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Siete investigadores del CSIC de los grupos de investigación especializados en acuicultura están incluidos en el listado de la Universidad de Stanford que recoge el 2% de los investigadores más influyentes en el mundo (World’s Top 2% Scientists 2022) en su última edición de 2022. [...]

Resumen del trabajo presentado en el Mucosal Health in Aquaculture, celebrado en Madrid (España) del 03 al 06 de octubre de 2022., Secreted mucins are highly O-glycosylated glycoproteins of high molecular mass, produced by goblet cells in mucosal epithelia. These complex biomolecules have a long peptide backbone, the apomucin of 5,000-13,000 amino acids, coated by hundreds of extremely diverse carbohydrate sidechains accounting for 80% of the mucin’s molecular mass. Mucin molecules multimerize in a mesh, constituting the viscous gel layer overlying the epithelia, which renders physical protection against erosion and dehydration and maintains homeostasis, offers the substrate for the resident and transient microbial communities, and is involved in pathogen recognition, adhesion and expulsion. The monogenean Sparicotyle chrysophrii is responsible for the widespread sparicotylosis in gilthead sea bream (GSB), invoking severe anaemia and compromising the fish’s immune condition. This ectoparasite attaches to the secondary lamellae of the gills with numerous haptor clamps and feeds on fish blood. This work aimed to identify changes in the mucus secretion of the gills of GSB suffering sparicotylosis, particularly changes related to mucus composition and goblet cell abundance and distribution. Understanding the underlying molecular mechanisms of goblet cell and mucin regulation will be addressed at a transcriptional level. Control unexposed (C, n = 23) and recipient (R, n = 28) GSB experimentally infected with S. chrysophrii were sampled at seven consecutive times: 0, 11, 20, 32, 41, 61 and 81 days post-exposure (dpe). Gills were sampled for paraffin histology (Bouin-fixed) and PCR array (RNAlater-preserved). Paraffin sections were stained with periodic acid Schiff (PAS) to demonstrate neutral mucins or with alcian blue (pH 2.5) to identify acidic mucins, and distinct terminal glycoconjugates were detected by lectin labelling (Concanavalina ensiformis, ConA; Ulex europaeus, UEA; Triticum vulgaris, WGA; Glycine max, SBA; Griffonia simplicifolia, BSL I; Sambucus nigra, SNA). Goblet cell abundance was estimated semiquantitatively using the different stainings at four different gill locations (filament tip, interlamellar pockets, lamellar epithelium and raker epithelium), and the intensity of each lectin labelling in these goblet cells was semiquantitatively registered. In addition, the presence and staining of discharged extracellular mucus were registered. Neutral goblet cells were ubiquitous in all gill locations. Their amount significantly increased at filament tips, interlamellar pockets and lamellar epithelia of R fish from 32 dpe on. Mucins did not acidify during the course of infection, but their glycosylation pattern varied depending on the specific gill site. The increase of SBA-label and decrease of SNA- and UEAlabel were signs of immature mucin secretion and a reduced capacity for microbial trapping. Histological observations demonstrated the onset of goblet cell hyperplasia after the first month of parasite exposure, matching a host response for adult worm expelling. The immature mucin features of the increased mucus secretion might limit the host’s capacity to regulate mucosal protection. The transcriptomic study of goblet cell and mucin regulation will shed some light on the underlying mechanisms.

Resumen del trabajo presentado en Mucosal Health in Aquaculture (MHA 2022), celebrado en Madrid (España) del 03 al 06 de octubre de 2022., In parallel with the growth of the aquaculture industry, an increasing interest in fish microbiota research has grown to improve fish welfare and nutrition, as it plays a critical role in protection against pathogens, nutrient digestion and absorption, excretion of nitrogenous wastes, and osmotic regulation. Thus, the characterization of microbial communities is pivotal for detecting potential alterations that lead to fish homeostatic imbalance. During intensive fish farming, high rearing densities can be achieved, and this can dramatically reduce water oxygen availability, especially with the summer increase of temperature. This study aimed to assess how mild hypoxia and relatively high stocking densities affect gilthead sea bream skin microbiota, and how these results could correlate with different operational welfare indicators (OWIs), in order to improve fish health and aquaculture productivity. Hereby, two-year-old gilthead sea bream (450-500 g) were tagged and distributed in 3,000 L tanks to achieve three different initial rearing densities (low, LD: 6 kg/m3; medium, MD: 12 kg/m3, high, HD: 22 kg/m3). Fish were fed close to satiety with a commercial diet from May to June (8 weeks) under natural photoperiod and temperature conditions at our latitude (40°5’N;0°10’E), varying the concentration of dissolved oxygen from 6-5 ppm in LD fish to 5-4 ppm in MD fish, and 4-3 ppm in HD fish. At the end of the trial, growth rates of HD fish were 40% lower in comparison to MD/LD fish. At this stage, randomly selected fish (12 fish per group) were evaluated for external tissue OWIs, using a scoring system from 1 to 5, which detected different levels of fin damage and skin epidermal lesions in HD fish and to a lower extent in MD fish. The same fish were used to obtain skin mucus samples for microbiota analysis, the 16S rRNA v1-v9 regions were sequenced with the Oxford Nanopore Technology (ONT) MinION device, and then processed with an in-house bioinformatics pipeline to analyze skin microbial composition. Discriminant analysis showed that the skin microbiome of the three different groups differed in bacterial abundance. However, in all the groups the dominant phyla were Proteobacteria (73-94%), Firmicutes (2-15 %), and Bacteroidetes (1-7%), and so as the family Alteromonadaceae (11-45%). Changes in bacterial composition, together with OWIs of external tissue damage can provide new insights into how crowding and hypoxia conditions have an effect on fish welfare and microbiota.

Resumen del trabajo presentado en Aquaculture Europe, celebrado en Rimini (Italia) del 27 al 30 de septiembre de 2022., [Introduction]: The AquaIMPACT H2020 EU project aims to integrate selective breeding and nutrition for producing more sustainable marine fish with a higher phenotypic plasticity to deal with environmental challenges. At this interplay between diet and genetics, gut microbiota emerge as a reliable criterion to assess the selection practices. However, the influence of the host genome on the composition and activity of gut microbiota is still scarcely investigated in gilthead sea bream (Piazzon et al., 2020). Thus, the aim of this study was to unravel the main effects of alternative diet formulations along the production cycle of gilthead sea bream selected for high growth (HG) compared to reference (REF) fish, establishing correlations between intestinal gene expression and gut microbiota abundance., [Methods]: HG and REF gilthead sea bream were fed CTRL (15% FM; 6-8% FO) and FUTURE (7.5% FM; 12% poultry sub-products and 3% DHA-rich algae oil) diets during 12 months at the experimental facilities of ECOAQUA, at ULPGC, under natural photoperiod and temperature conditions. Initial (t0; Nov-19; 10 g body weight; 18 fish), intermediate (Jul-20; t1; 200 g; 36 fish) and final (Nov-20; t2; 350 g; 40 fish) sampling points were established to assess the dynamics of anterior intestine (AI) mucosal adherent microbiota composition across the production cycle. As the most prominent differences in gut microbiota among groups were found in t1, fish of this sampling point were selected for RNA-sequencing. V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq, and taxonomy was assigned using the RDP database. RNA-seq libraries were sequenced by Illumina NovaSeq6000. Good-quality reads were mapped against CSIC gilthead sea bream genome and differential expressed transcripts (DET) were retrieved using DESeq2 (FDR < 0.05). Overrepresentation analyses of GO and KEGG terms were implemented in goseq and protein-protein association networks were retrieved using STRING (FDR < 0.05). DET expression and OTUs abundances of the equivalent fish of t1 were correlated using Spearman correlation tests (P < 0.001)., [Results]: Illumina sequencing and taxonomic assignment of the 94 samples allowed the identification of 1,897 OTUs at 97% identity threshold along the gilthead sea bream production cycle. Taking into account the entire population, Chao1 richness index progressively decreased with age, whereas the α-diversity Simpson index was significantly higher in t1 and t2 than in t0. Proteobacteria was the most abundant phylum (64-78%) in all the sampling times, followed by Firmicutes (13-15%), Actinobacteria (6-12%), and Bacteroidetes (~2%). The core microbiota of gilthead sea bream along the production cycle was constituted by 25 OTUs (>1% in at least one of the three sampling times). A validated PLS-DA (Figure 1A) rendered the separation of the three experimental time points. The FUTURE diet induced a shift in the gut microbiota composition of REF fish, validated by PLS-DA, but not in HG fish. The influence of the genetic background was already evidenced at t0, when discriminant analyses separated HG from REF fish. The magnitude of this separation and the number of discriminant taxa persisted along time, but progressively decreased in t1 and t2 in fish were fed CTRL diet. By contrast, HG and REF fish microbiota was not distinguished when fed the FUTURE diet. In t1, RNA-seq mapped reads were associated to 41,582 AI transcripts, with 1,429 DET (1,218 unique descriptions). Group comparisons displayed 1,057 and 459 DET differentially regulated between HG-CTRL and REF-CTRL fish, and between HG-FUTURE and HG-CTRL, respectively. A total of 1,025 significant associations were established between the abundance of 96 OTUs and the expression of 476 DET. A total of 35 correlations between 20 abundant (>1%) OTUs and 25 DET were stablished (Figure 1B). The 60% of the correlated OTUs were associated to DET related with immune system, with a clear presence of Bacilli linked to anti-inflammatory responses. The microbiota-independent list of DET (953) showed the over-representation of transcripts involved in β-oxidative processes of very long chain fatty acids (up-regulated in HG-CTRL) and smooth muscle contraction (upregulated in HG-FUTURE)., [Conclusions]: These results highlight the plasticity of gilthead sea bream gut microbiota along its production cycle, showing the effects of size and season. As in previous studies (Naya-Català et al., 2022), the genetic background strongly influenced the gut microbial composition and the intestinal response of this species, as selected fish exhibited a more plastic microbiota and more changes at the transcriptomic level, when facing dietary changes. Genes related with cell membrane fluency were up-regulated in HG-CTRL fish, whereas FUTURE diet boosted genes involved in intestinal motility in both fish groups. Correlation studies denoted the close link between immune system genes and the intestinal microbiota. Altogether, these results show how diet and genetics can affect the microbiota and the digestive process, and highlight that certain taxa could be interacting with the gilthead sea bream transcriptome., AquaIMPACT (H2020; #818367). RYC2018-024049-I & ESF.

Trabajo presentado en Aquaculture Europe, celebrado en Rimini (Italia) del 27 al 30 de septiembre de 2022., [Introduction]: Studies in gilthead sea bream evidenced that high stocking densities exacerbate the negative impact of reduced oxygen availability, showing transcriptional analyses a different contribution of analyzed tissues (liver ≥ heart > muscle > blood) to the hypoxic- and crowding stress-mediated responses to cope with a changing environment (Martos-Sitcha et al., 2019). Moreover, blood metabolic and muscle transcriptomic landmarks indicate that mild hypoxia induces a hypometabolic state, increasing the contribution of lipid metabolism to the whole energy supply to preserve the aerobic energy production during exercise (Naya-Català et al., 2021a). The intestinal microbiota also has key effects on host health and welfare, and these complex populations tightly interact with the host affecting local and systemic physiological functions (Naya-Català et al., 2021b). Thus, the aim of the current study was to assess how mild hypoxia and high stocking rearing conditions affect the intestinal health of gilthead sea bream by applying a hologenomic approach to determine the host-microbiota interactions., [Methods]. Two-year-old gilthead sea bream (450-500 g) were tagged and distributed in 3,000 L tanks to achieve three different initial rearing densities (low, LD: 6 kg/m3; medium, MD: 12 kg/m3; high, HD: 22 kg/m3). Fish were fed close to satiety with a commercial diet from May to June (8 weeks) under natural photoperiod and temperature conditions at our latitude (40°5’N;0°10’E), varying the concentration of dissolved oxygen from 6-5 ppm in LD fish to 5-4 ppm in MD fish, and 4-3 ppm in HD fish. At the end of the trial, the growth rate of HD fish was 40% lower than that of MD/LD fish, and randomly selected fish from each group were taken to obtain tissue and mucus samples from the anterior intestine. RNA extracted from the anterior intestine from 10 fish/group was sequenced using NovaSeq PE150 (50 M reads/sample), and reads were assembled and mapped against the gilthead sea bream genome. DESeq2 was used to extract genes with P < 0.05 in all comparisons, that were used to perform discriminant and cluster analysis to determine the number of differentially expressed genes among conditions. K-means, GO and KEGG enrichment analyses were subsequently performed. The V3V4 regions of the 16S rRNA of the mucus DNA samples from the same animals were 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. Correlations between differentially expressed genes and discriminant bacteria were studied using the corrplot R package., [Results]: RNAseq and discriminant analyses revealed that a total of 2,813 differentially expressed genes significantly separated the three groups (Fig. 1A). K-means analysis divided these genes into four different clusters according to their expression patterns in each group, separating one cluster of 800 genes with higher expression in LD, a second cluster of 1,103 genes with higher expression in MD, a third cluster of 688 genes with higher expression in HD, and a fourth cluster of 222 genes that gradually increased expression with the stocking density (LD, [Conclusions]: Crowding and mild hypoxia had a significant impact on gut health, evidenced by significant changes in host intestinal transcriptome and associated microbial population. These results offer the possibility of developing new tools and approaches for a more precise evaluation of welfare in farmed fish. The ultimate goal is to mitigate the negative impact of stressors related to intensive rearing and climate change on aquaculture production, promoting a more ethical and sustainable production., AQUAEXCEL3.0 (H2020 #871108), EATFISH (H2020 #956697).

Trabajo presentado en la 15th International Conference of Parasitology, celebrada en Copenhague (Dinamarca) del 21 al 26 de agosto de 2022., [Introduction]: Sparicotylosis is a ubiquitous disease affecting mainly gilthead sea bream (GSB; Sparus aurata) across the Mediterranean, and it is caused by the polyopisthocotylean monogenean gill parasite, Sparicotyle chrysophrii (Microcotylidae). The disease involves severe anaemia and white-gill syndrome. This study aims to elucidate how the parasite modulates the GSB health status throughout the infection., [Methods]: Experimental infections were conducted in a recirculating aquaculture system (RAS), exposing naïve GSB (R) to effluent water from S. chrysophrii-infected GSB. An additional tank with unexposed naïve fish (C), was maintained in parallel, with open water flow disconnected from the RAS. Haematological and infection parameters from sampled C and R fish were registered. Plasma samples grouped into 4 categories (high-H, medium-M, low-L and control-C) according to infection intensities, underwent a proteome analysis. Additional assays were performed to validate the proteome analysis findings., [Results]: The differential analysis of the plasma protein abundance revealed a clear separation into 3 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 homeostasis, complement system and lipid transport. Twenty-two proteins significantly correlated with the infection intensity, highlighting apolipoproteins, globins, biotinidases and complement C3. Validation assays in blood and plasma confirmed these correlations., [Conclusions]: The anaemic condition of severely S. chrysophrii-infected GSB is accompanied by innate immunity impairment, and altered lipid profile and biotin metabolism, besides the evident hypoxia.

Trabajo presentado en el 15th International Conference of Parasitology, celebrado en Copenhague (Dinamarca) del 21 al 26 de agosto de 2022., [Introduction]: Emaciative microsporidiosis due to Enterospora nucleophila is considered an emerging problem in gilthead sea bream farming. Its main impact is the severe growth retardation, normally accompanied by sustained trickling mortality, which is only noticed with high intensity of infection. The aim of the current study was to assess the possible points of entry of this intestinal parasite into the farming facilities, by using qPCR screening in a longitudinal epidemiological survey. [Methods]: Fish were sampled at different critical control points during two production cycles in Spain (C1) and Greece (C2), involving 2 hatcheries, 3 nursery conditions, 4 pre-fattening facilities, and 5 sea cage farms with different entering seasons, from egg to commercial size. Artemia and rotifers were also analysed. [Results]: After analysing 1,930 samples, the microsporidium was found in all the farms, but not in all farming events. Fish were negative for the parasite from the egg to the fry stage just after weaning in all sampled hatcheries-nurseries, no matter what type of water rearing system was used. Live prey was also negative. The first positive cases were detected in C2 when entering the nursery vs when leaving the nursery in C1. The smallest fish harbouring the parasite weighed 0.9 g. Before stocking into the sea cages, the highest prevalence of infection (100 %) was detected in a pre-fattening facility in which fish spent a longer period than usually, with an open seawater flow, without filtering. The parasite was found in all sea cages with prevalence ranging from 10 to 72.4%. [Conclusions]: The ubiquitous presence of E. nucleophila in the farms emphasize the importance of incorporating its routine detection into the management and biosecurity protocols of farms. [Funding]: ParaFishControl H2020 EU project (634429).

Trabajo presentado en el 15th International Conference of Parasitology, celebrado en Copenhague (Dinamarca) del 21 al 26 de agosto de 2022., Parasites are a major and recurrent problem in aquaculture, with heavy economic impact. These costs include those due to mortality, morbidity, poor growth, reduction of feed efficiency, parasitic castration, treatments and biosecurity measures, mort disposal, etc. A brief overview of the main fish parasites affecting marine aquaculture, with emphasis on sea cage farming in Europe will be presented. To understand the current situation some reflections will be provided about what should be done to set-up efficient means of control in order to hold parasites in a sustainable way. Aquaculture production often occurs in open or semi-open systems, where the level of containment and biosecurity applicable could never reach what is feasible in terrestrial farming. Sea cages pose a challenge for parasite control: 1) water cannot be controlled; 2) biofouling in nets and ropes favours entangling of some stages and the live of invertebrates that can be intermediate hosts; 3) juveniles are stocked sometimes from distant areas, bringing with them exotic parasites; 4) surrounding wild fish can be natural or reservoir hosts; and 5) non-controlled effluents (mucus casts, faeces, dead animals, escapees) allow parasite life cycles keep going. Sustainable control has to be based on three major pillars: 1) deep knowledge of the parasite, this means to know its life cycle, to achieve its in vitro culture, to develop an in vivo experimental infection, and to decipher all its biological weapons; 2) knowledge of the environment, including farm management, water conditions and diets; and 3) knowledge of the host, including the host-parasite interaction and the immune response, which will allow to introduce immunomodulatory measures and future vaccines. Funding: ParaFishControl H2020 EU project (634429).

Trabajo presentado en el XXII Congreso de Parasitología, celebrado en Madrid (España) del 05 al 08 de julio de 2022., En los últimos 25 años, la producción de peces marinos en España ha experimentado un constante y notable crecimiento, centrado primariamente en el cultivo de lubina, dorada y rodaballo. Además, se ha realizado un gran esfuerzo para la diversificación, llegando a una producción ya consolidada y creciente de otras especies de valor estratégico como lenguado, seriola, o corvina. Durante este tiempo, la búsqueda de mayores rendimientos productivos, junto a la exploración de distintos modelos de cultivo han puesto en evidencia el potencial de ciertas parasitosis para llegar a niveles epizoóticos y comprometer la viabilidad de numerosas instalaciones. Muchos de estos episodios han sido causados por especies de parásitos desconocidas hasta ese momento, que en algunos casos emergieron en la industria de modo catastrófico. Sin embargo, la posterior caracterización y estudio de estas parasitosis ha permitido comprobar que, con frecuencia, se trata de especies ubicuas, y que estos episodios están asociados a modelos de producción y circunstancias muy concretas, que en algunos casos son evitables. Una vez alcanzada cierta madurez en los modelos productivos para cada especie, la convivencia con las parasitosis es la norma, particularmente en sistemas abiertos y sin control de parámetros ambientales o tratamiento del agua. En este contexto, son varias las especies de ecto- y endo-parásitos responsables de mermas constantes y significativas en la producción, y que frecuentemente resultan invisibles. Junto con unos costes de operación incrementados y un potencial para generar brotes clínicos de importancia, la presión generada por las parasitosis sobre las explotaciones de acuicultura hace necesario un mayor esfuerzo para su prevención y control. Esta presentación hace un repaso a las infecciones parasitarias más relevantes en especies y sistemas de acuicultura marina españoles, haciendo hincapié en la problemática generada por algunas de ellas sobre etapas y sistemas de producción concretos. Se señalarán las principales carencias de conocimiento y necesidades de investigación, que es necesario abordar para generar herramientas que mitiguen el impacto de estas infecciones.