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

New insight on vertebral anomalies in cultured Senegalese sole (Solea senegalensis, Kaup) at early stages of development

Senegalese sole (Solea senegalensis, Kaup) is a promising flatfish species in aquaculture. However, skeletal anomalies are still a great concern in sole farming. Investigation of this issue is crucial to improving larval quality and optimizing production. The aim of this study was to thoroughly assess anomalies in the rachis of reared sole at early developmental stages. Sole (n = 507) were sampled at 31 or 32 days after hatching (dah). The specimens were stained with alcian blue and alizarin red and evaluated for the detection of vertebral deformities. Most fish presented 9:34:3 vertebrae in abdominal, caudal and caudal complex regions, respectively. Remarkably, all specimens showed at least one spinal anomaly. Alterations of neural/haemal elements, as well as deformities of hypurals, parhypural and epural, were recurrent. Vertebral body anomalies and/or vertebral column deviations were identified in 52% of the individuals. Vertebral deformations and fusions were common, especially in caudal complex. ‘Minor’ anomalies were predominant, and some of the detected disorders might be a result of non-/low-pathological processes. These results contribute a new insight into the main skeletal anomalies affecting cultured sole larvae. Further research is required to determine their impact on fish welfare and external appearances at commercial stagesConselleria de Economía e Industria - Xunta de Galicia (10MMA020E) Programa de Consolidación e Estructuración de Unidades de InvestigaciónCompetitivas GPC2015/034 A.M. de Azevedo held a University Professorship Formation (FPU) grant from the Spanish Ministry of Education.S

Skeletal Anomalies in Senegalese Sole (Solea senegalensis), an Anosteocytic Boned Flatfish Species

Skeletal anomalies affect animal welfare and cause important economic problems in aquaculture. Despite the high frequency of skeletal problems in reared Solea senegalensis, there is lack of information regarding the histological features of normal and deformed vertebrae in this flatfish. The aim of this study was to describe the histopathological and radiographical appearance of vertebral body anomalies. Sixty-seven juvenile fish were radiographically examined 104 or 105 days after hatching. Through radiographic images, vertebral segments were selected and processed for histopathological examination from 7 normal and 7 affected fish. Alterations in bone shape and vertebral fusion were the most significant anomalies in the vertebral bodies. These alterations occurred most frequently between the last 3 abdominal vertebrae and the first 10 caudal centra. Radiographically, deformed vertebrae showed flattening of the endplates and narrowing of the intervertebral spaces. The radiographic findings concurred with the histological lesions where affected vertebrae exhibited irregular endplates and changes in trabecular bone. Radiolucent cartilaginous tissue was evident in the endplates of the deformed vertebra and, in some cases, the cartilaginous material extended from the growth zone into the intervertebral space. These changes were likely the primary alterations that led to vertebral fusion. Fused vertebrae were often reshaped and showed a reorganization of the trabeculae. The formation of metaplastic cartilage is frequent in a variety of anomalies affecting teleost speciesThe author(s) received the following financial support for the research, authorship, and/or publication of this article: This work was funded by “Consellería de Economía e Industria” of Xunta de Galicia (10MMA020E) and by “Programa de Consolidación e Estructuración de Unidades de Investigación Competitivas GPC2015/034,” Spain. A. M. de Azevedo held a University Professorship Formation (FPU) grant from the Spanish Ministry of EducationS

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

Skeletal anomalies in reared Senegalese sole Solea senegalensis juveniles: a radiographic approach

Reared Senegalese sole Solea senegalensis Kaup show a high incidence of vertebral anomalies; however, little is known about its skeletal anomaly profile in the later farming phases. The purpose of this study was to provide a detailed description and quantification of the most common skeletal anomalies in reared Senegalese sole in the juvenile stage by means of computed radiography. A total of 374 Senegalese sole were classified according to the external morphology of the fish as normal or altered and then radiographed in latero-lateral and in dorso-ventral projections. Radiographic evaluation of anomalies focused especially on vertebral body anomalies (VBA) and vertebral column deviations (VCD). The 2 orthogonal projections provided a more complete visualization of the skeleton. Approximately 75% of the individuals showed at least 1 anomaly, while VBA and/or VCD were detected in 48.9% of the specimens. Regarding external morphology, 88% of the fish were categorized as normal, although about 72% of these normal fish displayed abnormalities in radiographies. The most frequent anomalies consisted of deformations of the caudal complex plates (hypurals, parhypural and epural), preurals and caudal vertebrae. Scoliosis was the most prevalent among VCD, affecting the caudal area in almost 15% of the individuals. The anomaly profile at the juvenile stages showed some differences compared to what has been reported previously in earlier stages of development. In light of these results, further investigation into the progression of skeletal anomalies over time and the causative factors at later stages is requiredThis work was supported by Consellería de Economía e Industria of Xunta de Galicia (10MMA020E) and Programa de Consolidación e Estructuración de Unidades de Investigación Competitivas GPC2015/034, Spain. A.M.A. held a University Professorship Formation (FPU) grant from the Spanish Ministry of EducationS

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

A combined strategy involving Sanger and 454 pyrosequencing increases genomic resources to aid in the management of reproduction, disease control and genetic selection in the turbot (Scophthalmus maximus)

Background: Genomic resources for plant and animal species that are under exploitation primarily for human consumption are increasingly important, among other things, for understanding physiological processes and for establishing adequate genetic selection programs. Current available techniques for high-throughput sequencing have been implemented in a number of species, including fish, to obtain a proper description of the transcriptome. The objective of this study was to generate a comprehensive transcriptomic database in turbot, a highly priced farmed fish species in Europe, with potential expansion to other areas of the world, for which there are unsolved production bottlenecks, to understand better reproductive- and immune-related functions. This information is essential to implement marker assisted selection programs useful for the turbot industry. Results: Expressed sequence tags were generated by Sanger sequencing of cDNA libraries from different immunerelated tissues after several parasitic challenges. The resulting database (“Turbot 2 database”) was enlarged with sequences generated from a 454 sequencing run of brain-hypophysis-gonadal axis-derived RNA obtained from turbot at different development stages. The assembly of Sanger and 454 sequences generated 52,427 consensus sequences (“Turbot 3 database”), of which 23,661 were successfully annotated. A total of 1,410 sequences were confirmed to be related to reproduction and key genes involved in sex differentiation and maturation were identified for the first time in turbot (AR, AMH, SRY-related genes, CYP19A, ZPGs, STAR FSHR, etc.). Similarly, 2,241 sequences were related to the immune system and several novel key immune genes were identified (BCL, TRAF, NCK, CD28 and TOLLIP, among others). The number of genes of many relevant reproduction- and immune-related pathways present in the database was 50–90% of the total gene count of each pathway. In addition, 1,237 microsatellites and 7,362 single nucleotide polymorphisms (SNPs) were also compiled. Further, 2,976 putative natural antisense transcripts (NATs) including microRNAs were also identified Conclusions: The combined sequencing strategies employed here significantly increased the turbot genomic resources available, including 34,400 novel sequences. The generated database contains a larger number of genes relevant for reproduction- and immune-associated studies, with an excellent coverage of most genes present in many relevant physiological pathways. This database also allowed the identification of many microsatellites and SNP markers that will be very useful for population and genome screening and a valuable aid in marker assisted selection programs.The current work was granted by the Spanish Government thanks to a Consolider Project (Project Aquagenomics, ref. CDS2007-0002) and to projects AGL2006-13158-C03 and AGL2009-13282-C01 and C02. LR was supported by an Aquagenomics postdoctoral contract and BGP was supported by an Isidro Parga Pondal research fellowship from the Xunta de Galicia (Spain).S

Morfopatología y recuperación branquial del salmón del Atlántico durante el desprendimiento parasitario de Margaritifera margaritifera

Trátase da versión previa á revisión enviada a Journal of Fish Diseases do seguinte artigo: Castrillo, PA; Varela-Dopico, C.; Bermúdez, R.; Ondina, P.& Quiroga, M.I (2021) Morphopathology and gill recovery of Atlantic salmon during the parasitic detachment of Margaritifera margaritifera que foi revisada e publicada finalmente en https://onlinelibrary.wiley.com/doi/full/10.1111/jfd.13372. Este artigo pode ser utilizado con uso non comercial segundo os termos e condicións da editorial Wiley para a utilización en versións autoarquivadas. O artigo forma forma parte dun proxecto multidisciplinar levado a cabo entre os grupos de investigación da USC: GI-1707_GAPAVET (Anatomía patológica veterinaria) e GI2040_COPEMOL (Conservación de Peixes e Moluscos), no que se desenvolveu a tese de doutoramento de Pedro Antonio Castrillo Arias. This is the peer reviewed version of the following article: Castrillo, PA; Varela-Dopico, C.; Bermúdez, R.; Ondina, P.& Quiroga, M.I (2021) Morphopathology and gill recovery of Atlantic salmon during the parasitic detachment of Margaritifera margaritifera which has been published in final form at https://onlinelibrary.wiley.com/doi/full/10.1111/jfd.13372. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. The article is part of a multidisciplinary project developed by the research groups of the USC: GI-1707_GAPAVET (Veterinary Pathological Anatomy) and GI2040_COPEMOL (Conservation of Fish and Molluscs), in which the doctoral thesis carried out by Pedro Antonio Castrillo Arias was developed.During the conservation aquaculture of the freshwater mussel Margaritifera margaritifera, fish health has become a concern due to the need of mussel larvae (glochidia) to parasitize the salmonid gills and metamorphose into juveniles. However, there is a lack of information about the impact on fish during the juvenile detachment and the subsequent gill healing. To evaluate the morphopathological changes and gill recovery after the parasitism of M. margaritifera, 51 Atlantic salmon fry (Salmo salar), infested with around 22 larvae/fish g, were necropsied during the synchronized detachment of the mussel juveniles, and gills were assessed by stereomicroscopy and by light and scanning electron microscopy. Salmon showed no clinical signs during the trial and gills recovered their normal morphology almost completely in a short time, suggesting a minimal impact on fish health after glochidiosis. In this sense, the non-erosive droplet detachment and the goblet cell hyperplasia favoured an effective gill remodelling mediated by apoptosis, polarization and cell shedding of the gill epithelia, providing insights to the defence, clearing and healing mechanisms of the gill. These morphopathological techniques could also be implemented to preserve fish welfare and to optimize the artificial breeding programmes of endangered freshwater mussels.The conservation programme was cofunded by the “Fundación Biodiversidad” within the MarMaCul and MargaSalmo Projects and by the Xunta de Galicia within “Programa de Consolidación e Estructuración de Unidades de Investigación Competitivas” (ED4313 2019/24 and ED431D 2017/22) for the regional development for scientific network. P. A. Castrillo held a University Professorship Formation (FPU) grant from the Spanish Ministry of Education, Culture and Sport (FPU17/02004). We also acknowledge the support of the research collaboration agreement with 30 the Consellería de Medio Ambiente, Territorio y Vivienda (Xunta de Galicia).S

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

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 lesionsThis work was supported by the Spanish Ministry of Science and Innovation project AGL2009-13282-C02-01 and -02S

Severe neurometabolic phenotype in npc1−/− zebrafish with a C-terminal mutation

Niemann Pick disease type C (NPC) is an autosomal recessive neurodegenerative lysosomal disorder characterized by an accumulation of lipids in different organs. Clinical manifestations can start at any age and include hepatosplenomegaly, intellectual impairment, and cerebellar ataxia. NPC1 is the most common causal gene, with over 460 different mutations with heterogeneous pathological consequences. We generated a zebrafish NPC1 model by CRISPR/Cas9 carrying a homozygous mutation in exon 22, which encodes the end of the cysteine-rich luminal loop of the protein. This is the first zebrafish model with a mutation in this gene region, which is frequently involved in the human disease. We observed a high lethality in npc1 mutants, with all larvae dying before reaching the adult stage. Npc1 mutant larvae were smaller than wild type (wt) and their motor function was impaired. We observed vacuolar aggregations positive to cholesterol and sphingomyelin staining in the liver, intestine, renal tubules and cerebral gray matter of mutant larvae. RNAseq comparison between npc1 mutants and controls showed 284 differentially expressed genes, including genes with functions in neurodevelopment, lipid exchange and metabolism, muscle contraction, cytoskeleton, angiogenesis, and hematopoiesis. Lipidomic analysis revealed significant reduction of cholesteryl esters and increase of sphingomyelin in the mutants. Compared to previously available zebrafish models, our model seems to recapitulate better the early onset forms of the NPC disease. Thus, this new model of NPC will allow future research in the cellular and molecular causes/consequences of the disease and on the search for new treatments