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[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.

Funding This work was funded by EU H2020 program through ParaFishControl Project (634429) and by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM). This publication reflects only the authors’ view and the European Union cannot be held responsible for any use that may be made of the information contained herein. MP was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I/AEI/10.13039/501100011033 %CITAVIPICKER£10.13039/501100011033££% & ESF), IE under APOSTD/2016/037 grant by the “Generalitat Valenciana” and RP was contracted under the PTA-Program from the Spanish Ministry of Science, Innovation and Universities (PTA2018-015315-I). We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., 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, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Funding This work was supported by the European Research Council (ERC Consolidator Grant 2016 725061 TEMUBLYM) and the European Commission under the H2020 Programme (Grant H2020-634429 ParaFishControl). IE was recipient of APOSTD/2016/037 grant by the “Generalitat Valenciana” and YH was recipient of a PhD Studentship from the Ministry of Education, Republic of China (Taiwan). JWH was supported by BBSRC grant BB/K009125/1 and SNSF grant CRSII3_147649-1. PDR was funded by grant T1-BIO-1672 from the “Comunidad de Madrid”., Proliferative kidney disease (PKD) is a widespread disease caused by the endoparasite Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea). Clinical disease, provoked by the proliferation of extrasporogonic parasite stages, is characterized by a chronic kidney pathology with underlying transcriptional changes indicative of altered B cell responses and dysregulated T-helper cell-like activities. Despite the relevance of PKD to European and North American salmonid aquaculture, no studies, to date, have focused on further characterizing the B cell response during the course of this disease. Thus, in this work, we have studied the behavior of diverse B cell populations in rainbow trout (Oncorhynchus mykiss) naturally infected with T. bryosalmonae at different stages of preclinical and clinical disease. Our results show a clear upregulation of all trout immunoglobulins (Igs) (IgM, IgD, and IgT) demonstrated by immunohistochemistry and Western blot analysis, suggesting the alteration of diverse B cell populations that coexist in the infected kidney. Substantial changes in IgM, IgD, and IgT repertoires were also identified throughout the course of the disease further pointing to the involvement of the three Igs in PKD through what appear to be independently regulated mechanisms. Thus, our results provide strong evidence of the involvement of IgD in the humoral response to a specific pathogen for the first time in teleosts. Nevertheless, it was IgT, a fish-specific Ig isotype thought to be specialized in mucosal immunity, which seemed to play a prevailing role in the kidney response to T. bryosalmonae. We found that IgT was the main Ig coating extrasporogonic parasite stages, IgT+ B cells were the main B cell subset that proliferated in the kidney with increasing kidney pathology, and IgT was the Ig for which more significant changes in repertoire were detected. Hence, although our results demonstrate a profound dysregulation of different B cell subsets during PKD, they po, Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Sparicotyle chrysophrii (Microcotylidae) is considered the most threatening pathogen affecting the gilthead seabream (GSB; Sparus aurata) off-shore farming due to its economic impact. This study explores the best experimental conditions to set up an in vivo infection model capable of mimicking the sparicotylosis signs observed in farmed diseased fish. The experimental setup for parasite transmission consisted of a recipient (R) fish tank with naïve GSB receiving water from two S. chrysophrii-infected donor tanks in a recirculating aquaculture system (RAS). Egg collectors, consisting of a polyester mesh in a supporting plastic frame, were placed in the R tank in order to monitor the progression of the parasitosis. An additional tank with control unexposed naïve fish was maintained in parallel, with open water flow and disconnected from the RAS. After a preliminary trial, infective pressure in the R tank was increased by placing an additional egg collector already loaded with entangled parasite eggs, and by maintaining the fish number throughout the experiment. Adult S. chrysophrii parasite load correlated with most of the evaluated biotic and abiotic factors. Haemoglobin and haematocrit significantly dropped around 40 days after exposing GSB to S. chrysophrii. Furthermore, the abundance of eosinophilic granular cells and goblet cells in gill filaments, and splenic melanomacrophagic centres increased. In contrast, hepatic fat was depleted in S. chrysophrii-infected GSB. This study provides an advancement not only for studying S. chrysophrii's biology and its interaction with its host, but also for further studying the disease under experimental conditions in search of treatment alternatives and prophylactic measures.

Sparicotyle chrysophrii (Microcotylidae) is considered the most threatening parasite affecting the gilthead seabream (Sparus aurata, GSB) in sea cage farming. There are no standardised in vivo models fully described in the scientific literature. This study aims to explore the best experimental conditions to set up an in vivo infection model capable of mimicking the sparicotylosis signs observed in diseased farmed fish. A recipient (R) fish tank (200L) with naïve GSB (N=28) received water from two S. chrysophrii-infected donor tanks (N=52; 200L) in a recirculating aquaculture system (RAS). Parasite egg collectors, consisting of a polyester mesh in a supporting plastic frame, were placed in the R tank in order to monitor the progression of the parasitosis. An additional tank with control unexposed naïve fish (C) was maintained in parallel (N=20) disconnected from the RAS. Five C were sampled at 0 days post exposure (dpe), whereas 3 C and 5 R were sampled at 14, 28, 42, 50, 58 dpe. Haemoglobin values were immediately measured and haematocrit values determined by microhematocrit capillary centrifugation. All right gill arches were dissected and infection intensities of juvenile and adult S. chrysophrii determined under a stereomicroscope. The left gill arches, head kidney, spleen and liver samples were fixed in Bouin¿s solution and processed for routine paraffin histology. R fish were successfully infected (100% prevalence after 14 dpe) and the parasite cycle was completed in the experimental setup. Adult parasite load was positively correlated with host biometric factors, temperature, dpe and tank biomass, whereas it negatively correlated with haemoglobin, haematocrit and condition factor. Egg counts peaked at 35 dpe and haemoglobin and haematocrit significantly dropped around 40 dpe. Furthermore, the abundance of eosinophilic granular cells and goblet cells in gill filaments, and splenic melanomacrophagic centres increased, whereas hepatic fat was depleted in infected

It is yet unknown whether gilthead seabream develops immunologic memory and protection after a first exposure to the gill ectoparasite Sparicotyle chrysophrii, known to feed on its host blood causing severe anemia. For this purpose, experimentally infected gilthead seabream (n=25) were kept during an entire year until they naturally lost the infection. These recovered fish (RE), and additional naïve fish (n=25, NAI), were exposed to S. chrysoprii. At three lethal samplings, blood was taken and hemoglobin and hematocrit values immediately registered, serum samples and gill mucus collected, and biometric and infection intensity data recorded. Before parasite exposure (t0), 5 RE and 5 NAI were sampled. At four- and eightweeks post exposure (wpe) (t1 and t2, respectively), 10 RE and 10 NAI were sampled. ELISAs for total and specific IgM and IgT detection in serum and mucus samples were carried out. Prevalence of infection was 100% in both recipient groups, but in t2, the RE group had significantly lower infection intensity and significantly higher hemoglobin concentration. Gill mucus IgT, total and specific, and specific IgM were significantly higher in RE than in NAI at t0, and were depleted at t2. Gill mucus total IgM of RE increased significantly at t2. Serum IgT was depleted at t2 in both experimental groups, whereas serum IgM, total and specific, was reestablished or increased only in RE at t2. Therefore, specific Ig levels found in the gill mucus of RE seem to mitigate sparicotylosis in gilthead seabream, pointing to a local protective acquired response.

The present study aimed to unravel the effects of partial (50%; 50LSAqua) and total (100%; 100LSAqua) replacement of fish meal (FM) by a commercial protein source (LSAqua SusPro) made of bacterial and processed animal proteins (PAP) in farmed juveniles of gilthead sea bream (Sparus aurata). The trial lasted 8 weeks, and the feasibility of replacement was assessed in terms of growth performance, histopathological scoring and composition of mucosal adherent microbiota from anterior intestine (AI). Specific growth rates (SGR) of 50LSAqua fish were undistinguishable from the CTRL group, whereas a slight but significant growth impairment was found with the total replacement. Histological signs of inflammation across the intestine were more evident at the highest level of FM replacement. Illumina sequencing of gut mucosal microbiota yielded a mean of 130,439 reads per sample assigned to 1,567 OTUs at 97% identity threshold. The bacterial richness was similar in all groups, but a significantly higher Simpson diversity index was found in 100LSAqua fish. At the phylum level, Proteobacteria were the most abundant in all groups, whereas Firmicutes decreased and Actinobacteria increased with the FM replacement. The inferred metagenome analysis suggested that these discriminant bacteria could be implicated in a counter-regulatory anti-inflammatory response. It also pointed to an over-representation of mucosal microbes that can potentially be involved in the natural production of antimicrobials in fish fed the experimental diets.

[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

This study aimed to highlight the relationship between diet, animal performance and mucosal adherent gut microbiota (anterior intestine) in fish fed plant-based diets supplemented with an egg white hydrolysate (EWH) with antioxidant and antiobesogenic activity in obese rats. The feeding trial with juveniles of gilthead sea bream (Sparus aurata) lasted 8 weeks. Fish were fed near to visual satiety with a fish meal (FM)/fish oil (FO) based diet (CTRL) or a plant-based diet with/without EWH supplementation. Specific growth rate decreased gradually from 2.16% in CTRL fish to 1.88% in EWH fish due to a reduced feed intake, and a slight impairment of feed conversion ratio. Plant-based diets feeding triggered a hyperplasic inflammation of the anterior intestine regardless of EWH supplementation.

The present study aimed to unravel the effects of partial (50%; 50LSAqua) and total (100%; 100LSAqua) replacement of fish meal (FM) by a commercial protein source (LSAqua SusPro) made of bacterial and processed animal proteins (PAP) in farmed juveniles of gilthead sea bream (Sparus aurata). The trial lasted 8 weeks, and the feasibility of replacement was assessed in terms of growth performance, histopathological scoring and composition of mucosal adherent microbiota from anterior intestine (AI). Specific growth rates (SGR) of 50LSAqua fish were undistinguishable from the CTRL group, whereas a slight but significant growth impairment was found with the total replacement. Histological signs of inflammation across the intestine were more evident at the highest level of FM replacement, and the total concentration of short chain fatty acids (SCFA) in stripped feces decreased in a dose dependent manner. Illumina sequencing of gut mucosal microbiota yielded a mean of 130,439 reads per sample assigned to 1,567 OTUs at 97% identity threshold. The bacterial richness was similar in all groups, but a significantly higher Simpson diversity index was found in 100LSAqua fish. At the phylum level, Proteobacteria were the most abundant in all groups, whereas Firmicutes decreased and Actinobacteria increased with the FM replacement. At a closer look, pro-inflammatory Gammaproteobacteria of the genus Psychrobacter, and Acinetobacter decreased with FM replacement, whereas the anti-inflammatory Paracoccus, Arthrobacter, and Actinomycetales increased, with a remarkable presence of the Propioniciclava genus in LSAqua groups. The inferred metagenome analysis suggested that these discriminant bacteria could be implicated in a counter-regulatory anti-inflammatory response. It also pointed to an over-representation of mucosal microbes that can potentially be involved in the natural production of antimicrobials in fish fed the experimental diets.

The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. The maximized replacement of fish meal (FM) and fish oil (FO) with plant ingredients has been extensively studied in the economically important species gilthead sea bream (Sparus aurata). Recently, major progress has been done with other alternative raw materials, though some non-pathological inflammatory response persisted with feed formulations that increased the circularity of resource utilization. In the present study, we evaluated the effects on growth performance, gene expression, intestinal microbiota and disease resistance of a FM-free diet (NoPAP SANA), based on plant ingredients, aquaculture by-products, algae oil, insect meal and bacterial fermentation biomasses as main dietary oil and protein sources, and supplemented with a commercially available health-promoting feed additive (SANACORE®GM). Juveniles of 21 g initial body weight were fed control or NoPAP SANA diets for 34 days, and head kidney, liver and posterior intestine were collected for gene expression analyses using customized PCR-arrays. Each tissue-specific PCR covered 96 genes in total and included markers of growth performance, lipid and energy metabolism, antioxidant defence, immune system, and intestinal function and integrity. From the same fish, the adherent bacteria of the posterior intestine were studied by Illumina sequencing of the V3-V4 region of the 16S rRNA. The remaining fish were challenged with the intestinal parasite Enteromyxum leei for 78 days and sampled for parasite diagnosis. Both control and NoPAP SANA fish grew efficiently considering gilthead sea bream standards. Before parasite challenge, the NoPAP SANA group showed differential expression of 17, 2 and 4 genes in liver, head kidney and posterior intestine, respectively. The intestinal bacterial composition showed no major differences in diversity or at the phylum level. However, 29

[Introduction]: The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. Tolerance to fish meal (FM) and fish oil replacement in the economically important gilthead sea bream (Sparus aurata) is being extensively studied with many products emerging as alternative feed ingredients. It has been demonstrated that alternative diets influence the composition of intestinal adherent microbial populations, which have a key role on host metabolism, health and disease resistance. In addition, low fish meal diets showed an increased susceptibility to enteric parasites (Piazzon et al., 2017). Clearly, differences in diet have an impact on the overall health and metabolism of the fish and many parameters have to be taken into account when studying alternative diets for their use in aquaculture. In this study we evaluated the effect of a novel feed formulation (NoPAP SANA) with total replacement of FM by insect meal and bacterial fermentation biomass, and supplemented with the health-promoter additive SANACORE®GM (Palenzuela et al., 2020), on growth performance, gene expression, intestinal microbiota and disease resistance in gilthead sea bream., [Methods]: Tagged gilthead sea bream of mean weight 21.3 g were distributed in two open-flow tanks (160 fish/tank) and fed ad libitum during 34 days with control or NoPAP SANA diets. Twelve fish/diet were sacrificed and head kidney (HK), liver (L) and posterior intestine (PI) were taken for RNA extraction. From the same fish, the adherent bacteria of PI were collected and immediately used for DNA extraction. RNA from HK, L and PI was used to run three customized PCR-arrays including genes of interest for each tissue, with markers of performance and metabolism (L), immune system (HK and PI), epithelial integrity, nutrient transport and mucins (PI). Using the bacterial DNA, the V3-V4 region of the 16S rRNA of each individual sample was amplified and sequenced by Illumina MiSeq. After quality filtering, taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq and beta diversity using PERMANOVA and PLS-DA models. Metagenome prediction and pathway analysis were performed using Piphillin. Differential gene expression and OTU presence and abundance correlations were studied using the corrplot R package. From the remaining fish, 70 fish/group were challenged with the intestinal parasite Enteromyxum leei by effluent exposure and the remaining fish were used as controls. The challenge lasted 78 days, including a non-lethal diagnosis sampling at day 40. At the end of the challenge all fish were sampled for histological and molecular diagnosis. Biometric values from all fish were taken in all sampling points., [Results]: A slight decrease in condition factor and specific growth rate was detected in the NoPAP SANA group. However, all fish grew efficiently considering gilthead sea bream standards. NoPAP SANA group showed differential expression of 17 out of 44 genes in L, two out of 29 in HK, and 4 out of 44 in PI. The bacterial composition at the PI showed no major differences in diversity or at the phylum level. However, 29 abundant (>1%) OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differential expression of genes in the different tissues, highlighting Pseudoxanthomonas which was positively correlated with the expression of seven L genes, or Actinomyces, significantly correlated with the expression of L and HK genes (Fig. 1). Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge was not significantly different between diets. In fact, infected fish from both groups showed similar recovery rates., [Conclusions]: NoPAP SANA promoted good growth parameters and efficient conversions arising as a good alternative for a FMbased diet in gilthead sea bream diets. This diet modulated the expression of several genes in L showing the capacity to reduce lipogenesis, mitochondrial activity and the risk of oxidative stress and, at the same time, promoting an antiinflammatory gene expression profile in HK and PI. Changes were also detected in the adherent bacterial populations of PI, with significant changes of OTUs that could potentially account for significant metabolic alterations. The correlations between presence and abundance of intestinal bacteria with changes in gene expression of different tissues, together with the pathway analysis results, show that microbiota changes can have an impact on host metabolism at a systemic level, and vice versa. Clearly, the changes induced by this novel FM-free diet supported an accelerated growth with an overall feed conversion ratio close to 1 and no increased susceptibility against this intestinal parasite, as often observed in studies when replacing a FM-based diet.

The present study aimed to unravel the effects of partial (50%; 50LSAqua) and total (100%; 100LSAqua) replacement of fish meal (FM) by a commercial protein source (LSAqua SusPro) made of bacterial and processed animal proteins (PAP) in farmed juveniles of gilthead sea bream (Sparus aurata). The trial lasted 8 weeks, and the feasibility of replacement was assessed in terms of growth performance, histopathological scoring and composition of mucosal adherent microbiota from anterior intestine (AI). Specific growth rates (SGR) of 50LSAqua fish were undistinguishable from the CTRL group, whereas a slight but significant growth impairment was found with the total replacement. Histological signs of inflammation across the intestine were more evident at the highest level of FM replacement, and the total concentration of short chain fatty acids (SCFA) in stripped feces decreased in a dose dependent manner. Illumina sequencing of gut mucosal microbiota yielded a mean of 130,439 reads per sample assigned to 1,567 OTUs at 97% identity threshold. The bacterial richness was similar in all groups, but a significantly higher Simpson diversity index was found in 100LSAqua fish. At the phylum level, Proteobacteria were the most abundant in all groups, whereas Firmicutes decreased and Actinobacteria increased with the FM replacement. At a closer look, pro-inflammatory Gammaproteobacteria of the genus Psychrobacter, and Acinetobacter decreased with FM replacement, whereas the anti-inflammatory Paracoccus, Arthrobacter, and Actinomycetales increased, with a remarkable presence of the Propioniciclava genus in LSAqua groups. The inferred metagenome analysis suggested that these discriminant bacteria could be implicated in a counter-regulatory anti-inflammatory response. It also pointed to an over-representation of mucosal microbes that can potentially be involved in the natural production of antimicrobials in fish fed the experimental diets.

Hake, mackerel, horse mackerel, anchovy and sardine larvae were identified from samples of ichthyoplankton collected during eight surveys in the Bay of Biscay between 2000 and 2005. Comparative analyses were carried out to explore the early growth patterns of these species based on differences in body proportion and the interpretation of the otolith microstructure to estimate their age. Statistical differences in development rates were observed for different body proportions. On the one hand, the larvae of hake, mackerel and horse mackerel prioritise the growth of those parts of the body related to feeding (body weight, head and jaw), while those of anchovy and sardine prioritise those related to swimming (body length). We propose that these different strategies are adaptations to reduce larval mortality, each of them focusing on the main factor that controls it during the early stages: starvation and predation. These growth strategies are interpreted from an environmental perspective mainly related to the characteristics of the food and the abundance of predators in the environment in which the larvae develop during this vulnerable life stage.

[Introduction]: Sparicotyle chrysophrii is a polyopisthocotylean monogenean (Microcotylidae) parasite of gilthead sea bream (Sparus aurata) (GSB). It attaches to the gills and it can cause Sparicotylosis, which often involves severe anaemia and white-gill syndrome. The disease is ubiquitous across sea cages in the Mediterranean and it causes direct and indirect economic losses to the GSB farming sector. S. chrysophrii infection intensity determinations are necessary for appropriate application and coordination of farms’ health management plans, aiming at keeping parasite presence below threatening thresholds. In the farms, these counts are tedious and time-consuming. Parasite loads are usually extrapolated from partial counts, e.g., of a number of gill arches from one or both sides. However, different protocols and indexes are used by the industry. In previous studies, we have demonstrated the hematophagous nature of S. chrysophrii and its direct role in GSB anaemia. The aim of the current study is to explore for a fast, non-lethal approach to predict the parasitic burden of affected GSB using haematological parameters. Therefore, a large data set on infection intensity and parasite distribution, as well as biometrical and haematological data from laboratory experimental infections and from commercial cages, have been gathered and analysed., [Materials and methods]: Fish sampling, Hematological data and Parasite Counts Fish kept under experimental (N = 150) and farming conditions (N = 388) were euthanized and bled from the caudal vein. Haematocrit values were recorded after standard microhematocrit capillary tube centrifugation, and haemoglobin values obtained using the HemoCue ® Hb 801 System. Gill arches were dissected and either inspected thoroughly under stereomicroscopes, or processed by a high-throughput method based on an incubation of the gill arches in a diluted formalin solution, followed by concentration and final counting of parasites in a Sedgewick-Rafter chamber. The number and type of life stages (juveniles and adults) were registered and the performance of both counting methods compared to assess the total parasite burden. Sparicotyle chrysophrii distribution A Kruskal-Wallis H test was performed in order to analyse the distribution of adults, juveniles and the total parasite population throughout the gill arches. Haematology analysis Haematocrit and haemoglobin values were correlated using Pearson correlation coefficient and the R2 value was calculated. Prediction models Partial counts sensitivity was assessed for farm data (N = 2 fresh gill arches per fish counted with scope) and laboratory data (N = 6 remaining arches processed for Sedgewick-Rafter counts) from commercial GSB cages. Several generalised linear models (GLMs) including haematocrit and haemoglobin variables were explored in order to predict the number of S. chrysophrii adults in affected GSB. All the statistical analyses, were conducted using R Statistical Software (version 3.6.1; R Foundation for Statistical Computing, Vienna, Austria)., [Results]: Sparicotyle chrysophrii distribution Parasite presence in the different arches presented a non-normal distribution in the different arches of the left and right gills. No significant differences were found in neither adult, juvenile, nor total parasite counts among the different gill arches. Moreover, no significant differences were found in total parasite counts between the left and right gill arches. Haematology analyses Correlations between haematocrit and haemoglobin values were highly significant in experimental, farm and in combined experimental and farm data. Prediction models. Different iterations were explored with subsets of data extrapolated from total or partial counts. Significant differences were found in the predicted burden values calculated from partial count data from two and six gill arches, being the latter the most reliable. Correlations between adult parasite load and haematocrit and between adult parasite load and haemoglobin values were highly significant for data from experimental and farming conditions (Fig. 1). For our entire dataset, a negative binomial regression model was obtained, [Discussion and Conclusions]: No bias associated to specific gill arches of GSB could be found in the distribution of juveniles or adults of S. chrysophrii, and therefore, extrapolation of the total parasite load can be performed from parasite counts in a limited number of arches. However, the sensitivity of the partial counts in two gill arches was significantly inferior, showing up to 66.67% of false negatives in the earliest stages of infection. A high-throughput method was developed for S. chrysophrii counting from GSB gill arches, which delivers highly sensitive and reliable intensity values. Haemoglobin and haematocrit values proved to be reliable parameters, which are strongly correlated with the parasite load in GSB infected with S. chrysophrii. Furthermore, haematological data will allow the assessment of parasite burden in a fast and non-lethal manner by means of the prediction models developed.

Background: Myxozoan parasites infect fish worldwide causing significant disease or death in many economically important fish species, including rainbow trout and steelhead trout (Oncorhynchus mykiss). The myxozoan Ceratonova shasta is a parasite of salmon and trout that causes ceratomyxosis, a disease characterized by severe inflammation in the intestine resulting in hemorrhaging and necrosis. Populations of O. mykiss that are genetically fixed for resistance or susceptibility to ceratomyxosis exist naturally, offering a tractable system for studying the immune response to myxozoans. The aim of this study was to understand how steelhead trout that are resistant to the disease respond to C. shasta once it has become established in the intestine and identify potential mechanisms of resistance. Results: Sequencing of intestinal mRNA from resistant steelhead trout with severe C. shasta infections identified 417 genes differentially expressed during the initial stage of the infection compared to uninfected control fish. A strong induction of interferon-gamma and interferon-stimulated genes was evident, along with genes involved in cell adhesion and migration. A total of 11,984 genes were differentially expressed during the late stage of the infection, most notably interferon-gamma, interleukin-6, and immunoglobulin transcripts. A distinct hardening of the intestinal tissue and a strong inflammatory reaction in the intestinal submucosa including severe hyperplasia and inflammatory cell infiltrates were observed in response to the infection. The massive upregulation of caspase-14 early in the infection, a protein involved in keratinocyte differentiation might reflect the rapid onset of epithelial repair mechanisms, and the collagenous stratum compactum seemed to limit the spread of C. shasta within the intestinal layers. These observations could explain the ability of resistant fish to eventually recover from the infection. Conclusions: Our results suggest that resistance

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.

This study aimed to highlight the relationship between diet, animal performance and mucosal adherent gut microbiota (anterior intestine) in fish fed plant-based diets supplemented with an egg white hydrolysate (EWH) with antioxidant and anti-obesogenic activity in obese rats. The feeding trial with juveniles of gilthead sea bream (Sparus aurata) lasted 8 weeks. Fish were fed near to visual satiety with a fish meal (FM)/fish oil (FO) based diet (CTRL) or a plant-based diet with/without EWH supplementation. Specific growth rate decreased gradually from 2.16% in CTRL fish to 1.88% in EWH fish due to a reduced feed intake, and a slight impairment of feed conversion ratio. Plant-based diets feeding triggered a hyperplasic inflammation of the anterior intestine regardless of EWH supplementation. However, EWH ameliorated the goblet cell depletion, and the hepatic and intestinal lipid accumulation induced by FM/FO replacement. Illumina sequencing of gut mucosal microbiota yielded a mean of 136,252 reads per sample assigned to 2,117 OTUs at 97% identity threshold. The bacterial diversity was similar in all groups, but a significantly lower richness was found in EWH fish. At the phylum level, Proteobacteria reached the highest proportion in CTRL and EWH fish, whereas Firmicutes were decreased and Actinobacteria increased with the FM/FO replacement. The proportion of Actinobacteria was restored by dietary EWH supplementation, which also triggered a highest amount of Bacteroidetes and Spirochaetes. At a closer look, a widespread presence of Lactobacillales among groups was found. Otherwise, polysaccharide hydrolases secretors represented by Corynebacterium and Nocardioides were increased by the FM/FO replacement, whereas the mucin-degrading Streptococcus was only raised in fish fed the plant-based diet without EWH. In addition, in EWH fish, a higher abundance of Propionibacterium was related to an increased concentration of intestinal propionate. The antagonism of gut health-pr

[Introduction]: The sustainability and physiological effects of high inclusion levels of new feed ingredients is often questioned, and their potential application requires the use of conventional methodologies, but also cutting-edge tools, for unravelling the close talk between diets, host metabolism and gut microbiota (Fontinha et al., 2020). In this challenging scenario, the aim of the present study was to evaluate, in a high valuable farmed fish model (gilthead sea bream), the effects on growth performance and gut microbiota of partial (50%) and total (100%) replacement of fish meal (FM) by a combination of processed animal proteins and single cell proteins., [Methods]: The feeding trial (8 weeks, May-July) with control (CTRL) and experimental diets (50LSAqua, 1000LSAqua) was conducted in triplicate 500 L tanks under natural photoperiod and temperature conditions in a flow through system with well aerated sea water (O2 concentration > 5.5 ppm). Fish with an initial body weight of 23 g were fed near to visual satiety one time per day, six days per week. Nine fish per group were sacrificed, and the adherent bacteria from the anterior intestinal portion were collected and immediately used for DNA extraction. The V3-V4 region of the 16S rRNA of each individual sample was amplified and sequenced by Illumina MiSeq. After quality filtering, taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq, and beta diversity using PERMANOVA and PLS-DA models. Metagenome prediction and pathway analysis were performed using Piphillin., [Results]: All fish grew efficiently, with feed conversion ratios between 1.09 and 1.19 (no statistical differences). Specific growth rates did not vary significantly between CTRL and 50LSAqua fish, though a slight decrease of growth rate (5%) was found with the total FM replacement (100LSAqua). Organosomatic weight indexes of liver, mesenteric fat and intestine were not altered by dietary treatment, but intestine length was shortened by the strategy of FM replacement. In addition, the concentration of lactic acid, was increased in stripped faeces of fish fed LSAqua diets. When fish of fish fed LS-Aqua diets were put together (50/100LSAqua), the bacterial richness was similar in both fish groups, but a remarkably lower diversity was found in CTRL fish. At the phylum level, Proteobacteria was the most abundant phylum, constituting ≥ 55% of the total resident bacteria in the anterior intestine. Firmicutes was the second most abundant phylum in the CTRL group (~20%) with a significant decrease (10.3%) in the 50/100LSAqua group. Conversely, Actinobacteria raised from 11.9% in CTRL fish to 23.2% in 50/100LSAqua fish, whereas the abundance of Bacteroidetes remained almost constant (~4.5%) in both fish groups. PLS-DA analyses showed significant differences in the microbial composition among dietary groups. For these discriminant bacteria, a first type of response was mediated by 27 OTUs overrepresented in fish fed 50/100LSAqua diets. In this group, it was remarkable the presence of Verrucomicrobia and Chlamydiae phyla, the class Betaproteobacteria and the genera Paracoccus, Omithinimicrobium, Tetrasphaera, Rubellimicrobium and Butivibrio. A second type of response grouped 20 OTUs with a significantly higher proportion in CTRL fish. This group included Bacteroidetes and Cyanobacteria phyla, Chromatiales, Bacillales and Methylococcales orders and Aggregatibacter, Clostridium sensu stricto, Acinetobacter, Rhodolyulum, Albimonas, Propioniclava and Psychrobacter genera. Inferre, [Conclusions]: These results point to a reduced activity of IL-17 pathways in fish fed LSAqua diets, which, among their functions, is to limit proliferation of resident bacteria (Douzanez-Mobarrez et al., 2019). This anti-inflammatory response would be reinforced by the over-representation in the gut microbiota of OTUs involved in flavones and flavonoids biosynthetic processes. At the same time, host defence and inflammation processes would be counter-regulated by the overrepresentation of mucosal microbes involved in the natural production of antimicrobials, which would contribute to control proliferation of specific bacteria in our model of carnivorous fish fed with FM-free diets.