Chronic aeromoniasis in turbot (Scophthalmus maximus), caused by Aeromonas salmonicida, provokes cutaneous lesions that occasionally expose dermal tubercles, which may act as unconventional ecological niches. A storm in February 2024 caused severe damage at a turbot farm in Galicia (Northwestern Spain), leaving various tanks uncovered and subsequently exposing fish with chronic aeromoniasis to several days of intense solar irradiation. Afterward, farm staff observed green tufts over the animals. Two affected turbots were submitted for necropsy. Morphological features identified the epibiont as the alga Ulva spp., covered by a biofilm containing A. salmonicida DNA (detected by real-time polymerase chain reaction). The algae did not trigger additional host responses beyond those induced by aeromoniasis, indicating true epibiosis rather than infection. However, algal colonization physically obstructs re-epithelialization and may prolong lesion chronicity, while the associated biofilm could serve as an environmental reservoir for the pathogen. This case illustrates how environmental disruptions can generate unexpected host-pathogen-epibiont interactions. To our knowledge, this is the first report of Ulva epibiosis on fish.

Thousands tons of discards of blue whiting (BW) and tuna heads (YT) by-products are generated each year in Europe. BW is the species most discarded by European fishing fleet and, in some canning factories, YT are processed for the retrieval of oil rich in omega-3, but producing a huge amount of solid remains and effluents disposal as wastes. The development of optimal and sustainable processes for both substrates is mandatory in order to reach clean solutions under the circular economy precepts. This work focused on the mathematical optimization of the production of tailored fish protein hydrolysates (FPH), from blue whiting and tuna residues, in terms of controlling average molecular weights (Mw) of proteins. For the modeling of the protein depolymerization time -course, a pseudo -mechanistic model was used, which combined a reaction mechanistic equation affected, in the kinetic parameters, by two non -lineal equations (a first -order kinetic and like-Weibull formulae). In all situations, experimental data were accurately simulated by that model achieving R- 2 values higher than 0.96. The validity of the experimental conditions obtained from modeling were confirmed performing productions of FPH at scale of 5 L -reactor, without pH -control in most of cases, at the different ranges of Mw selected (1 -2 kDa, 2 -5 kDa and 5 -10 kDa). The results showed that FPH from BW with lower Mw led to a remarkable yield of production (12 % w /w of substrate), largest protein contents (77 % w/w of BW hydrolysate), greatest in vitro digestibility ( >95 %), highest essential amino acid presence (43 %) and the best antioxidant (DPPH = 62 %) and antihypertensive (IC 50 -ACE = 80 mg/L) properties. Our results prove that the proposed procedure to produce sustainable FPH, with specific Mw characterisitics, could be extended to other fish waste substrates. Tailored FPH may have the potential to serve as valuable ingredients for functional foods and highquality aquaculture feed.

The larval development of the endangered freshwater mussel Margaritifera margaritifera (L.) represents one of the most unique parasitism among naiads, in which larva parasite the fish gills for several months. Despite the importance of this parasitic phase to successfully culture the freshwater mussel, the larval morphogenesis remains understudied. To describe the parasitic larval development and metamorphosis, Atlantic salmon (Salmo salar L.) were exposed to glochidia, sampled periodically to visualize the gills by stereomicroscopy and light microscopy and results were summarized throughout three developmental stages. Once attached to the fish gills, glochidia changed their morphology within the first days and acquired an intermediate stage termed mushroom larva due to the presence of the mushroom body and the zip membrane, both structures are transitory and distinctive of this long-lasting parasitism. The zip membrane, located at the valve cleft, may play a unique role in the isolation and acquisition of non-particulate nutrients from the fish, while the mushroom body of the mantle accumulates abundant intracytoplasmic lipid droplets. After 200 days, a successful metamorphosis was evidenced by the formation of a complete set of post-larval organs, pointing to the acquisition of different functionality, which will be essential for the settlement and deposit-feeding into the riverbed. Among the post-larval organs, the byssal complex of the post-larval foot was described for the first time at the end of the parasitic stage of naiads. In conclusion, this study provides an overview of the larval morphogenesis of M. margaritifera, from glochidium to post-larva, essential for understanding the parasitic interaction between the freshwater mussel larva and the fish host. Moreover, the morphological techniques and the hallmarks described might be applicable to optimize and monitor the larval developmental status during one of the most critical stages of the captive breeding programmes of endangered freshwater mussels.

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.

Melanomacrophage centres (MMCs) are aggregates of macrophages accumulating various pigments. They have been proposed as an indicator of fish immune response. Blood flukes are common parasites in farmed fish. Two cohorts of wild Southern Bluefin Tuna (Thunnus maccoyi) were examined at transfer, before treatment against blood flukes (pre-treatment) and at harvest. MMCs were assessed in histological sections using image analysis, while Cardicola forsteri and Cardicola orientalis infection severity was determined using qPCR, count of adult flukes in heart flushes and count of eggs in gill filaments. Fish from both cohorts showed the same pattern in the changes in the surface area of MMCs. The surface area of splenic MMCs increased over the ranching duration and was positively correlated to the PCR determined copy numbers of Cardicola forsteri ITS2 rDNA in the gills of those fish. However, the infection with blood fluke was more variable, both between cohorts and individuals within the same cohort. Eggs of blood fluke were detected in renal MMCs using histology. Cardicola forsteri had a higher prevalence than Cardicola orientalis. This study contributes to our understanding of blood fluke infections in Southern Bluefin Tuna and their interactions with MMCs.

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.

Enteromyxoses 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 picture.

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.

Enteromyxosis, caused by Enteromyxum scophthalmi, is one of the most devastating diseases stemming from myxozoan parasites in turbot (Scophthalmus maximus L.), being a limiting factor for its production. The disease develops as a cachectic syndrome, associated to catarrhal enteritis and leukocytic depletion, with morbidity and mortality rates usually reaching 100%. To date, no effective treatment exists and there are different unknown issues concerning its pathogenesis. The gross and microscopic lesions associated to enteromyxosis have been thoroughly described, and several morphopathological studies have been carried out to elucidate the mechanisms of this host-parasite interaction. More recently, efforts have been focused on a multidisciplinary approach, combining histopathology and transcriptome analysis, which has provided significant advances in the understanding of the pathogenesis of this parasitosis. RNA-Seq technology was applied at early and advanced stages of the disease on fishes histologically evaluated and classified based on their lesional degree. In the same way, the transcriptomic data were analyzed in relation to the morphopathological picture and the course of the disease. In this paper, a comprehensive review of turbot enteromyxosis is presented, starting from the disease description up to the most novel information extracted by an integrated approach on the infection mechanisms and host response. Further, we discuss ongoing strategies toward a full understanding of host-pathogen interaction and the identification of suitable biomarkers for early diagnosis and disease management strategies.

Freshwater mussels of the order Unionida encyst into the fish mucosa to metamorphose and complete their life cycle, causing a parasitic disease known as glochidiosis. This parasitic stage represents a bottleneck for the survival of naiads, particularly for critically endangered species as Margaritifera margaritifera; however, little is known about the events occurring during this critical stage. Therefore, this study aimed to histologically characterize the development of M. margaritifera glochidiosis in Atlantic salmon to get insight into the pathogenesis of this interaction. Fish exposed to glochidia were sampled during the first 44 days post-exposure, and organs were observed by stereomicroscopy and light microscopy. Glochidia attached to the gills by pinching the lamellar epithelium, whereupon an acute proliferative branchitis engulfed most of the larvae. However, during the first 14 days, a severe detachment of unviable glochidia occurred, associated with the presence of pleomorphic inflammatory infiltrate and epithelial degeneration. In the cases where larvae remained attached, a chronification of the lesions with none to scarce inflammation was observed. These results provide key information to better understand the complex host-parasite interaction during the early stages of glochidiosis and provide valuable information to optimize artificial rearing of naiads in conservation of threatened freshwater mussel populations.

Zebrafish has become a popular research model in the last years, and several diseases affecting zebrafish research facilities have been reported. However, only one case of naturally occurring viral infections was described for this species. In 2015, infectious spleen and kidney necrosis virus (ISKNV) was detected in zebrafish from a research facility in Spain. Affected fish showed lethargy, loss of appetite, abnormal swimming, distention of the coelomic cavity and, in the most severe cases, respiratory distress, pale gills and petechial haemorrhages at the base of fins. Cytomegaly was the most relevant histopathological finding in organs and tissues, sometimes associated to degenerative and necrotic changes. ISKNV belongs to the relatively newly defined genus Megalocytivirus, family Iridoviridae, comprising large, icosahedral cytoplasmic DNA viruses. This is the first case of naturally occurring Megalocytivirus infection in zebrafish research facilities, associated with morbidity. The virus has been identified based on both pathologic and genetic evidence, to better understand the pathogenesis of the infection in zebrafish and the phylogenetic relationship with other iridoviruses. Given the ability of megalocytiviruses to cross-species boundaries, it seems necessary to implement stringent biosecurity practices as these infections may invalidate experimental data and have major impact on laboratory and cultured fish.

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 research.

The modulation of the immune system can have multiple applications such as cancer treatment, and a wide type of processes involving inflammation where the potent chemotactic agent cyclophilin A (Cyp A) is implicated. The Porifera phylum, in which Spongionella is encompassed, is the main producer of marine bioactive compounds. Four secondary metabolites obtained from Spongionella (Gracilin H, A, L, and Tetrahydroaplysulphurin-1) were described to hit Cyp A and to block the release of inflammation mediators. Based on these results, some role of Spongionella compounds on other steps of the signaling pathway mediated by this chemotactic agent can be hypothesized. In the present paper, we studied the effect of these four compounds on the surface membrane CD147 receptor expression, on the extracellular levels of Cyp A and on the ability to migrate of concanavalin (Con A)-activated T lymphocytes. Similar to a well-known immunosuppressive agent cyclosporine A (CsA), Gracilin H, A, L, and tetrahydroaplysulphurin-1 were able to reduce the CD147 membrane expression and to block the release of Cyp A to the medium. Besides, by using Cyp A as chemotactic agent, T cell migration was inhibited when cells were previously incubated with Gracilin A and Gracilin L. These positive results lead us to test the in vivo effect of Gracilin H and L in a mouse ear delayed hypersensitive reaction. Thus, both compounds efficiently reduce the ear swelling as well as the inflammatory cell infiltration. These results provide more evidences for their potential therapeutic application in immune-related diseases of Spongionella compounds.

Aeromonas salmonicida subsp. salmonicida represents one of the major threats in aquaculture, especially in salmonid fish and turbot farming. In order to fight bacterial infections, fish have an immune system composed by innate and specific cellular and humoral elements analogous to those present in mammals. However, innate immunity plays a primordial role against bacterial infections in teleost fish. Among these non-specific mechanisms, the production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) pathway and the tumour necrosis factor-alpha (TNFα) produced by mononuclear phagocytes, are two of the main immune effectors to eliminate bacterial pathogens. In this study, the distribution and kinetic of iNOS and TNFα-producing cells of kidney and spleen of turbot experimentally inoculated with A. salmonicida was assessed by immunohistochemistry. In control and challenged fish, individual iNOS(+) and TNFα(+) cells, showing a similar pattern of distribution, were detected. In challenged fish, the number of immunoreactive cells was significantly increased in the evaluated organs, as well as the melanomacrophage centres showed variable positivity for both antigens. These results indicate that A. salmonicida induced an immune response in challenged turbot, which involved the increase of the activity of iNOS and TNFα in the leukocytic population from kidney and spleen.

Bluetongue virus (BTV) replicates in lymphoid tissues where infected mononuclear leukocytes secrete proinflammatory and vasoactive mediators that can contribute to bluetongue (BT) pathogenesis. Using the well-characterized IFNAR(-/-) mice animal model, we have now studied the histopathology and dynamics of leukocyte populations in different target tissues (spleen, thymus, and lung) during BTV-4 infection by histological and immunohistochemical techniques. The spleen and thymus of BTV-4 infected mice showed severe lymphoid depletion on H&E stained sections. This finding was confirmed by IHC, showing moderate decreased immunopositivity against CD3 in the thymus, and scarce immunoreactivity against CD3 and CD79 in the rest of the white pulp in the spleen, together with an increase in MAC387 immunostaining. BTV-4 infection also induced the expression of active caspase-3 in the spleen, where apoptotic debris was observed by H&E. A dramatic increase in iNOS immunoreactivity associated to necrotic areas of the white pulp was observed, being less noticeable in the thymus and the lung. The induction of pro-inflammatory cytokines in tissues where BTV replicates was evaluated by measuring transcript levels by RT-qPCR. BTV-4 infection led to enhance transcription of IFN-γ, TNF, IL-6, IL-12-p40, and IL-1β mRNA in the thymus, spleen and lung, correlating with the level of virus replication in these tissues. Disease progression and pathogenesis in IFNAR(-/-) mice closely mimics hallmarks of bluetongue disease in ruminants. IFNAR(-/-) mice are a good choice to facilitate a faster advance in the field of orbiviruses.

Domoic acid (DA) is one of the best known marine toxins, causative of important neurotoxic alterations. DA effects are documented both in wildlife and experimental assays, showing that this toxin causes severe injuries principally in the hippocampal area. In the present study we have addressed the long-term toxicological effects (30 days) of DA intraperitoneal administration in rats. Different histological techniques were employed in order to study DA toxicity in heart, an organ which has not been thoroughly studied after DA intoxication to date. The presence of DA was detected by immunohistochemical assays, and cellular alterations were observed both by optical and transmission electron microscopy. Although histological staining methods did not provide any observable tissue damage, transmission electron microscopy showed several injuries: a moderate lysis of myofibrils and loss of mitochondrial conformation. This is the first time the association between heart damage and the presence of the toxin has been observed.

Trabajo presentado en la XXVIII Reunion de la Sociedad Espanola de Anatomia Patologica Veterinaria, celebrada en Cordoba (Espana), del 8 al 10 de junio de 2016

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.

Aeromonas salmonicida subspecies salmonicida (A. salmonicida), is one of the major pathogens of fish farm, being particularly harmful for salmonid and turbot culture. Vaccination against A. salmonicida generally provides a protective response, especially in Atlantic salmon and rainbow trout. However, there are few data there about vaccine against this pathogen in turbot. In order to evaluate the efficacy of the experimental vaccines against A. salmonicida on this species, we have assessed the degree of protection and the side-effects of different vaccinal formulations. Two groups of fish were intracoelomically injected with aqueous and oil based vaccines containing formalin inactivated A. salmonicida as immunogen. The analysis of lesions was performed by gross and microscopic studies, as well as immunohistochemical methods using anti-cytokeratin, -TNF-α and -iNOS antibodies. Immunohistochemistry was also employed to describe the distribution of vaccinal antigen in turbot tissues.