Gene expression profiling reveals functional specialization along the intestinal tract of a carnivorous teleostean fish (Dicentrarchus labrax)

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.This work was funded by the EU seventh Framework Programme by the ARRAINA (Advanced Research Initiatives for Nutrition and Aquaculture; KBBE-2011-288925) project. It does not necessarily reflect the views of the EU and in no way anticipates the Commission's future policy in this area. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Additional funding was obtained from the Spanish Ministerio de Economía y Competitividad through the MI2-FISH project (Unraveling Metabolic, Intestinal and Immunopathological Fish Status; AGL2013-48560) and Generalitat Valenciana (PROMETEO FASE II-2014/085).Peer Reviewe

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

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

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

Skin mucus of gilthead sea bream (sparus aurata l.). protein mapping and regulation in chronically stressed fish

publishedVersio

Metabolic and transcriptional responses of gilthead sea bream (Sparus aurata L.) to environmental stress: New insights in fish mitochondrial phenotyping

The aim of the current study was to phenotype fish metabolism and the transcriptionally-mediated response of hepatic mitochondria of gilthead sea bream to intermittent and repetitive environmental stressors: (i) changes in water temperature (T-ST), (ii) changes in water level and chasing (C-ST) and (iii) multiple sensory perception stressors (M-ST). Gene expression profiling was done using a quantitative PCR array of 60 mitochondria-related genes, selected as markers of transcriptional regulation, oxidative metabolism, respiration uncoupling, antioxidant defense, protein import/folding/assembly, and mitochondrial dynamics and apoptosis. The mitochondrial phenotype mirrored changes in fish performance, haematology and lactate production. T-ST especially up-regulated transcriptional factors (PGC1α, NRF1, NRF2), rate limiting enzymes of fatty acid β-oxidation (CPT1A) and tricarboxylic acid cycle (CS), membrane translocases (Tim/TOM complex) and molecular chaperones (mtHsp10, mtHsp60, mtHsp70) to improve the oxidative capacity in a milieu of a reduced feed intake and impaired haematology. The lack of mitochondrial response, increased production of lactate and negligible effects on growth performance in C-ST fish were mostly considered as a switch from aerobic to anaerobic metabolism. A strong down-regulation of PGC1α, NRF1, NRF2, CPT1A, CS and markers of mitochondrial dynamics and apoptosis (BAX, BCLX, MFN2, MIRO2) occurred in M-ST fish in association with the greatest circulating cortisol concentration and a reduced lactate production and feed efficiency, which represents a metabolic condition with the highest allostatic load score. These findings evidence a high mitochondrial plasticity against stress stimuli, providing new insights to define the threshold level of stress condition in fish. © 2014 Elsevier Inc.This work was funded by the EU AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research, FP7/2007/2013; grant agreement No. 262336), and the Spanish AQUAGENOMICS (CSD2007-00002, Improvement of aquaculture production by the use of biotechnological tools) projects. Additional funding was obtained by Generalitat Valenciana (research grant PROMETEO 2010/006).Peer Reviewe

Long-term epidemiological survey of Kudoa thyrsites (Myxozoa) in Atlantic salmon (Salmo salar L.) from commercial aquaculture farms

Kudoa thyrsites (Myxozoa) encysts within myocytes of a variety of fishes. While infected fish appear unharmed, parasite-derived enzymes degrade the flesh post-mortem. In regions of British Columbia (BC), Canada, up to 4–7% of fillets can be affected, thus having economic consequences and impacting the competitiveness of BC's farms. K. thyrsites was monitored in two farms having high (HP) or low (LP) historical infection prevalence. At each farm, 30 fish were sampled monthly for blood and muscle during the first year followed by nine samplings during year two. Prevalence and intensity were measured by PCR and histology of muscle samples. In parallel, fillet tests were used to quantify myoliquefaction. Infections were detected by PCR after 355 and 509 degree days at LP and HP farms, respectively. Prevalence reached 100% at the HP farm by 2265 degree days and declined during the second year, whereas it plateaued near 50% at the LP farm. Infection intensities decreased after 1 year at both farms. Blood was PCR-positive at both farms between 778 and 1113 degree days and again after 2000 degree days. This is the first monitoring project in a production environment and compares data between farms with different prevalence.This project was funded by Marine Harvest Canada and an NSERC IRDF Fellowship to WL Marshall.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

High levels of vegetable oils in plant protein-rich diets fed to gilthead sea bream (Sparus aurata L.): growth performance, muscle fatty acid profiles and histological alterations of target tissues

The feasibility of fish oil (FO) replacement by vegetable oils (VO) was investigated in gilthead sea bream (Sparus aurata L.) in a growth trial conducted for the duration of 8 months. Four isolipidic and isoproteic diets rich in plant proteins were supplemented with l-lysine (0ú55 %) and soya lecithin (1 %). Added oil was either FO (control) or a blend of VO, replacing 33 % (33VO diet), 66 % (66VO diet) and 100 % (VO diet) of FO. No detrimental effects on growth performance were found with the partial FO replacement, but feed intake and growth rates were reduced by about 10 % in fish fed the VO diet. The replacement strategy did not damage the intestinal epithelium, and massive accumulation of lipid droplets was not found within enterocytes. All fish showed fatty livers, but signs of lipoid liver disease were only found in fish fed the VO diet. Muscle fatty acid profiles of total lipids reflected the diet composition with a selective incorporation of unsaturated fatty acids in polar lipids. The robustness of the phospholipid fatty acid profile when essential fatty acid requirements were theoretically covered by the diet was evidenced by multivariate principal components analysis in fish fed control, 33VO and 66VO diets