Mucosal affairs: glycosylation and expression changes of gill goblet cells and mucins in a fish-polyopisthocotylidan interaction.

Introduction: Secreted mucins are highly O-glycosylated glycoproteins produced by goblet cells in mucosal epithelia. They constitute the protective viscous gel layer overlying the epithelia and are involved in pathogen recognition, adhesion and expulsion. The gill polyopisthocotylidan ectoparasite Sparicotyle chrysophrii, feeds on gilthead seabream (Sparus aurata) blood eliciting severe anemia. Methods: Control unexposed and recipient (R) gill samples of gilthead seabream experimentally infected with S. chrysophrii were obtained at six consecutive times (0, 11, 20, 32, 41, and 61 days post-exposure (dpe)). In histological samples, goblet cell numbers and their intensity of lectin labelling was registered. Expression of nine mucin genes (muc2, muc2a, muc2b, muc5a/c, muc4, muc13, muc18, muc19, imuc) and three regulatory factors involved in goblet cell differentiation (hes1, elf3, agr2) was studied by qPCR. In addition, differential expression of glycosyltransferases and glycosidases was analyzed in silico from previously obtained RNAseq datasets of S. chrysophrii-infected gilthead seabream gills with two different infection intensities. Results and Discussion: Increased goblet cell differentiation (up-regulated elf3 and agr2) leading to neutral goblet cell hyperplasia on gill lamellae of R fish gills was found from 32 dpe on, when adult parasite stages were first detected. At this time point, acute increased expression of both secreted (muc2a, muc2b, muc5a/c) and membrane-bound mucins (imuc, muc4, muc18) occurred in R gills. Mucins did not acidify during the course of infection, but their glycosylation pattern varied towards more complex glycoconjugates with sialylated, fucosylated and branched structures, according to lectin labelling and the shift of glycosyltransferase expression patterns. Gilthead seabream gill mucosal response against S. chrysophrii involved neutral mucus hypersecretion, which could contribute to worm expulsion and facilitate gas exchange to counterbalance parasite-induced hypoxia. Stress induced by the sparicotylosis condition seems to lead to changes in glycosylation characteristic of more structurally complex mucins.

An inside out journey: biogenesis, ultrastructure and proteomic characterisation of the ectoparasitic flatworm Sparicotyle chrysophrii extracellular vesicles.

BACKGROUND: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture. METHODS: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry. RESULTS: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL-1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14-3-3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes. CONCLUSIONS: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

Genetics and Nutrition Drive the Gut Microbiota Succession and Host-Transcriptome Interactions through the Gilthead Sea Bream (Sparus aurata) Production Cycle.

Fish genetically selected for growth (GS) and reference (REF) fish were fed with CTRL (15% FM, 5-7% FO) or FUTURE (7.5% FM, 10% poultry meal, 2.2% poultry oil + 2.5% DHA-algae oil) diets during a 12-months production cycle. Samples from initial (t0; November 2019), intermediate (t1; July 2020) and final (t2; November 2020) sampling points were used for Illumina 16S rRNA gene amplicon sequencing of the adherent microbiota of anterior intestine (AI). Samples from the same individuals (t1) were also used for the gene expression profiling of AI by RNA-seq, and subsequent correlation analyses with microbiota abundances. Discriminant analyses indicated the gut bacterial succession along the production cycle with the proliferation of some valuable taxa for facing seasonality and different developmental stages. An effect of genetic background was evidenced along time, decreasing through the progression of the trial, namely the gut microbiota of GS fish was less influenced by changes in diet composition. At the same time, these fish showed wider transcriptomic landmarks in the AI to cope with these changes. Our results highlighted an enhanced intestinal sphingolipid and phospholipid metabolism, epithelial turnover and intestinal motility in GS fish, which would favour their improved performance despite the lack of association with changes in gut microbiota composition. Furthermore, in GS fish, correlation analyses supported the involvement of different taxa with the down-regulated expression of pro-inflammatory markers and the boosting of markers of extracellular remodelling and response to bacterium. Altogether, these findings support the combined action of the gut microbiome and host transcriptionally mediated effects to preserve and improve gut health and function in a scenario of different growth performance and potentiality.

A bloody interaction: plasma proteomics reveals gilthead sea bream (Sparus aurata) impairment caused by Sparicotyle chrysophrii.

BACKGROUND: Sparicotylosis is an enzootic parasitic disease that is well established across the Mediterranean Sea. It is caused by the polyopisthocotylean monogenean Sparicotyle chrysophrii and affects the gills of gilthead sea bream (GSB; Sparus aurata). Current disease management, mitigation and treatment strategies are limited against sparicotylosis. To successfully develop more efficient therapeutic strategies against this disease, understanding which molecular mechanisms and metabolic pathways are altered in the host is critical. This study aims to elucidate how S. chrysophrii infection modulates the plasma proteome of GSB and to identify the main altered biological processes involved. METHODS: Experimental infections were conducted in a recirculating aquaculture system (RAS) in which naïve recipient GSB ([R]; 70 g; n = 50) were exposed to effluent water from S. chrysophrii-infected GSB (98 g; n = 50). An additional tank containing unexposed naïve fish (control [C]; 70 g; n = 50) was maintained in parallel, but with the open water flow disconnected from the RAS. Haematological and infection parameters from sampled C and R fish were recorded for 10 weeks. Plasma samples from R fish were categorised into three different groups according to their infection intensity, which was based on the number of worms fish-1: low (L: 1-50), medium (51-100) and high (H: > 100). Five plasma samples from each category and five C samples were selected and subjected to a SWATH-MS proteome analysis. Additional assays on haemoglobin, cholesterol and the lytic activity of the alternative complement pathway were performed to validate the proteome analysis findings. RESULTS: The discriminant analysis of plasma protein abundance revealed a clear separation into three groups (H, M/L and C). A pathway analysis was performed with the differentially quantified proteins, indicating that the parasitic infection mainly affected pathways related to haemostasis, the immune system and lipid metabolism and transport. Twenty-two proteins were significantly correlated with infection intensity, highlighting the importance of apolipoproteins, globins and complement component 3. Validation assays of blood and plasma (haemoglobin, cholesterol and lytic activity of alternative complement pathway) confirmed these correlations. CONCLUSIONS: Sparicotylosis profoundly alters the haemostasis, the innate immune system and the lipid metabolism and transport in GSB. This study gives a crucial global overview of the pathogenesis of sparicotylosis and highlights new targets for further research.

Diet and Host Genetics Drive the Bacterial and Fungal Intestinal Metatranscriptome of Gilthead Sea Bream.

The gut microbiota is now recognised as a key target for improving aquaculture profit and sustainability, but we still lack insights into the activity of microbes in fish mucosal surfaces. In the present study, a metatranscriptomic approach was used to reveal the expression of gut microbial genes in the farmed gilthead sea bream. Archaeal and viral transcripts were a minority but, interestingly and contrary to rRNA amplicon-based studies, fungal transcripts were as abundant as bacterial ones, and increased in fish fed a plant-enriched diet. This dietary intervention also drove a differential metatranscriptome in fish selected for fast and slow growth. Such differential response reinforced the results of previously inferred metabolic pathways, enlarging, at the same time, the catalogue of microbial functions in the intestine. Accordingly, vitamin and amino acid metabolism, and rhythmic and symbiotic processes were mostly shaped by bacteria, whereas fungi were more specifically configuring the host immune, digestive, or endocrine processes.

To React or Not to React: The Dilemma of Fish Immune Systems Facing Myxozoan Infections.

Myxozoans are microscopic, metazoan, obligate parasites, belonging to the phylum Cnidaria. In contrast to the free-living lifestyle of most members of this taxon, myxozoans have complex life cycles alternating between vertebrate and invertebrate hosts. Vertebrate hosts are primarily fish, although they are also reported from amphibians, reptiles, trematodes, mollusks, birds and mammals. Invertebrate hosts include annelids and bryozoans. Most myxozoans are not overtly pathogenic to fish hosts, but some are responsible for severe economic losses in fisheries and aquaculture. In both scenarios, the interaction between the parasite and the host immune system is key to explain such different outcomes of this relationship. Innate immune responses contribute to the resistance of certain fish strains and species, and the absence or low levels of some innate and regulatory factors explain the high pathogenicity of some infections. In many cases, immune evasion explains the absence of a host response and allows the parasite to proliferate covertly during the first stages of the infection. In some infections, the lack of an appropriate regulatory response results in an excessive inflammatory response, causing immunopathological consequences that are worse than inflicted by the parasite itself. This review will update the available information about the immune responses against Myxozoa, with special focus on T and B lymphocyte and immunoglobulin responses, how these immune effectors are modulated by different biotic and abiotic factors, and on the mechanisms of immune evasion targeting specific immune effectors. The current and future design of control strategies for myxozoan diseases is based on understanding this myxozoan-fish interaction, and immune-based strategies such as improvement of innate and specific factors through diets and additives, host genetic selection, passive immunization and vaccination, are starting to be considered.

Cross-Talk Between Intestinal Microbiota and Host Gene Expression in Gilthead Sea Bream (Sparus aurata) Juveniles: Insights in Fish Feeds for Increased Circularity and Resource Utilization.

New types of fish feed based on processed animal proteins (PAPs), insect meal, yeast, and microbial biomasses have been used with success in gilthead sea bream. However, some drawback effects on feed conversion and inflammatory systemic markers were reported in different degrees with PAP- and non-PAP-based feed formulations. Here, we focused on the effects of control and two experimental diets on gut mucosal-adherent microbiota, and how it correlated with host transcriptomics at the local (intestine) and systemic (liver and head kidney) levels. The use of tissue-specific PCR-arrays of 93 genes in total rendered 13, 12, and 9 differentially expressed (DE) genes in the intestine, liver, and head kidney, respectively. Illumina sequencing of gut microbiota yielded a mean of 125,350 reads per sample, assigned to 1,281 operational taxonomic unit (OTUs). Bacterial richness and alpha diversity were lower in fish fed with the PAP diet, and discriminant analysis displayed 135 OTUs driving the separation between groups with 43 taxa correlating with 27 DE genes. The highest expression of intestinal pcna and alpi was achieved in PAP fish with intermediate values in non-PAP, being the pro-inflammatory action of alpi associated with the presence of Psychrobacter piscatorii. The intestinal muc13 gene was down-regulated in non-PAP fish, with this gene being negatively correlated with anaerobic (Chloroflexi and Anoxybacillus) and metal-reducing (Pelosinus and Psychrosinus) bacteria. Other inflammatory markers (igm, il8, tnfα) were up-regulated in PAP fish, positively correlating the intestinal igm gene with the inflammasome activator Escherichia/Shigella, whereas the systemic expression of il8 and tnfα was negatively correlated with the Bacilli class in PAP fish and positively correlated with Paracoccus yeei in non-PAP fish. Overall changes in the expression pattern of il10, galectins (lgals1, lgals8), and toll-like receptors (tlr2, tlr5, tlr9) reinforced the anti-inflammatory profile of fish fed with the non-PAP diet, with these gene markers being associated with a wide range of OTUs. A gut microbiota-liver axis was also established, linking the microbial generation of short chain fatty acids with the fueling of scd1- and elovl6-mediated lipogenesis. In summary, by correlating the microbiome with host gene expression, we offer new insights in the evaluation of fish diets promoting gut and metabolism homeostasis, and ultimately, the health of farmed fish.

Ceratothoa oestroides Infection in European Sea Bass: Revealing a Long Misunderstood Relationship.

Ceratothoa oestroides (Cymothoidea, Isopoda) is a generalist crustacean parasite that negatively affects the economic sustainability of European sea bass (Dicentrarchus labrax) aquaculture in the North-East Mediterranean. While mortalities are observed in fry and fingerlings, infection in juvenile and adult fish result in approximately 20% growth delay. A transcriptomic analysis (PCR array, RNA-Seq) was performed on organs (tongue, spleen, head kidney, and liver) from infected vs. Ceratothoa-free sea bass fingerlings. Activation of local and systemic immune responses was detected, particularly in the spleen, characterized by the upregulation of cytokines (also in the tongue), a general reshaping of the immunoglobulin (Ig) response and suppression of T-cell mediated responses. Interestingly, starvation and iron transport and metabolism genes were strongly downregulated, suggesting that the parasite feeding strategy is not likely hematophagous. The regulation of genes related to growth impairment and starvation supported the growth delay observed in infected animals. Most differentially expressed (DE) transcripts were exclusive of a specific organ; however, only in the tongue, the difference between infected and uninfected fish was significant. At the attachment/feeding site, the pathways involved in muscle contraction and intercellular junction were the most upregulated, whereas the pathways involved in fibrosis (extracellular matrix organization, collagen formation, and biosynthesis) were downregulated. These results suggest that parasite-inflicted damage is successfully mitigated by the host and characterized by regenerative processes that prevail over the reparative ones.

Experimental Horizontal Transmission of Enterospora nucleophila (Microsporea: Enterocytozoonidae) in Gilthead Sea Bream (Sparus aurata).

Enterospora nucleophila is a microsporidian enteroparasite that infects mainly the intestine of gilthead sea bream (Sparus aurata), leading to an emaciative syndrome. Thus far, the only available information about this infection comes from natural outbreaks in farmed fish. The aim of the present study was to determine whether E. nucleophila could be transmitted horizontally using naturally infected fish as donors, and to establish an experimental in vivo procedure to study this host-parasite model without depending on natural infections. Naïve fish were exposed to the infection by cohabitation, effluent, or intubated either orally or anally with intestinal scrapings of donor fish in four different trials. We succeeded in detecting parasite in naïve fish in all the challenges, but the infection level and the disease signs were always milder than in donor fish. The parasite was found in peripheral blood of naïve fish at 4 weeks post-challenge (wpc) in oral and effluent routes, and up to 12 wpc in the anal transmission trial. Molecular diagnosis detected E. nucleophila in other organs besides intestine, such as gills, liver, stomach or heart, although the intensity was not as high as in the target tissue. The infection tended to disappear through time in all the challenge routes assayed, except in the anal infection route.

Genetic selection for growth drives differences in intestinal microbiota composition and parasite disease resistance in gilthead sea bream.

BACKGROUND: The key effects of intestinal microbiota in animal health have led to an increasing interest in manipulating these bacterial populations to improve animal welfare. The aquaculture sector is no exception and in the last years, many studies have described these populations in different fish species. However, this is not an easy task, as intestinal microbiota is composed of very dynamic populations that are influenced by different factors, such as diet, environment, host age, and genetics. In the current study, we aimed to determine whether the genetic background of gilthead sea bream (Sparus aurata) influences the intestinal microbial composition, how these bacterial populations are modulated by dietary changes, and the effect of selection by growth on intestinal disease resistance. To that aim, three different groups of five families of gilthead sea bream that were selected during two generations for fast, intermediate, or slow growth (F3 generation) were kept together in the same open-flow tanks and fed a control or a well-balanced plant-based diet during 9 months. Six animals per family and dietary treatment were sacrificed and the adherent bacteria from the anterior intestinal portion were sequenced. In parallel, fish of the fast- and slow-growth groups were infected with the intestinal parasite Enteromyxum leei and the disease signs, prevalence, intensity, and parasite abundance were evaluated. RESULTS: No differences were detected in alpha diversity indexes among families, and the core bacterial architecture was the prototypical composition of gilthead sea bream intestinal microbiota, indicating no dysbiosis in any of the groups. The plant-based diet significantly changed the microbiota in the intermediate- and slow-growth families, with a much lower effect on the fast-growth group. Interestingly, the smaller changes detected in the fast-growth families potentially accounted for more changes at the metabolic level when compared with the other families. Upon parasitic infection, the fast-growth group showed significantly lower disease signs and parasite intensity and abundance than the slow-growth animals. CONCLUSIONS: These results show a clear genome-metagenome interaction indicating that the fast-growth families harbor a microbiota that is more flexible upon dietary changes. These animals also showed a better ability to cope with intestinal infections. Video Abstract.

Passive Immunization Delays Disease Outcome in Gilthead Sea Bream Infected With Enteromyxum leei (Myxozoa), Despite the Moderate Changes in IgM and IgT Repertoire.

Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that gilthead sea bream (GSB, Sparus aurata) that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific IgM in serum. Thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in GSB and to study in detail the nature of these protective antibodies. Serum from a pool of resistant (SUR) or naïve (NAI) animals was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). Effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. In parallel, we performed an immunoglobulin repertoire analysis of the fish generating SUR and NAI sera. The results showed that, fish injected with parasite-specific antibodies (spAbs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. Repertoire analysis revealed that E. leei induced a polyclonal expansion of diverse IgM and IgT subsets that could be in part an evasion strategy of the parasite. Nonetheless, GSB was able to produce sufficient levels of parasite-spAbs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. These results highlight the crucial role of spAb responses against E. leei and set the basis for the development of effective treatment or prophylactic methods for aquaculture.

Transcriptome sequencing supports a conservation of macrophage polarization in fish.

Mammalian macrophages can adopt polarization states that, depending on the exact stimuli present in their extracellular environment, can lead to very different functions. Although these different polarization states have been shown primarily for macrophages of humans and mice, it is likely that polarized macrophages with corresponding phenotypes exist across mammals. Evidence of functional conservation in macrophages from teleost fish suggests that the same, or at least comparable polarization states should also be present in teleosts. However, corresponding transcriptional profiles of marker genes have not been reported thus far. In this study we confirm that macrophages from common carp can polarize into M1- and M2 phenotypes with conserved functions and corresponding transcriptional profiles compared to mammalian macrophages. Carp M1 macrophages show increased production of nitric oxide and a transcriptional profile with increased pro-inflammatory cytokines and mediators, including il6, il12 and saa. Carp M2 macrophages show increased arginase activity and a transcriptional profile with increased anti-inflammatory mediators, including cyr61, timp2b and tgm2b. Our RNA sequencing approach allowed us to list, in an unbiased manner, markers discriminating between M1 and M2 macrophages of teleost fish. We discuss the importance of our findings for the evaluation of immunostimulants for aquaculture and for the identification of gene targets to generate transgenic zebrafish for detailed studies on M1 and M2 macrophages. Above all, we discuss the striking degree of evolutionary conservation of macrophage polarization in a lower vertebrate.

Enterospora nucleophila (Microsporidia) in Gilthead Sea Bream (Sparus aurata): Pathological Effects and Cellular Immune Response in Natural Infections.

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.

Sex, Age, and Bacteria: How the Intestinal Microbiota Is Modulated in a Protandrous Hermaphrodite Fish.

Intestinal microbiota is key for many host functions, such as digestion, nutrient metabolism, disease resistance, and immune function. With the growth of the aquaculture industry, there has been a growing interest in the manipulation of fish gut microbiota to improve welfare and nutrition. Intestinal microbiota varies with many factors, including host species, genetics, developmental stage, diet, environment, and sex. The aim of this study was to compare the intestinal microbiota of adult gilthead sea bream (Sparus aurata) from three groups of age and sex (1-year-old males and 2- and 4-year-old females) maintained under the same conditions and fed exactly the same diet. Microbiota diversity and richness did not differ among groups. However, bacterial composition did, highlighting the presence of Photobacterium and Vibrio starting at 2 years of age (females) and a higher presence of Staphylococcus and Corynebacterium in 1-year-old males. The core microbiota was defined by 14 Operational Taxonomic Units (OTUs) and the groups that showed more OTUs in common were 2- and 4-year-old females. Discriminant analyses showed a clear separation by sex and age, with bacteria belonging to the phyla Firmicutes, Proteobacteria and Actinobacteria driving the separation. Pathway analysis performed with the inferred metagenome showed significant differences between 1-year-old males and 4-year-old females, with an increase in infection-related pathways, nitrotoluene degradation and sphingolipid metabolism, and a significant decrease in carbohydrate metabolism pathways with age. These results show, for the first time, how intestinal microbiota is modulated in adult gilthead sea bream and highlight the importance of reporting age and sex variables in these type of studies in fish.

Disruption of gut integrity and permeability contributes to enteritis in a fish-parasite model: a story told from serum metabolomics.

BACKGROUND: In the animal production sector, enteritis is responsible for serious economic losses, and intestinal parasitism is a major stress factor leading to malnutrition and lowered performance and animal production efficiency. The effect of enteric parasites on the gut function of teleost fish, which represent the most ancient bony vertebrates, is far from being understood. The intestinal myxozoan parasite Enteromyxum leei dwells between gut epithelial cells and causes severe enteritis in gilthead sea bream (Sparus aurata), anorexia, cachexia, growth impairment, reduced marketability and increased mortality. METHODS: This study aimed to outline the gut failure in this fish-parasite model using a multifaceted approach and to find and validate non-lethal serum markers of gut barrier dysfunction. Intestinal integrity was studied in parasitized and non-parasitized fish by immunohistochemistry with specific markers for cellular adhesion (E-cadherin) and tight junctions (Tjp1 and Cldn3) and by functional studies of permeability (oral administration of FITC-dextran) and electrophysiology (Ussing chambers). Serum samples from parasitized and non-parasitized fish were analyzed using non-targeted metabolomics and some significantly altered metabolites were selected to be validated using commercial kits. RESULTS: The immunodetection of Tjp1 and Cldn3 was significantly lower in the intestine of parasitized fish, while no strong differences were found in E-cadherin. Parasitized fish showed a significant increase in paracellular uptake measured by FITC-dextran detection in serum. Electrophysiology showed a decrease in transepithelial resistance in infected animals, which showed a diarrheic profile. Serum metabolomics revealed 3702 ions, from which the differential expression of 20 identified compounds significantly separated control from infected groups in multivariate analyses. Of these compounds, serum inosine (decreased) and creatine (increased) were identified as relevant and validated with commercial kits. CONCLUSIONS: The results demonstrate the disruption of tight junctions and the loss of gut barrier function, a metabolomic profile of absorption dysfunction and anorexia, which further outline the pathophysiological effects of E. leei.

Acquired protective immune response in a fish-myxozoan model encompasses specific antibodies and inflammation resolution.

The myxozoan parasite Enteromyxum leei causes chronic enteritis in gilthead sea bream (GSB, Sparus aurata) leading to intestinal dysfunction. Two trials were performed in which GSB that had survived a previous infection with E. leei (SUR), and naïve GSB (NAI), were exposed to water effluent containing parasite stages. Humoral factors (total IgM and IgT, specific anti-E. leei IgM, total serum peroxidases), histopathology and gene expression were analysed. Results showed that SUR maintained high levels of specific anti-E. leei IgM (up to 16 months), expressed high levels of immunoglobulins at the intestinal mucosa, particularly the soluble forms, and were resistant to re-infection. Their acquired-type response was complemented by other immune effectors locally and systemically, like cell cytotoxicity (high granzyme A expression), complement activity (high c3 and fucolectin expression), and serum peroxidases. In contrast to NAI, SUR displayed a post-inflammatory phenotype in the intestine and head kidney, characteristic of inflammation resolution (low il1ß, high il10 and low hsp90α expression).

Acting locally - affecting globally: RNA sequencing of gilthead sea bream with a mild Sparicotyle chrysophrii infection reveals effects on apoptosis, immune and hypoxia related genes.

BACKGROUND: Monogenean flatworms are the main fish ectoparasites inflicting serious economic losses in aquaculture. The polyopisthocotylean Sparicotyle chrysophrii parasitizes the gills of gilthead sea bream (GSB, Sparus aurata) causing anaemia, lamellae fusion and sloughing of epithelial cells, with the consequent hypoxia, emaciation, lethargy and mortality. Currently no preventive or curative measures against this disease exist and therefore information on the host-parasite interaction is crucial to find mitigation solutions for sparicotylosis. The knowledge about gene regulation in monogenean-host models mostly comes from freshwater monopysthocotyleans and almost nothing is known about polyopisthocotyleans. The current study aims to decipher the host response at local (gills) and systemic (spleen, liver) levels in farmed GSB with a mild natural S. chrysophrii infection by transcriptomic analysis. RESULTS: Using Illumina RNA sequencing and transcriptomic analysis, a total of 2581 differentially expressed transcripts were identified in infected fish when compared to uninfected controls. Gill tissues in contact with the parasite (P gills) displayed regulation of fewer genes (700) than gill portions not in contact with the parasite (NP gills) (1235), most likely due to a local silencing effect of the parasite. The systemic reaction in the spleen was much higher than that at the parasite attachment site (local) (1240), and higher than in liver (334). NP gills displayed a strong enrichment of genes mainly related to immune response and apoptosis. Processes such as apoptosis, inflammation and cell proliferation dominated gills, whereas inhibition of apoptosis, autophagy, platelet activation, signalling and aggregation, and inflammasome were observed in spleen. Proteasome markers were increased in all tissues, whereas hypoxia-related genes were down-regulated in gills and spleen. CONCLUSIONS: Contrasting forces seem to be acting at local and systemic levels. The splenic down-regulation could be part of a hypometabolic response, to counteract the hypoxia induced by the parasite damage to the gills and to concentrate the energy on defence and repair responses. Alternatively, it can be also interpreted as the often observed action of helminths to modify host immunity in its own interest. These results provide the first toolkit for future studies towards understanding and management of this parasitosis.

Gene expression analysis of Atlantic salmon gills reveals mucin 5 and interleukin 4/13 as key molecules during amoebic gill disease.

Amoebic gill disease (AGD) is one of the main diseases affecting Atlantic salmon (Salmo salar L.) mariculture. Hallmarks of AGD are hyperplasia of the lamellar epithelium and increased production of gill mucus. This study investigated the expression of genes involved in mucus secretion, cell cycle regulation, immunity and oxidative stress in gills using a targeted 21-gene PCR array. Gill samples were obtained from experimental and natural Neoparamoeba perurans infections, and sampling points included progressive infection stages and post-freshwater treatment. Up-regulation of genes related to mucin secretion and cell proliferation, and down-regulation of pro-inflammatory and pro-apoptotic genes were associated with AGD severity, while partial restoration of the gill homeostasis was detected post-treatment. Mucins and Th2 cytokines accoun ted for most of the variability observed between groups highlighting their key role in AGD. Two mucins (muc5, muc18) showed differential regulation upon disease. Substantial up-regulation of the secreted muc5 was detected in clinical AGD, and the membrane bound muc18 showed an opposite pattern. Th2 cytokines, il4/13a and il4/13b2, were significantly up-regulated from 2 days post-infection onwards, and changes were lesion-specific. Despite the differences between experimental and natural infections, both yielded comparable results that underline the importance of the studied genes in the respiratory organs of fish, and during AGD progression.

Hints on T cell responses in a fish-parasite model: Enteromyxum leei induces differential expression of T cell signature molecules depending on the organ and the infection status.

BACKGROUD: Enteromyxum leei is a myxozoan parasite that produces a slow-progressing intestinal disease. This parasite invades the paracellular space of the intestinal epithelium and progresses from the posterior to the anterior intestine. The aim of the present study was to gain insights into fish T cell responses in the gilthead sea bream-E. leei infection model using a PCR-array with 30 signature molecules for different leukocyte responses in head kidney, spleen, anterior and posterior intestine. RESULTS: The PCR-array results suggest that E. leei induced migration of T cells from head kidney to intestines where TH1, CTL and TH17 profiles were activated and kept in balance by the upregulation of regulatory cytokines. These results were partially validated by the use of cross-reacting antibodies and BrdU immunostaining to monitor proliferation. Zap70 immunostaining supported the increased number of T cells in the anterior intestine detected by gene expression, but double staining with BrdU did not show active proliferation of this cell type at a local level, supporting the migration from lymphohaematopoietic tissues to the site of infection. Global analyses of the expression profiles revealed a clear separation between infected and exposed, but non-infected fish, more evident in the target organ. Exposed, non-infected animals showed an intermediate phenotype closer to the control fish. CONCLUSIONS: These results evidence a clear modulation of the T cell response of gilthead sea bream upon E. leei infection. The effects occurred both at local and systemic levels, but the response was stronger and more specific at the site of infection, the intestine. Altogether, this research poses a promising basis to understand the response against this important parasite and establish effective preventive or palliative measures.

Sodium salt medium-chain fatty acids and Bacillus-based probiotic strategies to improve growth and intestinal health of gilthead sea bream (Sparus aurata).

BACKGROUND: The increased demand for fish protein has led to the intensification of aquaculture practices which are hampered by nutritional and health factors affecting growth performance. To solve these problems, antibiotics have been used for many years in the prevention, control and treatment against disease as well as growth promoters to improve animal performance. Nowadays, the use of antibiotics in the European Union and other countries has been completely or partially banned as a result of the existence of antibiotic cross-resistance. Therefore, a number of alternatives, including enzymes, prebiotics, probiotics, phytonutrients and organic acids used alone or in combination have been proposed for the improvement of immunological state, growth performance and production in livestock animals. The aim of the present study was to evaluate two commercially available feed additives, one based on medium-chain fatty acids (MCFAs) from coconut oil and another with a Bacillus-based probiotic, in gilthead sea bream (GSB, Sparus aurata), a marine farmed fish of high value in the Mediterranean aquaculture. METHODS: The potential benefits of adding two commercial feed additives on fish growth performance and intestinal health were assessed in a 100-days feeding trial. The experimental diets (D2 and D3) were prepared by supplementing a basal diet (D1) with MCFAs in the form of a sodium salt of coconut fatty acid distillate (DICOSAN®; Norel, Madrid, Spain), rich on C-12, added at 0.3% (D2) or with the probiotic Bacillus amyloliquefaciens CECT 5940, added at 0.1% (D3). The study integrated data on growth performance, blood biochemistry, histology and intestinal gene expression patterns of selected markers of intestinal function and architecture. RESULTS: MCFAs in the form of a coconut oil increased feed intake, growth rates and the surface of nutrient absorption, promoting the anabolic action of the somatotropic axis. The probiotic (D3) induced anti-inflammatory and anti-oxidant effects with changes in circulating cortisol, immunoglobulin M, leukocyte respiratory burst, and mucosal expression levels of cytokines, lymphocyte markers and immunoglobulin T. DISCUSSION: MCFA supplementation showed positive effects on GSB growth and intestinal architecture acting mainly in the anterior intestine, where absorption takes place. The probiotic B. amyloliquefaciens CECT 5940 exhibited key effects in the regulation of the immune status inducing anti-inflammatory and anti-oxidant effects which can be potentially advantageous upon infection or exposure to other stressors. The potential effects of these feed additives in GSB are very promising to improve health and disease resistance in aquaculture.