la IgM fue el tiempo de exposición al parásito (que a su vez determina el nivel de infección) y este fenotipo fue más pronunciado a nivel intestinal local en los peces alimentados con 66VO. Se analizó el efecto modulador ejercido por E. leei sobre otras poblaciones de leucocitos en doradas infectadas experimentalmente por vía anal tras tiempos de exposición más cortos (alimentadas únicamente con una dieta comercial FO). En los infiltrados inflamatorios intestinales y órganos linfohematopoyéticos (riñón anterior y bazo) de peces parasitados, el número de plasmacitos, linfocitos B y mastocitos fue significativamente mayor, mientras que los granulocitos acidófilos disminuyeron, en comparación con peces no parasitados y con no expuestos. Esta respuesta fue más intensa en el intestino posterior, no se detectó en el timo y, en su conjunto, apareció antes, tanto a nivel local como sistémico, que en las infecciones de larga duración por efluente. El reclutamiento desde los reservorios sistémicos al lugar de la infección pareció ocurrir a través de la circulación sanguínea. El aparente comienzo de una respuesta inmunitaria adaptativa estuvo asociado al mayor porcentaje de superficie esplénica ocupada por los centros melanomacrofágicos en los peces no parasitados, en comparación con los parasitados y con los no expuestos. Sorprendentemente, los mastocitos y granulocitos acidófilos, ambos células acidófilas/eosinófilas, presentaron patrones opuestos de respuesta a la enteromixosis y no se descarta un posible efecto inmunosupresor de E. leei sobre los granulocitos acidófilos, que se consideran el principal tipo de granulocito en la dorada. Además, la heterogeneidad de las células acidófilas/eosinófilas de la dorada se manifestó por las distintas morfologías celulares y densidades de gránulos observadas.The current thesis includes several aspects of enteromyxosis in gilthead sea bream, dealing mainly with the parasite transmission and characterization, with factors that affect parasite establishment and host-parasite interaction, and with the immune response elicited by the parasite. First of all, a new method of transmission of E. leei, that improved the previous available ones, was established. The parasite was successfully transmitted to gilthead sea bream by peranal intubation with intestinal scrapings of infected fish. By the anal route, the progression of the infection was faster than by effluent and cohabitation, and more effective and uniform than by the oral route. In terms of infection intensity and parasite maturation, the progression of the infection followed a posterior-anterior intestinal gradient. The histopathological damage caused by the parasite included the usual disruption of the mucosa integrity and epithelial desquamation and the inflammatory host reaction involved lymphocyte and eosinophilic granulocyte infiltration, all at earlier time points than by the other infection routes. By anal route, the achieved prevalence and intensity of infection were therefore comparable with the infection level of E. leei in the more susceptible species sharpsnout sea bream Diplodus puntazzo and of the more pathogenic Enteromyxum scophthalmi in turbot Psetta maxima. Peranal intubation of gilthead sea bream is a fast and effective method for the experimental transmission of E. leei, assuring for each individual simultaneously a uniform infective dose to the main target site for the parasite. In regard to the effectiveness of the parasite transmission, water temperature was shown to play an important role, having a clear relationship prevalence and water temperature during experimental infections. The inhibitory effect of the low temperatures on the development of enteromyxosis was confirmed since infection prevalence decreased gradually from high summer temperatures (22-25 °C), to autumn temperatures (19-22 °C) and under a constant temperature of 18 °C. Interestingly, under low winter temperature (11-12°C) no fish was infected, but infection appeared later on when water temperature increased in spring. This re-emergence of E. leei indicates that the parasite is capable of remaining latent and undetectable during cooler periods, thus false negatives become reservoirs for the parasite entailing epidemiological consequences. In order to develop immunoassays that allow the detection of the parasite, its localization and functional characterization of antigens, rabbit polyclonal antibodies were raised against E. leei (aPab-Eleei) and E. scophthalmi (aPab-Escoph). Their sensitivity and specificity were characterized by ELISA and immunohistochemistry. After adsorption with non-infected intestinal scrapings of the corresponding host, both had high specificity for the corresponding proliferative (primary and secondary cells) and sporogonic parasite stages (sporoblasts, spore valves) and presented different titres in ELISA (aPab-Eleei 1:32,000; aPab-Escoph 1:16,000) and immunohistochemistry (aPab-Eleei 1:8,000; aPab-Escoph 1:16,000). The presence of more diverse parasitic stages in E. leei immunogens injected to the rabbits, probably accounted for the cross-reactivity detected with aPab-Eleei, which immunolabeled also Sphaerospora dicentrarchi and S. testicularis, whereas aPab-Escoph did not cross-react with any of the additionally tested myxozoans. Cross-reactivity among myxozoans is attributed to the share of antigenic epitopes, particularly carbohydrate moieties. These antisera enable Enteromyxum spp. detection at any phase of their life cycle. For the antigenic characterization of the parasite, partially purified E. leei extracts were obtained, which contained large amounts of spores together with some disporous sporoblasts. Parasite extracts were separated by SDS-PAGE and compared with healthy intestinal extracts, leading to the visualization of the E. leei protein profile in Coomassie Brilliant blue stained gels (six reduced and denatured antigenic bands ranging from 10 to 49 kDa). In Western blots, the aPab-Eleei and the heterologous polyclonal antibody raised against the polar filament of Myxobolus pendula (PabMPPF) detected proteic epitopes (ranging from 15 to 165 kDa with aPab-Eleei; from 15 to >209 kDa with PabMPPF) on five E. leei antigenic bands and on an immunoreactive smear. These antigens were compared with results obtained in Lectin blots, and particularly, the 15 kDa and the 165 kDa glycoproteic bands were detected with both antibodies, suggesting shared antigens among myxozoans. In addition, both contained glucose and mannose moieties, a common trait of myxozoans. The 165 kDa parasite antigen also presented galactose, N-acetyl-galactosamine and N-acetyl-glucosamine residues pointing to its possible origin on chitin-built spore valves. The 15 kDa glycoproteic parasite antigen would match for its molecular weight with the minicollagen-homologue found in myxozoans. A 34 kDa glycoproteic antigen was also detected by aPab-Eleei (positive for glucose and mannose moieties), which might be a further common antigen between E. leei and M. pendula. In zymographies of E. leei extracts, several functional gelatinolytic proteases were detected (ranging between 43 and 245 kDa), which may have a potential role in the parasite’s pathogenesis. The effect of dietary factors on gilthead sea bream enteromyxosis was studied in fish fed during 9 months a plant protein-based diet containing a blend of vegetable oils as major lipid source (at 66% of replacement) (66VO diet) and then exposed to E. leei by effluent, and compared with gilthead sea bream fed a fish oil (FO) commercial diet. The 66VO (vs FO) fish presented higher progression of enteromyxosis (higher prevalence and intensity of infection, broader and faster intestinal invasion) and severer disease signs (growth, condition factor, specific growth rate and haematocrit were all lower). However, body mass loss in recipient fish was mainly due to anorexia and probably enhanced by impairment of nutrient absorption due to the parasite-derived intestinal damage, osmoregulatory failure and metabolic cost of the host’s immune response. The nutritional background by itself did not produce any detrimental effect on the fish biometric and haematological parameters since the 66VO control, unexposed group did not exhibit any harmful effect. However, this group presented significantly lower values of serum nitric oxide and lysozyme, whereas the alternative complement pathway of serum (ACH50) was significantly enhanced. In any case, for recipient fish of both diet groups, the intensity of infection was negatively correlated with growth parameters and haematocrit. Thus, the 66VO replacement in gilthead sea bream can be a predisposing factor for enteromyxosis. This was also suggested by the significant decrease of goblet cells with neutral and acidic mucins at the anterior and middle intestine sections, and of those with carboxylic mucins and sialic acid at the middle intestine in 66VO control fish, since these intestinal sections presented higher prevalence of infection in recipient fish of this diet group (compared to FO). The lower content of such mucin types could therefore be responsible for a deficient protective mucous barrier in 66VO fish. However, in FO fed fish, enteromyxosis accounted for a significant decrease of most goblet cell types, i.e. neutral, acidic, car

Dysregulation of B Cell Activity During Proliferative Kidney Disease in Rainbow Trout

This work was supported by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM) and the European Commission under the H2020 Programme (Grant H2020-634429 ParaFishControl). IE was recipient of APOSTD/2016/037 grant by the “Generalitat Valenciana” and YH was recipient of a PhD Studentship from the Ministry of Education, Republic of China (Taiwan). JWH was supported by BBSRC grant BB/K009125/1 and SNSF grant CRSII3_147649-1. PDR was funded by grant T1-BIO-1672 from the “Comunidad de Madrid”.Peer reviewedPublisher PD

Novel horizontal transmission route for Enteromyxum leei (Myxozoa) by anal intubation of gilthead sea bream Sparus aurata

11 p., il., bibliografíaThe aim of the present study was to determine whether Enteromyxum leei, one of the most threatening parasitic diseases in Mediterranean fish culture, could be transmitted by peranal intubation in gilthead sea bream Sparus aurata L. Fish were inoculated either orally or anally with intestinal scrapings of infected fish in 3 trials. Oral transmission failed, but the parasite was efficiently and quickly transmitted peranally. Prevalence of infection was 100% at 60 d post inoculation (p.i.) in Trial 1 under high summer temperature (22 to 25°C; fish weight = 187.1 g), and 85.7 % in just 15 d p.i. in Trial 3 using smaller fish (127.5 g) at autumn temperature (19 to 22°C). In Trial 2, prevalence reached 60% at 60 d p.i. in the group reared at constant temperature (18°C), whereas no fish was infected in the group that was kept at low winter temperature (11 to 12°C), although infection appeared (46.1 % at 216 d p.i.) when temperature increased in spring. The arrested development at low temperature has important epidemiological consequences, as fish giving false negative results in winter can act as reservoirs of the parasite. Histopathological examination showed a posterior-anterior intestinal gradient in the progression of the infection, in terms of both intensity and parasite maturation. Thus, peranal intubation provides a very uniform, reliable and faster mode of transmission of E. leei than the commonly used transmission methods (cohabitation, exposure to infected effluent and oral inoculation), which require long exposure times or give variable and unpredictable results.This work was funded by the Spanish Ministerio de Educación y Ciencia (MEC) (AGL2006-13158-C03-01) and Ministerio de Ciencia e Innovación (AGL2009-13282-C02-01). Additional funding was obtained from the ‘Generalitat Valenciana’ (research grant PROMETEO 2010/006). I.E. received a Spanish FPI-PhD fellowship from MEC.Peer reviewe

Modulation of the IgM gene expression and IgM immunoreactive cell distribution by the nutritional background in gilthead sea bream (Sparus aurata) challenged with Enteromyxum leei (Myxozoa)

The aim of the present work was to determine if a plant protein-based diet containing vegetable oils (VO) as the major lipid source could alter the distribution of IgM immunoreactive cells (IRCs) and the IgM expression pattern in the intestine and haematopoietic tissues of gilthead sea bream (GSB) (. Sparus aurata) challenged with the myxosporean Enteromyxum leei. In a first trial (T1), GSB fed for 9 months either a fish oil (FO) diet or a blend of VO at 66% of replacement (66VO diet) was challenged by exposure to parasite-contaminated water effluent. All fish were periodically and non-lethally sampled to know their infection status. After 102 days of exposure, samples of intestine and head kidney were obtained for IgM expression and immunohistochemical detection (IHC). Additional samples of spleen were taken for IHC. Fish were categorized as control (C, not exposed), and early (E), or late (L) infected. The 66VO diet had no effect on the number of IgM-IRCs in any of the tissues or on IgM expression in C fish, whereas the infection with E. leei had a strong effect on the intestine. A combined time-diet effect was also observed, since the highest expression and IRCs values were registered in the posterior intestine (Pi) of E-66VO fish. A positive correlation was found between IgM expression and the presence of IgM-IRCs in the Pi. The effect of the time of infection was studied more in detail in a second trial (T2) in which samples of Pi were taken at 0, 24, 51, 91 and 133 days after exposure to the parasite. A significant increase of the IgM expression was detected only in parasitized fish, and very late after exposure. These results show that the duration of the exposure to the parasite is the most determinant factor for the observed intestinal IgM increased phenotype which gets magnified by the feeding of a high VO-based diet. © 2012 Elsevier Ltd.This work was funded by EU through projects AQUAMAX (FOOD-CT-2006-16249; Sustainable Aquafeeds to Maximise the Health Benefits of Farmed Fish for Consumers) and ARRAINA (Advanced Research Initiatives for Nutrition & Aquaculture, FP7/2007/2013; grant agreement n° 288925), and by the Spanish Ministry of Science and Innovation (MICINN) through the project AGL2009-13282-C02-01. Additional funding was obtained from the “Generalitat Valenciana” (research grant PROMETEO 2010/006). I. E. received a Spanish PhD fellowship (FPI) from MICINN.Peer Reviewe

Assembled PTO based on an array of double-acting hydraulic cylinders for WECs: From Conceptual Design to an Adjusted Detailed Model

All Wave Energy Converters (WECs) based on wave activated bodies comprises a Power Take- Off (PTO) system among other subsystems like the reaction mechanism, the supervisor of the system and the electrical generator. One of the most applied PTO systems consists of oil high pressure hydraulic devices. These devices are able to apply high forces, to store large quantities of energy through accumulators and to provide smoother power output to the motor coupled to a generator. In these systems the poor efficiency and the oil leakages contaminating the environment are considered main drawbacks. Despite of this, they are widely used in several promising WECs with the aim of optimizing the harvested wave energy along the time. The initial challenge to absorb an oscillating movement of ±30º at 5rad/s as a maximum angular speed absorbing up to 16000Nm from a specific WEC, led to the development of a simplified hydraulic model before manufacturing a PTO prototype to be verified in a Test Bench (Figure 1). The experimental results of PTO under different conditions have been used to adjust a full detailed PTO Model using Mathworks® software platform. This work presents a patented oil high pressure hydraulic PTO prototype based on an array of four double-acting hydraulic cylinders. This prototype has been designed and completely modelled as a proof concept at 1:4 scale being able to apply a variable Coulomb type damping torque through the activation of each hydraulic cylinder independently and through the modification of geometrical parameters easily. The complete model of the PTO has been accurately tuned up through adjustment of model parameters using the results of the experimental tests. This will allow the study of control strategies to optimize the extracted wave energy from a specific WEC, like point-absorbers

Modeling Innovative Power Take-Off Based on Double-Acting Hydraulic Cylinders Array for Wave Energy Conversion

One of the key systems of a Wave Energy Converter for extraction of wave energy is the Power Take-Off (PTO) device. This device transforms the mechanical energy of a moving body into electrical energy. This paper describes the model of an innovative PTO based on independently activated double-acting hydraulic cylinders array. The model has been developed using a simulation tool, based on a port-based approach to model hydraulics systems. The components and subsystems used in the model have been parameterized as real components and their values experimentally obtained from an existing prototype. In fact, the model takes into account most of the hydraulic losses of each component. The simulations show the flexibility to apply different restraining torques to the input movement depending on the geometrical configuration and the hydraulic cylinders on duty, easily modified by a control law. The combination of these two actions allows suitable flexibility to adapt the device to different sea states whilst optimizing the energy extraction. The model has been validated using a real test bench showing good correlations between simulation and experimental testsSpanish Government PS-440000-2008-1, Regional Government of the Basque Country IT719-13, UPV/EHU UFI11/2

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.)

Gilthead sea bream juveniles were fed different doses (0, 50, 100, 200, 300ppm) 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 100ppm. 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 100ppm 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. © 2015 Elsevier Ltd.This work has been carried out with financial support from the Commission of the European Communities, specific RTD programme of Framework Programme 7, (FP7/2007-2013) under grant projects ARRAINA (KBBE-2011-288925) and AQUAEXCEL (262336) under TNA project 0019/02/04/14 to ANDROMEDA. It does not necessarily reflect the EU views and in no way anticipates the Commission's future policy in this area. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Additional funding has been received by Spanish MINECO project no. AGL2013-48560 and Generalitat Valenciana (PROMETEOII/2014/085 and ISIC/2012/003).Peer Reviewe

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

This is the peer reviewed version of the following article: Ronza, P, Estensoro, I, Bermúdez, R, et al. Effects of Enteromyxum spp. (Myxozoa) infection in the regulation of intestinal E‐cadherin: Turbot against gilthead sea bream. J Fish Dis. 2020; 43: 337– 346, which has been published in final form at https://doi.org/10.1111/jfd.13130. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsEnteromyxoses 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 pictureThis work has been funded by the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) through the projects AGL2015‐67039‐C3‐1‐R, AGL2015‐67039‐C3‐3‐R and AGL‐2013‐48560‐R and by the Horizon 2020 Framework Programme through ParaFishControl Project (634429). I.E. was contracted under APOSTD/2016/037 grant by the “Generalitat Valenciana” and G.P.‐C. under the “Juan de la Cierva” programme, granted by the Spanish Ministry of Science and Innovation (JCI‐2011‐09438)S

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

[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.This work has been carried out with financial support from the European Union under grant projects ARRAINA (FP7-KBBE-2011-288,925) to JPS, MP and VK and ParaFishControl (H2020-634429) to ASB. Additional funding has been received from Spanish Ministry of Economy and Competitiveness (MINECO) project no. AGL2013- 48560-R to JPS and ASB, and Generalitat Valenciana (PROMETEOII/2014/085) to ASB. MCP was contracted under CSIC PIE project no. 201740E013 and MINECO FPDI-2013-15741, and IE under APOSTD/2016/037 grant by the “Generalitat Valenciana”.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

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

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