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

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.This study was funded by the Spanish Ministry of Economy and Competitiveness (AGL 2009-13282-C02-01 and -02; AGL2015-67039-C3-1-R and AGL2015-67039-C3-3-R), the European Regional Development Fund (ERDF, European Union) and Xunta de Galicia (Spain) local government (GRC2014/010 and GPC2015/34). Diego Robledo was supported by a FPU fellowship funded by the Spanish Ministry of Education, Culture and Sport. Paolo Ronza was supported by a grant from the scientific network “INMUNOGENOM”, funded by Xunta de Galicia (REDES GI-1251).Peer Reviewe

The Teleost Thymus in Health and Disease: New Insights from Transcriptomic and Histopathological Analyses of Turbot, Scophthalmus maximus

The thymus is a primary lymphoid organ that plays a pivotal role in the adaptive immune system. The immunobiology of the thymus in fish is considered to be similar to that of mammals, but it is actually poorly characterized in several cultured teleost species. In particular, while investigations in human and veterinary medicine have highlighted that the thymus can be affected by different pathological conditions, little is known about its response during disease in fish. To better understand the role of the thymus under physiological and pathological conditions, we conducted a study in turbot (Scophthalmus maximus), a commercially valuable flatfish species, combining transcriptomic and histopathological analyses. The myxozoan parasite Enteromyxum scophthalmi, which represents a major challenge to turbot production, was used as a model of infection. The thymus tissues of healthy fish showed overrepresented functions related to its immunological role in T-cell development and maturation. Large differences were observed between the transcriptomes of control and severely infected fish. Evidence of inflammatory response, apoptosis modulation, and declined thymic function associated with loss of cellularity was revealed by both genomic and morphopathological analyses. This study presents the first description of the turbot thymus transcriptome and provides novel insights into the role of this organ in teleosts’ immune responsesThis research was funded by THE SPANISH MINISTRY OF ECONOMY, INDUSTRY AND COMPETITIVENESS AND THE EUROPEAN REGIONAL DEVELOPMENT FUND (ERDF) through the projects AGL2015–67039–C3–1–R and AGL2015–67039–C3–3–R. DR is supported by BBSRC INSTITUTE STRATEGIC PROGRAM GRANTS to the Roslin Institute (BB/P013732/1, BB/P013740/1, BB/P013759/1)S

Immunohistochemical expression of E–cadherin in different tissues of the teleost fish Scophthalmus maximus

E–cadherin is an evolutionary conserved protein, whose main role as the principal component of adherens junctions is supporting epithelial cell–cell adhesion. It is an essential molecule for the maintenance of the epithelial barrier function and the analysis of its immunohistochemical expression is a valuable resource in morphopathological, ontogenetic and pathogenesis studies in mammals. As well, there is an increasing understanding of the importance of E-cadherin in the physiology of the immune system and the development of the immune response. Mucosal health is a primary issue in aquaculture research; nevertheless, there is a lack of immunohistochemical studies of cell junction proteins in fish species. In this work, an immunohistochemical technique was optimized in Bouin- and formalin-fixed paraffin-embedded tissues of turbot Scophthalmus maximus, employing a commercial antibody raised against human E-cadherin. The specificity of the antibody in recognizing the molecule in this teleost species was tested by western blot and mass spectrometry-based proteomic analyses. The assays showed a good specificity and indicated that the antibody recognizes the well conserved cytoplasmic domain of the protein. Immunohistochemistry showed the localisation of E-cadherin at cell-cell contact in the epithelia of the different organs, between the hepatocytes and the pancreatic acinar cells, as well as in the reticulo-epithelial stroma of the thymus. Also, the immunoreaction was observed in the cells constituting the melano-macrophage centres in the spleen and kidney. No immunostaining was detected, as expected, only in the heart and brain. No significant difference was noticed between the two fixative used for collecting the tissues samples. This is the first description of E-cadherin immunohistochemical expression in several tissues of a teleost. The immunohistochemical technique represents a useful tool to be used in the different areas of fish health researchThis work was supported by the Spanish Ministry of Economy, Industry and Competitiveness, and the European Regional Development Fund (ERDF) through the projects AGL2015–67039–C3–1–R and AGL2015–67039–C3–3–RS

Blood Transcriptomics of Turbot Scophthalmus maximus:A Tool for Health Monitoring and Disease Studies

Blood transcriptomics is emerging as a relevant tool to monitor the status of the immune system and assist in diagnosis, prognosis, treatment and pathogenesis studies of diseases. In fish pathology, the potential of transcriptome profiling of blood is still poorly explored. Here, RNA sequencing was applied to analyze the blood transcriptional profile of turbot (Scophthalmus maximus), the most important farmed flatfish. The study was conducted in healthy specimens and specimens parasitized by the myxozoan Enteromyxum scophthalmi, which causes one of the most devastating diseases in turbot aquaculture. The blood of healthy turbot showed a transcriptomic profile mainly related to erythrocyte gas transportation function, but also to antigen processing and presentation. In moderately infected turbot, the blood reflected a broad inhibition of the immune response. Particularly, down-regulation of the B cell receptor signaling pathway was shared with heavily parasitized fish, which showed larger transcriptomic changes, including the activation of the inflammatory response. Turbot response to enteromyxosis proved to be delayed, dysregulated and ineffective in stopping the infection. The study evinces that blood transcriptomics can contribute to a better understanding of the teleost immune system and serve as a reliable tool to investigate the physiopathological status of fish

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

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

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 parasitosisThis study was funded by the Spanish Ministry of Economy and Competitiveness (AGL 2009-13282-C02-01 and -02; AGL2015-67039-C3-1-R and AGL2015-67039-C3-3-R), the European Regional Development Fund (ERDF, European Union) and Xunta de Galicia (Spain) local government (GRC2014/010 and GPC2015/34). Diego Robledo was supported by a FPU fellowship funded by the Spanish Ministry of Education, Culture and Sport. Paolo Ronza was supported by a grant from the scientific network “INMUNOGENOM”, funded by Xunta de Galicia (REDES GI-1251)S

New insights into the Manila clam – Perkinsus olseni interaction based on gene expression analysis of clam hemocytes and parasite trophozoites through in vitro challenges

The Manila clam (Ruditapes philippinarum) is the bivalve species with the highest global production from both fisheries and aquaculture, but its production is seriously threatened by perkinsosis, a disease caused by the protozoan parasite Perkinsus olseni. To understand the molecular mechanisms underlying R. philippinarum–P. olseni interactions, we analysed the gene expression profiles of in vitro challenged clam hemocytes and P. olseni trophozoites, using two oligo-microarray platforms, one previously validated for R. philippinarum hemocytes and a new one developed and validated in this study for P. olseni. Manila clam hemocytes were in vitro challenged with trophozoites, zoospores, and extracellular products from P. olseni in vitro cultures, while P. olseni trophozoites were in vitro challenged with Manila clam plasma along the same time-series (1 h, 8 h, and 24 h). The hemocytes showed a fast activation of the innate immune response, particularly associated with hemocyte recruitment, in the three types of challenges. Nevertheless, different immune-related pathways were activated in response to the different parasite stages, suggesting specific recognition mechanisms. Furthermore, the analyses provided useful complementary data to previous in vivo challenges, and confirmed the potential of some proposed biomarkers. The combined analysis of gene expression in host and parasite identified several processes in both the clam and P. olseni, such as redox and glucose metabolism, protease activity, apoptosis and iron metabolism, whose modulation suggests cross-talk between parasite and host. This information might be critical to determine the outcome of the infection, thus highlighting potential therapeutic targets. Altogether, the results of this study aid understanding the response and interaction between R. philippinarum and P. olseni, and will contribute to developing effective control strategies for this threatening parasitosisThis work was funded by the Ministerio de Economía y Competitividad of the Spanish Government, through the projects (AGL2011-30449-C02-01 and AGL2012-37981), the European Regional Development Funds (FEDER 2007–2013) and the Regional Government of Galicia, Xunta de Galicia, Spain, through the projects ED431C 2018/28 and ED431D 2017/21. The study was also supported by the Centro de Supercomputación de Galicia (CESGA). The first author would like to acknowledge the PhD scholarship awarded by the EXPERTS III Consortium of the European Community Mobility Programme “Erasmus Mundus Action 2, Strand 1” (EMA2). SFB was supported by a scholarship of the Consellería do Mar da Xunta de GaliciaS

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

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

The Teleost Thymus in Health and Disease: New Insights from Transcriptomic and Histopathological Analyses of Turbot, Scophthalmus maximus

The thymus is a primary lymphoid organ that plays a pivotal role in the adaptive immune system. The immunobiology of the thymus in fish is considered to be similar to that of mammals, but it is actually poorly characterized in several cultured teleost species. In particular, while investigations in human and veterinary medicine have highlighted that the thymus can be affected by different pathological conditions, little is known about its response during disease in fish. To better understand the role of the thymus under physiological and pathological conditions, we conducted a study in turbot (Scophthalmus maximus), a commercially valuable flatfish species, combining transcriptomic and histopathological analyses. The myxozoan parasite Enteromyxum scophthalmi, which represents a major challenge to turbot production, was used as a model of infection. The thymus tissues of healthy fish showed overrepresented functions related to its immunological role in T-cell development and maturation. Large differences were observed between the transcriptomes of control and severely infected fish. Evidence of inflammatory response, apoptosis modulation, and declined thymic function associated with loss of cellularity was revealed by both genomic and morphopathological analyses. This study presents the first description of the turbot thymus transcriptome and provides novel insights into the role of this organ in teleosts’ immune responses