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A new amoeba species pathogenic for Senegalese sole is described based on ultrastructural analysis and SSU rDNA phylogenetic inference. The parasite presents round to ovoid trophozoites (<5 ¿m) with a high degree of intracellular simplification. No mitochondria were observed, but mitosome-like organelles were present. No cysts could be detected. Phylogenetic analysis confirmed the Senegalese sole parasite as an amitochondriate Archamoeba related to Endolimax nana and Iodamoeba spp., and we tentatively describe it as a new species in the genus Endolimax, Endolimax piscium. However, the genetic distance with E. nana is quite large, with only 60% pairwise identity between both SSU rDNA genotypes. Although the overall topology of the Archamoebae cladograms containing E. piscium was consistent, the support for the branching of Endolimax spp. relative to its closest neighbours was variable, being higher with distance or parsimony-based inference methods than with ML or Bayesian trees. The use of stringent alignment sampling masks also caused instability and reduced support for some branches, including the monophyly of Endolimax spp. in the most conservative data sets. The characterization of other Archamoebae parasitizing fish could help to clarify the status of E. piscium and to interpret the large genetic distance observed between Endolimax species. ; This work was funded by the Spanish Ministry of Science and Innovation and by aquaculture companies through CSIC Internal Research Programmes (Intramural projects). Additional funding was granted by the regional Government (Generalitat Valenciana PROMETEO 2010/006 and ISIC 2012/003). Constenla was supported by a Ph.D. student grant of the Universitat Autònoma de Barcelona ('P.I.F. Programme'). ; Peer Reviewed

We determined the in vitro effect of the azol-derivative antifungal ketoconazole (KZ) on the morphology, growth, and development of teleost fish parasite Ichthyophonus sp. The KZ was delivered to culture medium using liposomes (L) or a lipid emulsion (E) at five different doses (i.e. 5, 50, 100, 200, and 400 μg/ml) for both L and E formulations. Controls consisted of Eagle's minimum essential medium (MEM) supplemented with 10% foetal bovine serum (MEM-10) alone (C-MEM) or containing amounts of L or E equivalent to those used in the KZ100 and KZ400 treatments (i.e. 100L, 400L, 100E, and 400E, respectively). Morphological alterations, such as a decrease in the number of dividing spores and nuclei, and condensation or even destruction of the cytoplasm, were observed using light and electron microscopy in the MEM-cultured organisms receiving KZ formulations, especially with KZ400L preparations, at both 7- and 14-d postinoculation. The KZ treatments also demonstrated a statistically significant inhibition of Ichthyophonus growth in MEM. These treatments also had an inhibitory effect on subsequent Ichthyophonus germination in Earle's fish saline agar (EFSA) medium, which was more evident for L formulations when the organism was treated for 7 d and for E formulations at 14 d. Our results endorse the potential use of KZ for the treatment for ichthyophonosis and provide support to proceed to in vivo assays. ; Funding for this work was obtained from the project PTR93-0073 of the PETRI Program(Spanish Government R&D National Plan). ; Peer reviewed

[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. ; This work was supported by the Spanish Projects Bream-AquaINTECH: From Nutrition and Genetics to Sea Bream Aquaculture Intensification and Technological Innovation, RTI2018-094128-B-100; PROGENSA III: Mejora de la Competitividad del Sector de la Dorada a Través de la Selección Genética, JACUMAR program with support from the European Maritime and Fisheries. Additional funding was obtained from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 818367; AquaIMPACT - Genomic and nutritional innovations for genetically superior farmed fish to improve efficiency in European aquaculture. 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. MCP was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I/AEI/ 10.13039/501100011033 & ESF). EP was funded by a Postdoctoral Research Fellowship (Juan de la Cierva-Incorporación, Reference IJCI-2016-27637) from MINECO. We acknowledge the support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed

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. ; This work has been carried out with financial support from the European Union and the Spanish MINECO under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. APS was contracted under ParaFishControl project, RDP was contracted under the PTA-Program from the Spanish Ministry of Science, Innovation and Universities (PTA2018-015315-I), and MCP under CSIC PIE Project No. 201740E013. NHA was the recipient of a fellowship from the Egyptian Cultural Office in Spain. ; Peer reviewed

© 2021 by the authors. ; Gill monogenean Sparicotyle chrysophrii is considered the most detrimental fish parasite to the Mediterranean aquaculture. Treatment of sparicotylosis relies on frequent gill inspections correlated with the seasonal increase in seawater temperature, application of functional feeds, and treatments with formalin baths where permitted. While the latter is bound to be banned in Europe, other synthetic anthelminthics, such as praziquantel and ivermectin, are prone to induce resistance in the parasites. Therefore, we investigated, in vitro, 14 synthetic and natural compounds against adult S. chrysophrii, developing dose–response modelsm and estimated toxicity levels at 20%, 50%, and 80% parasite mortality. Bactericidal activity of target compounds was also tested in two important aquaculture bacteria; Vibrio harveyi and V. anguillarum, while their potential host toxicity was evaluated in gilthead seabream SAF-1 cell line. Synthetic compound bithionate sodium exerted the most potent toxicity against the monogenean, no host cytotoxicity, and a medium and high potency against two bacterial pathogens. In comparison, target natural compounds were approximately 20 (cedrol) or up to 154 times (camphor) less toxic for the monogenean. Rather than completely dismissing natural compounds, we suggest that their application in combination with synthetic drugs, especially if administered in the feed, might be useful in sparicotylosis treatment. ; This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 634429 (ParaFishControl) and additional support from the Spanish Ministry of Science, Innovation and Universities to the project Sparicontrol (RTI2018-098664-B-100A). This publication reflects the views of the authors only, and the European Commission cannot be held responsible for any usewhichmay bemade of the information contained therein. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Animal Welfare Committee of Institute of Aquaculture Torre de la Sal (IATS-CSIC),with the permit number 2018/VSC/PEA/0240 by the "GeneralitatValenciana".

© 2021 by the authors. ; 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 work has been carried out with financial support from the European Union and the Spanish MINECO under grant projects ParaFishControl (H2020-634429) and AGL2013-R-48560-C2-2-R, respectively. A.P.-S. was contracted under ParaFishControl project. This publication reflects only the authors' view, and the European Commission cannot be held responsible for any use which may be made of the information contained herein. M.C.P. was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I/AEI/10.13039/501100011033 and ACOND/2020 Generalitat Valenciana), and R.P. was contracted under the PTA-Program from the Spanish Ministry of Science, Innovation and Universities (PTA2018-015315-I). Both contracts have been co-funded by the European Social Fund (ESF).We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

[Background]: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested. [Results]: The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome. [Conclusions]: This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach. ; This work has been carried out with financial support from the European Union under grant projects ARRAINA (FP7-KBBE-2011-288,925) to JPS, MP and VK and ParaFishControl (H2020-634429) to ASB. Additional funding has been received from Spanish Ministry of Economy and Competitiveness (MINECO) project no. AGL2013- 48560-R to JPS and ASB, and Generalitat Valenciana (PROMETEOII/2014/085) to ASB. MCP was contracted under CSIC PIE project no. 201740E013 and MINECO FPDI-2013-15741, and IE under APOSTD/2016/037 grant by the "Generalitat Valenciana". ; We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).