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DEMERSTEM- Objectives of WP 1: -Improve the knowledge on the selected demersal stocks with a particular effort to solve the problems of stock identification and improvement of the quality of the data used for the assessment. -This follows the recommendations regularly made by CECAF.

Investigations of population structuring in wild species are fundamental to complete the bigger picture defining their ecological and biological roles in the marine realm, to estimate their recovery capacity triggered by human disturbance and implement more efficient management strategies for fishery resources. The Blackspot Seabream (Pagellus bogaraveo, Brünnich 1768) is a commercially valuable deep-water fish highly exploited over past decades. Considering its exploitation status, deepening the knowledge of intraspecific variability, genetic diversity, and differentiation using high-performing molecular markers is considered an important step for a more effective stock assessment and fishery management. With one of the largest efforts conceived of and completed by countries overlooking the Atlantic and Mediterranean coasts in recent years, a total of 320 individuals were collected from different fishing grounds in the Mediterranean Sea and Atlantic Ocean and analysed using 29 microsatellite loci. We applied multiple statistical approaches to investigate the species’ connectivity and population structure across most of its described distribution area. Considering the incomplete knowledge regarding the migratory behaviour of adults, here we suggest the importance of egg and larval dispersal in sustaining the observed genetic connectivity on such a large geographical scale.

[ES] La conservación y adecuada gestión de los recursos vivos marinos precisa caracterizar genéticamente las poblaciones. La genética de poblaciones y evolución emplea marcadores moleculares y modelos matemáticos para conocer los cambios en la composición genética de las poblaciones debidos a factores evolutivos. La diferenciación entre poblaciones de una especie es clave en su evolución y en ella influyen tanto factores físicos como biológicos. En los ecosistemas marinos factores físicos como la batimetría o la hidrografía, condicionan la estructura de las poblaciones y la conservación de la diversidad genética, que subyace a la biodiversidad marina. El objetivo de este capítulo es realizar una revisión de algunos trabajos que han investigado la estructura genética de animales del mar de Alborán y de los factores que favorecen o limitan el intercambio genético entre poblaciones. La conectividad genética entre poblaciones del mar de Alborán está influenciada por dos importantes factores físicos: el estrecho de Gibraltar y el frente Almería-Orán, que pueden tanto obstaculizar como favorecer el intercambio genético entre poblaciones. En especies de animales invertebrados, los factores físicos pueden favorecer la aparición de poblaciones diferenciadas o pueden dar lugar a patrones de aislamiento por distancia. Factores biológicos como el comportamiento migratorio o reproductor de una especie, a su vez, también condicionan la conectividad genética, y en algunas especies de animales vertebrados podrían posibilitar la diferenciación genética entre poblaciones atlánticas y mediterráneas., [EN] The conservation and management of marine living resources requires the genetic characterization of populations. Populations genetics and evolution uses molecular markers and mathematical models to understand the changes in the genetic composition of popu lations due to evolutionary factors. The differentiation between populations of a species is key to its evolution and can be influenced by both physical or biological factors. In marine ecosystems, physical factors such as bathymetry or hydrography influence population struc ture and conservation of genetic diversity, which underlies marine biodiversity. The objective of this chapter is to review some of the works that have investigated the genetic structure of animals of the Alboran Sea and the factors that favour or limit genetic exchange between populations. Genetic connectivity between populations in the Alboran Sea is influenced by two important physical barriers, the Strait of Gibraltar and the Almería-Oran oceanographic front, which can hinder or favour genetic exchange between populations. In invertebrate animal species, physical barriers may favour the appearance of differentiated populations or may lead to patterns of isolation by distance. Biological factors such as the migratory or reproductive behaviour of a species, in turn, also influence genetic connectivity, and in some species of vertebrate animals enable genetic differentiation between Atlantic and Mediterranean populations.

Introduction: The diversification of species in aquaculture requires the continuous study of animal welfare parameters and the susceptibility to pathogens. An inadequate welfare state results in stress, poor health and increased severity of infectious diseases. In fact, several stress conditions have been related with increased mortalities and betanodavirus (NNV) spread. The shi drum (Umbrina cirrosa) has attracted attention due to its good growth rates and great adaptability to culture conditions together with a great flesh quality. However, this species is susceptible to the four genotypes of NNV and displays stress behavior at high rearing densities. Methodology: In order to determine the response of shi drum upon NNV infection at different stressed conditions, specimens reared at low or high density were experimentally infected. A transcriptomic study was performed comparing the gene expression pattern of several tissues of infected and non-infected specimens from the group showing mortalities. The presence of NNV in the gonad of survivors was analyzed. Results: The mortalities observed were low but only occurred at the group reared at high density. The transcriptomic study showed that the number of tissue-specific genes expressed increased in head-kidney, liver and brain, and decreased in spleen. In the four tissues, cell adhesion, leukocyte migration, cytokine interaction, cell proliferation, cell survival and autophagy related pathways were differentially expressed. NNV was detected in the gonad of surviving fish at all rearing densities analyzed. Conclusions: No differences in the viral capacity of colonizing the gonad were observed between low- and high-density conditions. At this last condition, NNV infection produced a deep systemic alteration in shi drum. The main pathways altered were related with immune response, cell cycle and autophagy, accordingly with studies performed in other fish species.

Autophagy is an intracellular degradation process playing an important role in homeostasis. Viral infections can modulate autophagy and, when induced, can be host-beneficial. To date evidence points to the interest of investigating strategies that upregulate autophagy as fish virus preventive treatments. Nodavirus (NNV) is a great threat for Mediterranean aquaculture as it produces severe natural outbreaks leading to great economic losses to the industry. Regarding autophagy, the NNV capsid protein is able to induce autophagic processes. Antimicrobial peptides (AMPs) are short proteins with lytic activity against a broad range of pathogens, and exert great regulatory effects on the immune response. Recently, in insects, a positive relationship between the AMP and autophagy-related genes has been successfully established. Thus, the objective of this work was to analyze whether the administration of synthetic AMPs might regulate the NNV-induced expression of autophagy-related genes. To accomplish our objective, hepcidin and dicentracin peptides were intramuscularly administered to European sea bass juveniles and a day later infected with NNV. Brain, the NNV target tissue, and muscle, the site of infection, were removed in order to ascertain NNV replication in both tissues as well as analyze the transcription levels of autophagy-related genes. Mortality rates were recorded daily. Our data show that NNV replicates in both studied tissues and AMPs administration resulted in lower mortality rates. The transcription profile observed in brain suggests that the treatment with AMPs prior to NNV infection induces autophagy in the main target site of the virus. Therefore, the autophagy induced by AMPs could be an effective mode of viral clearance and could contribute to the increase of survival during NNV infection. Funded by MCIN/AEI 10.13039/501100011033 (grants RTI2018-096625-B-C33, PID2019-105522GB-100 and PRE2020-093771 to L.C) and “Fundación Séneca” (grant 19883/GERM/15).

Spain leads the aquaculture production in the European Union; however the appearance of new competitors in the Mediterranean area resulted in lower prices for most cultured species. In this framework shi drum (Umbrina cirrosa) has attracted attention due to its good growth rates and great adaptability to culture conditions together with a great flesh quality. An inadequate welfare state results in stress, poor health and increased severity of infectious diseases, including those caused by viral pathogens. The betanodavirus (NNV) is the causative agent of viral encephalopathy and retinopathy and infects more than 170 fish species. Nowadays, NNV is a great concern due to its high prevalence in the Mediterranean basin. In addition, several kind of physiological and induced stress conditions have been related with increased mortalities and NNV spread. The diversification of species in aquaculture requires the continuous study of the physiological process of stress to determine appropriate animal welfare parameters for each species together with the study of the susceptibility to pathogens. Shi drum is susceptible to the four genotypes of NNV, although with different clinical signs and replication rates. To increase the knowledge on the biological aspects of fish welfare in general and of shi drum in particular, we have studied different rearing densities and their impact on the response to stress and to NNV infection. Our data show an increase in stress behavior in the highest density that in turn affects the physiological performance of fish. This study was financed by the project OWI TWO (Operational welfare indicators in two aquaculture species) cofunded by the European Maritime and Fisheries Fund (EMFF) and IEO-CSIC, and by MCIN/AEI 10.13039/501100011033 (grants RTI2018-096625-B-C33 and PID2019-105522GB-100) and “Fundación Séneca” (grant 19883/GERM/15).

Infectious diseases are a burden for aquaculture. Antigen processing and presentation (APP) to the immune effector cells that fight pathogens is key in the adaptive immune response. At the core of the adaptive immunity that appeared in lower vertebrates during evolution are the variable genes encoding the major histocompatibility complex (MHC). MHC class I molecules mainly present peptides processed in the cytosol by the proteasome and transported to the cell surface of all cells through secretory compartments. Professional antigen-presenting cells (pAPC) also express MHC class II molecules, which normally present peptides processed from exogenous antigens through lysosomal pathways. Autophagy is an intracellular self-degradation process that is conserved in all eukaryotes and is induced by starvation to contribute to cellular homeostasis. Self-digestion during autophagy mainly occurs by the fusion of autophagosomes, which engulf portions of cytosol and fuse with lysosomes (macroautophagy) or assisted by chaperones (chaperone-mediated autophagy, CMA) that deliver proteins to lysosomes. Thus, during self-degradation, antigens can be processed to be presented by the MHC to immune effector cells, thus, linking autophagy to APP. This review is focused on the essential components of the APP that are conserved in teleost fish and the increasing evidence related to the modulation of APP and autophagy during pathogen infection.

The production of the aquaculture industry has increased to be equal to that of the world fisheries in recent years. However, aquaculture production faces threats such as infectious diseases. Betanodaviruses induce a neurological disease that affects fish species worldwide and is caused by nervous necrosis virus (NNV). NNV has a nude capsid protecting a bipartite RNA genome that consists of molecules RNA1 and RNA2. Four NNV strains distributed worldwide are discriminated according to sequence homology of the capsid protein encoded by RNA2. Since its first description over 30 years ago, the virus has expanded and reassortant strains have appeared. Preventive treatments prioritize the RGNNV (red-spotted grouper nervous necrosis virus) strain that has the highest optimum temperature for replication and the broadest range of susceptible species. There is strong concern about the spreading of NNV in the mariculture industry through contaminated diet. To surveil natural reservoirs of NNV in the western Mediterranean Sea, we collected invertebrate species in 2015 in the Alboran Sea. We report the detection of the RGNNV strain in two species of cephalopod mollusks (Alloteuthis media and Abralia veranyi), and in one decapod crustacean (Plesionika heterocarpus). According to RNA2 sequences obtained from invertebrate species and reported to date in the Mediterranean Sea, the strain RGNNV is predominant in this semienclosed sea. Neither an ecosystem- nor host-driven distribution of RGNNV were observed in the Mediterranean basin.

Highly migratory Atlantic bluefin tuna (ABFT) is managed as two stocks, Western and Eastern. Western ABFT spawn mainly in the Gulf of Mexico (GOM) and Eastern ABFT in the Mediterranean Sea (MED) (1). Understanding connectivity between ABFT populations is important for conservation and management of this valuable fishery resource that has been exploited for centuries. ABFT are highly mixed, with multiple disciplines supporting weak structuring between Western and Eastern stocks (1). Concerning genetics, subtle structuring of ABFT populations across the Atlantic Ocean has been the conclusion of studies describing genetic tools for traceability (2,3). Larval fish provide the genetic signal of successful breeders and have occasionally been genetically characterized with juveniles (young-of-the-year, YOY) collected in nursery areas. For the first time, cooperative field collection of tuna larvae during 2014 in the main spawning area for each stock enabled us to assess the structuring of ABFT genetic diversity in a precise temporal and spatial frame exclusively through larvae (5). Partitioning of genetic diversity at nuclear microsatellite loci and in the mitochondrial control region resulted in low significant fixation indices. Individual-based clustering analysis of larval ABFT genetic diversity indicate apparent connectivity between the GOM and MED spawning grounds that could support the hypothesis of mixing of breeders belonging to different stocks.

The spawning grounds of the Atlantic bluefin tuna (Thunnus thynnus) are traditionally considered to be the Gulf of Mexico (Gulf of Mexico) and the Mediterranean Sea (Mediterranean Sea). However, for the western Atlantic, unequivocal evidence of bluefin spawning outside the Gulf of Mexico has been shown. In this study we present the first records of genetically confirmed bluefin larvae in the southern Bay of Biscay (eastern Atlantic). These findings provide evidence of bluefin spawning activity outside the Mediterranean Sea, in the north-eastern Atlantic. However, our results suggest that the bluefin spawning in the Bay of Biscay is a sporadic phenomenon.

The diversity of protists was researched in the Alboran Sea (SW Mediterranean Sea) by means of high-throughput sequencing technologies based on the amplification of the V9 region of 18S rRNA. Samples were collected at different depths in seven stations following an environmental gradient from a coastal upwelling zone to the core of an oligotrophic anticyclonic gyre (AG). Sampling was performed during summer, when the water column was stratified. The superphyla Alveolata, Stramenopila and Rhizaria accounted for 84% of the total operational taxonomic units (OTUs). The most diverse groups were Dinophyceae (21% of OTUs), Marine Alveolates-II (MALV-II; 20%), Ciliophora (9%) and MALV-I (6%). In terms of read abundance, the predominant groups were Dinophyceae (29%), Bacillariophyta (14%), MALV-II (11%) and Ciliophora (11%). Samples were clustered into three groups according to the sampling depth and position. The shallow community in coastal stations presented distinguishable patterns of diatoms and ciliates compared with AG stations. These results indicate that there was a strong horizontal coupling between phytoplankton and ciliate communities. Abundance of Radiolaria and Syndiniales increased with depth. Our analyses demonstrate that the stratification disruption produced by the AG caused shifts in the trophic ecology of the plankton assemblages inducing a transition from bottom-up to top-down control.

The heavy exploitation rates of Atlantic bluefin tuna (ABFT) during the nineties propitiated research into the larval ecology of ABFT and its associated species. The transition from a planktivorous to a piscivorous diet is considered a major bottleneck in the survival of ABFT larvae observed in aquaculture experiments. Although larval piscivory was reported in the Gulf of Mexico (GOM), the most important spawning grounds of this species in the W Atlantic, trophic studies have not been able to reveal piscivory in Mediterranean ABFT larvae. This study analyzes the trophic behavior of Mediterranean ABFT larvae by stomach content analysis. The results show that more than 90% of the larvae had at least one prey in their stomachs. The diet shifted from copepods and cladocerans to gastropod larvae in pre-flexion stages and to ABFT larvae in post-flexion stages. This is the first time that cannibalism is reported for wild ABFT larvae from the Mediterranean Sea. Intracohort cannibalistic feeding was observed when the requisite density-dependent processes aligned, namely the spatio/temporal overlap of a wide range of ABFT larval cohorts of different size class. Moreover, stomach contents of ABFT larvae revealed the ingestion of microplastic fibres. Whether these plastic contaminants were passively or actively ingested, they may affect the condition of larvae. The presence of microplastic strands in fish larvae undoubtedly raises concern because its impact on the survival of ABFT larvae still remains uncertain and is open to scientific experimentation.

The spawning grounds of the Atlantic Thunnus thynnus are traditionally considered to be the Gulf of Mexico (GOM) and the Mediterranean Sea (MED). However, for the western Atlantic, unequivocal evidence of T. thynnus spawning outside the GOM has been presented. In this study, we show evidence for T. thynnus reproduction outside the MED in the eastern Atlantic. Using opportunistic ichthyoplankton samplings, 9 T. thynnus larvae were collected during July 2010 and 2015, in the Southern Bay of Biscay (SBB). The microscopic identification of Atlantic bluefin larvae, based on morphologic and meristic characters and pigmentation patterns was confirmed by genetic analysis. As for Sarda sarda, the spawning grounds for this species in the eastern Atlantic are located in the neritic regions of the tropical and subtropical Atlantic and of the MED. However, 6 larvae of this species were collected in the SBB since 2001. Therefore, the SBB should be evaluated as a spawning ground for T. thynnus, since its larvae appeared in 33% of the stations sampled during July 2010 in the slope-oceanic region, where this species reproduces.

Nowadays, the production of marine aquaculture is already the first source for the human supply of marine products. In order to guarantee that the aquaculture production meet its objectives, further studies on health and welfare are mandatory. Between them, the distribution, prevalence and infective capacities of the main pathogens of cultivable species are worthy to be performed. In this work we have studied the prevalence of nodaviruses in two species of marine invertebrates (a crustacean and a cephalopod) in the Mediterranean Sea. As far as we are concern, this work demonstrates, for the first time, the presence of the virus in cephalopods in the Mediterranean Sea. It is worthy to highlight the great similarity of the sequences obtained with different isolates of the genotype RGNNV.