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.

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.

Effects of Enteromyxum scophthalmi experimental infection on the neuroendocrine system of turbot, Scophthalmus maximus (L.).

Enteromyxum scophthalmi is an intestinal myxosporean parasite responsible for serious outbreaks in turbot Scophthalmus maximus (L.) culture, in North-western Spain. The disease affects the digestive tract, provokes severe catarrhal enteritis, emaciation and high rates of mortality. The digestive parasitization triggers a response with the coordinate participation of immune and neuroendocrine systems through the action of peptides released by enteroendocrine cells and present in nervous elements, acting as neuro-immune modulators. The present study was designed to assess the response of the turbot neuroendocrine system against E. scophthalmi infection. Immunohistochemical tests were applied to sections of the gastrointestinal tract of uninfected and E. scophthalmi-infected turbot to characterize the presence of bombesin (BOM), glucagon (GLUC), somatostatin (SOM), leu-enkephalin (LEU) and met-enkephalin (MET). The occurrence of E. scophthalmi in the turbot gastrointestinal tract increased the number of enteroendocrine cells immunoreactive to SOM, LEU and MET. On the other hand, BOM and GLUC immunoreactive cells were less numerous in the gastrointestinal tract of the parasitized turbot. Scarce immunoreactivity to BOM, GLUC and SOM was observed in nerve fibres and neurons of the myenteric plexus of control and infected fish. The results indicate that E. scophthalmi infection in turbot induced changes in the neuroendocrine system, with the diminution of the anorexigenic peptides BOM and GLUC; the increase of enkephalins, related to pro-inflammatory processes; and the increase of SOM, which may cause inhibitory effects on the immune response, constituting a compensatory mechanism to the exacerbated response observed in E. scophthalmi-infected turbot.

Study of the distribution of active caspase-3-positive cells in turbot, Scophthalmus maximus (L.), enteromyxosis.

Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria-submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti-(active caspase-3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi-infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi-infected turbot compared with non-infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase-3-positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.