Author: marina02

Unraveling the mechanisms of freeze-dried Lactiplantibacillus plantarum in strengthening the immunity of Penaeus vannamei shrimp against acute hepatopancreatic necrosis disease

The lack of a classical adaptive immunity in shrimp necessitates a multi-pronged approach in the development of the disease control and prevention measures in shrimp. In this study, we implemented conventional and omics-based strategies to investigate the potential of freeze-dried whole culture of Lactiplantibacillus plantarum (FD-LAB) as a feed supplement in strengthening the immunity of the Penaeus vannamei shrimp against the acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus. Shrimp was fed with feed supplemented with FD-LAB for 15 days and tissues were collected at specific time points during the feeding period for RNA sequencing using Illumina platform. Transcriptome analyses identified several differentially-expressed genes reported to be related to innate immunity and immune memory. Additionally, differentially-expressed immune-related circular RNAs were also explored as these noncoding regulatory RNAs may also play a role in host immunity. Aside from the impact of FD-LAB feed supplementation on host innate immune system, its effects on the bacterial communities of the shrimp were also investigated. 16S rRNA metagenome sequencing using Illumina platform also revealed that FD-LAB feed supplementation induces specific changes in the gill and gut microbiota without causing dysbiosis as shown in the nonsignificant changes in alpha and beta diversity indices. Several taxa previously reported to be beneficial on shrimp, such as members of Rhodobacteraceae, Rubritaleaceae, actinomycetes, and lactic acid bacteria were enriched in both tissues. Additionally, in vitro analyses showed that FD-LAB interferes with the virulence and pathogenicity of V. parahaemolyticus AHPND strain (VP_AHPND). Lastly, artificial challenge tests using conducted immediately after FD-LAB feeding yielded protection on shrimp against VP_AHPND. Significant increase in survival rates were also observed upon VP_AHPND challenge 7 days and 14 days after the last intake of FD-LAB, indicating trained immunity in shrimp. These findings unraveled the mechanisms of FD-LAB in improving the immune status of the shrimp, emphasizing its potential application as an immunostimulant inducing lasting enhanced immunity against infections in shrimp.

1. Guzman, John Paul Matthew, Tokyo University of Marine Science and Technology (JPN) & Industrial Technology Development Institute (PHL), Presenter
2. Nozaki, Reiko, Tokyo University of Marine Science and Technology (JPN), Author
3. Konishi, Kayo, Tokyo University of Marine Science and Technology (JPN), Author
4. Mwamburi, Samuel Mwakisha, Tokyo University of Marine Science and Technology (JPN) & Kenya Marine and Fisheries Research Institute (KEN), Author
5. Aoki, Mikio, Sumitomo Chemicals Co. Ltd. (JPN), Author
6. Kuwahara, Hiroshi, Sumitomo Chemicals Co. Ltd. (JPN), Author
7. Mikata, Kazuki, Sumitomo Chemicals Co. Ltd. (JPN), Author
8. Koiwai, Keiichiro, Tokyo University of Marine Science and Technology (JPN), Author
9. Kondo, Hidehiro, Tokyo University of Marine Science and Technology (JPN), Author
10. Hirono, Ikuo, Tokyo University of Marine Science and Technology (JPN), Author

Transcriptomic analyses in European seabass during Vibrio anguillarum infection: a beneficial modulation of disease response induced by marine synbiotic

Introduction

The intensification of aquaculture, coupled with climate change, heightens infection risks in fish farms. Probiotics are a promising strategy to mitigate biotic/abiotic stressors, enhance fish health, and – consequently – reduce environmental impact. A two-month long dietary supplementation of juvenile seabass with a synbiotic of marine probiotics bio-encapsulated in algae (MA_N1) improved resistance to Vibrio anguillarum (+18% survival) combined with a beneficial modulation of their intestinal microbiota. Gene expression in spleen, a key organ of the immune system, was analyzed by RNA-Seq to (1) study the disease response in non-supplemented seabass and (2) identify mechanisms potentially involved in survival gain induced by synbiotic supplementation.

Methodology

RNA extractions were realized on spleens from 12 fish per condition (Control and MA_N1) at Day 0 (end of the dietary supplementation) and 24 hours after the V. anguillarum intraperitoneal injection. RNA extracts were sequenced with polyA enrichment method and Illumina 150-paired-ends sequencing. Sequences were trimmed and mapped to the European seabass reference genome to identify Differentially Expressed Genes (DEGs) between sampling times (D0 vs 24h) and between Control and MA_N1 synbiotic conditions. Graphical output and Gene Ontology (GO) annotations were analyzed using the AskoR package.

Results

After 24 hours of infection, 4,173 DEGs were identified in non-supplemented fish (Control) compared to D0 (Control 24h vs Control D0), for which GO annotation revealed their link to innate immune activation and disease biomarkers. On contrast, downregulation of some genes related to antigen recognition and cytokine activity suggests that the pathogen may disturbed the immune response. Fish supplemented with marine synbiotic also showed innate immune activation, including complement components and pro-inflammatory cytokines. However, a total of 217 DEGs were significantly downregulated post-infection compared to non-supplemented fish (MA_N1 24h vs Control 24h). These genes are linked to immune response, ion homeostasis, bacterial defense, blood coagulation or negative regulation of apoptosis pathways, indicating a modulation of the disease response. The pathways targeted by the synbiotic will be described with the objective to elucidate the possible mechanisms of protection against Vibrio anguillarum.

Conclusion

These results suggest that marine synbiotic supplementation, in addition to a beneficial modulation of the gut microbiota, modifies significantly the disease response with a more controlled innate immune response in juvenile seabass 24h after V. anguillarum infection, resulting in significant survival gain. This study highlights the potential of marine synbiotics as a sustainable strategy to improve fish health in aquaculture.

1. Le Bihan, Claire, Marine Akwa, Presenter
2. Grippon, Pauline, ANSES VIMEP, Author
3. Lannuzel, Guillaume, ANSES VIMEP, Author
4. Danion, Morgane, ANSES VIMEP, Author
5. Fleury, Yannick, LBCM, Author
6. Morin, Thierry, ANSES VIMEP, Author
7. Gautier, Camille, Marine Akwa, Author
8. Giudicelli, Fanny, Marine Akwa, Author

does timing matter: effects of β-glucan supplementation duration on the gut microbiome and health of juvenile nile tilapia

supplementing fish diet with prebiotics, e.g., β-glucans, has been used as an alternative to antibiotic treatment to boost the non-specific immune system and modulate the gut microbiota profile, thereby enhancing gut mucosal health and improving resistance against diseases. however, such benefits are mostly studied from older fish, and little is known on early life stages. in this study, we aimed to evaluate the effects of β-glucan supplementation periods during early-life feeding of nile tilapia (oreochromis niloticus) on growth performance, gut microbiota profile, and the regulation of immune responses.

we fed 10-day-old nile tilapia with six diet combinations over a period of six weeks by switching between a diet containing insoluble β-glucans—at low-(l) or high-(h) concentration—and a control diet (c), without supplementation. the diet was switched after the first two weeks (lcc, lcl, clc, hcc), or the fish were fed the same diet throughout the experiment (ccc, lll). gut samples were collected every two weeks and used for dna and rna extraction to analyze gut microbiota profile and immunomodulation, respectively. gut microbiota profile was assessed using oxford nanopore technology sequencing, targeting full-length 16s-rrna genes. immunomodulation was assessed by measuring the relative expression of cytokine genes (il1b, tnfa, ifnγ, il10, il12p40) and tight junction protein (tjp) genes (claudin3 and marveld) using rt-qpcr. following the feeding trial, fish were immune-challenged with inactivated streptococcus iniae for 48h, after which the expression of cytokine and tjp-genes was analyzed.

our results showed no significant effects of supplementation on fish growth. we also found no effects of supplementation on gut microbial diversity at the end of feeding trial. however, regardless of the administration protocol, we observed an increased abundance of potential fermentative bacteria (romboutsia and paraclostridium) in all β-glucan-fed groups. the ongoing 16s-rrna sequencing analyses of gut samples collected at week 2 and 4 are expected to provide further understanding how different administration protocols alter gut microbiota composition. following the immune challenge with inactivated bacteria, we observed a downregulation of expression of the cytokine il1b and tjp marveld genes in fish from the lll and hcc groups, suggesting that continuous supplementation, or short high-dose exposure, may induce more pronounced effects.

finally, our results underscore the potential of β-glucans supplementation in modulating gut microbiota of juvenile tilapia towards beneficial taxa and suggest that different administration protocols can lead to different patterns of expression of health-related genes, although further (ongoing) analysis is needed to support these conclusions.

1. FINO, HANISWITA AMRIZAL, WAGENINGEN UNIVERSITY, Presenter
2. WIEGERTJES, GEERT F, WAGENINGEN UNIVERSITY, Author
3. KOKOU, FOTINI, WAGENINGEN UNIVERSITY, Author

Vector Optimization for Efficient Expression of Oplegnathus fasciatus Interferon-Gamma in Chlorella vulgaris

Red sea bream iridoviral disease (RSIVD) remains a persistent challenge in the aquaculture industry, particularly affecting Oplegnathus fasciatus, where it causes high mortality and significant economic losses. To develop an effective oral vaccine against RSIVD, we aimed to express O. fasciatus interferon-gamma (Of IFN-γ), a key cytokine involved in antiviral immune responses. Microalgae have emerged as promising platforms for oral vaccine delivery to combat this issue. Chlorella vulgaris is a strong candidate as a platform for an oral vaccine because it is GRAS (Generally Recognized as Safe), easy to culture at low cost, and is a eukaryotic organism that enables post-translational modification. However, the low efficiency of recombinant protein expression via genetic transformation in C. vulgaris limits practical vaccine development.

Thus, we created various vector constructs to enhance the expression of recombinant protein from C. vulgaris. These vectors were constructed by combining three promoters with different strengths (SIP, 35S, Psad) and three terminators (RBCS2, NOS, Psad). Each vector was transformed into the C. vulgaris PKVL7422. In the case of vectors containing the SIP promoter, an inducible promoter, recombinant protein expression was induced by exposing transformed C. vulgaris cells to 250 mM NaCl for three days. Protein expression levels in each C. vulgaris strain transformed with the different vector constructs were assessed by SDS-PAGE followed by Western blotting.

Our results revealed that different combinations of promoters and terminators significantly affected the levels of recombinant protein expression. Notably, vectors containing the SIP promoter, which is inducible under salt stress conditions, exhibited increased expression following treatment with 250 mM NaCl for three days. By optimizing the combination of promoters and terminators, we were able to enhance the expression of O. fasciatus interferon-gamma, a key immune-related protein. Overall, this study presents a vector optimization strategy that improves the efficiency of recombinant protein production in C. vulgaris, thereby enhancing the potential of microalgae-based oral vaccine platforms for aquaculture.

1. Sohyeon, Jo, PUKYONG NATIONAL UNIVERSITY, Author
2. Sohyeon, Jo, PUKYONG NATIONAL UNIVERSITY, Presenter
3. Heejin, Ahn, PUKYONG NATIONAL UNIVERSITY, Author
4. Min-Kwan, Sung, PUKYONG NATIONAL UNIVERSITY, Author
5. TeaJin, Choi, PUKYONG NATIONAL UNIVERSITY, Author
6. SuJin, Jang, PUKYONG NATIONAL UNIVERSITY, Author
7. SuYeon, Kim, PUKYONG NATIONAL UNIVERSITY, Author

High-quality genome sequences enable in-depth studies of the genomic structure and pathogenic potential of Aphanomyces astaci genotypes

Aphanomyces astaci, the causal agent of crayfish plague, is a devastating oomycete pathogen that causes high mortalities in ecologically important European crayfish species such as noble crayfish, white-clawed crayfish, and stone crayfish. To date, at least five A.astaci genotype groups (A-E) with a pathogenic potential ranging from causing only chronic infections to rapid death have been identified. It is assumed that the differences in pathogenicity have a genomic component. It is also known from other oomycete pathogens that these organisms often have highly plastic genomes with a high percentage of repetitive elements. The A.astaci reference sequence is made from short-read data and thus quite fragmented (un-gapped length of 58Mb, N50 of 657kb).To study phenomena such as chromosomal rearrangements or effector expansion, high quality genome sequences are required, ideally with telomere to telomere (T2T) contigs.

To this end, we cultivated the five A.astaci genotype groups A-E in liquid culture and sequenced the extracted DNA with PacBio long reads. The data was assembled using Hifiasm and for gene prediction and functional annotation we made use of publicly available resources such as A.astaci transcriptomic data (PRJNA187375) and the Companion server (10.1093/nar/gkae378).

The resulting reference sequences show a high level of completeness and continuity. The assembly of genotype D for example includes all BUSCO genes of Stramenopiles (stramenopiles_odb10: C:100.0%[S:98.0%,D:2.0%], n:100), has an N50 and L50 of 3.7Mb and 12 contigs, respectively. More than 80Mb are contained in contigs with at least one telomer and the six T2T contigs comprise 30Mb. The high continuity of these genome assemblies allows comparative analyses of the five genotypes on the single nucleotide level, but more importantly also enables the assessment of the genomic structures, in particular long genomic or chromosomal re-arrangements. Differences in the genetic make-up of the genotypes, for example in the composition of effector genes, might help us to better understand the differences in pathogenicity. They are also a valuable resource for large scale gene expression studies in A.astaci aiming at shedding light on the mechanism of infection.

1. OBERHAENSLI, SIMONE, UNVERSITY OF BERN, Presenter
2. JEMMI, ELIANE, UNVERSITY OF BERN, Author
3. NICHOLSON, PAMELA, UNVERSITY OF BERN, Author
4. PISANO, SIMONE, UNVERSITY OF BERN, Author
5. SCHMIDT-POSTHAUS, HEIKE, UNVERSITY OF BERN, Author

16S rRNA NGS-based characterization of a Flavobacterium-driven skin pathology in rainbow trout

Introduction

Columnaris disease, a group of Flavobacterium species known collectively as Columnaris Causing Bacteria (CCB), is a well-recognized threat in freshwater fish farming worldwide, including salmonids. In recent years, unexplained peracute skin disease outbreaks with similarities to CCB have been reported in rainbow trout (Oncorhynchus mykiss) farms in Northern Italy, with cumulative mortality rates of up to 80% within a week. Affected fish displayed distinctive dorsal skin discoloration and scale lifting, but no internal organ involvement. Despite successful control using oxytetracycline, standard bacteriological and virological diagnostics failed to consistently identify a causative agent, raising the suspicion of an undescribed or overlooked bacterial pathogen.

Methodology

An integrated approach combining necropsy, histopathology, scanning electron microscopy (SEM), and 16S rRNA next-generation sequencing (NGS) was used to clarify the etiology. During an outbreak, skin samples and organ pools were collected from healthy fish (Healthy T0), moribund fish (Moribund T0), and survivors after antibiotic treatment (T1). Microbial DNA was extracted from skin samples and sequenced targeting the 16S rRNA V3-V4 regions. The resulting data were processed through standard bioinformatic pipelines to assess microbial composition, diversity, and taxonomic profiles. Comparative analyses were carried out to evaluate shifts in microbial communities across groups and to compare detected Flavobacterium species with known CCB.

Results

Histological examination revealed sharply demarcated areas of epidermal necrosis, scale pocket dilation, and filamentous bacteria within the denuded superficial dermis. SEM confirmed the presence of bacterial aggregates within scale pockets. NGS results showed a striking microbial shift; diseased fish were overwhelmingly dominated (≈68% of reads) by Flavobacterium spp., particularly Flavobacterium bernardetii, F. aquicola, and F. hiemivividum. Sequence alignments confirmed that these species differed from the classical CCB group, reducing the likelihood of misidentification. Antibiotic treatment successfully halted mortality and reduced the presence of Flavobacterium spp. down to<1>

Conclusions

This study describes a previously unrecognized, high-mortality skin disease in rainbow trout closely resembling CCB but likely caused by an uncharacterized Flavobacterium species. By integrating molecular, histopathological, and ultrastructural analyses, it was possible to gain critical insight into the nature of the lesions and identify putative bacterial agents not detectable through conventional diagnostics. These findings highlight how NGS can provide an essential contribution to the study of aquaculture pathogens and stress the importance of sustained efforts to isolate and characterize these organisms to guide the development of precise and effective control measures.

1. Brocca, Ginevra, Aquatic Diagnostic Services, Atlantic Veterinary College, University of Prince Edward Island, Presenter
2. Zamparo, Samuele, Azienda Agricola Erede Rossi Silvio di Rossi Niccola, Author
3. Marroni, Fabio, Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Author
4. Radovic, Slobodanka, IGA Technology Services Srl., Author
5. Castellano, Ciro, Azienda Agricola Erede Rossi Silvio di Rossi Niccola, Author
6. Torge, Diana, Department of Life, Health and Environmental Sciences, University of L’Aquila, Author
7. Bianchi, Serena, Department of Life, Health and Environmental Sciences, University of L’Aquila, Author
8. Groman, David, Aquatic Diagnostic Services, Atlantic Veterinary College, University of Prince Edward Island, Author
9. Macchiarelli, Guido, Department of Life, Health and Environmental Sciences, University of L’Aquila, Author
10. Muscatello, Luisa Vera, Department of Veterinary Medical Sciences, University of Bologna, Author
11. Volpatti, Donatella, Department of Agricultural, Food, Environmental and Animal Sciences, University of Udine, Author
12. Orioles, Massimo, AULSS 2 Marca Trevigiana, Author

Longitudinal study of molecular and functional characteristics of Saprolegnia isolates from Scottish fish farms and investigation into control methods

Saprolegniosis in salmon is caused by Saprolegnia, which is an oomycete or water mould that can cause serious losses in the aquaculture industry. Saprolegnia only infects fish in the freshwater stage. Therefore, our study aims to provide insight into what Saprolegnia strains are prevalent in fish farms across Scotland and characterizing their distinct phenotypic features, while exploring potential control methods. Samples were collected from water and infected fish from fish farms across the Scotland over the period November 2017 to January 2020. Following purification, species identification was done by PCR amplification analysis targeting the ITS regions. We identified several Saprolegnia species these include S. parasitica, S. ferax, S. diclina and S. australis. Within the S. parasitica clade, we identified 6 different phylotypes of S. parasitica (S1-S6) present in Scottish fish farms based on the ITS sequencing. Our current focus is to do a longitudinal study and compare these isolates with those isolated from the same farms over 5 years later. We are comparing the growth rates of these ‘modern’ versus ‘older’ isolates in response to temperature and other parameters. We will present a comparative phylogenetic analysis to determine if the modern farm isolates have evolved traits over time which would contribute to an increase in pathogenicity. In addition, we are investigating biocontrol methods for Saprolegnia parasitica we tested a novel Pseudomonas bacterium isolated from a fish farm and some other bacteria on up to 41 distinct isolates of Saprolegnia collected from all over the world to investigate its efficiency in killing Saprolegnia species. Here we present our latest results.

1. TUMPA, ISRAT JAHAN, UNIVERSITY OF ABERDEEN, Presenter
2. SHREVES, KYPHER, UNIVERSITY OF ABERDEEN, Author
3. McLAGGAN, DEBBIE, UNIVERSITY OF ABERDEEN, Author
4. VAN WEST, PIETER, UNIVERSITY OF ABERDEEN, Author

Genomics of Tenacibaculum maritimum in British Columbia (Canada) and in-silico investigations into immunogenics and virulence.

Tenacibaculum maritimum, a Gram-negative, obligate marine bacteria, is a causative agent of tenacibaculosis globally, inducing several regional clinical presentations. In British Columbia (BC) Canada, the disease is colloquially known as ‘yellow mouth’ or ‘mouthrot’ in Atlantic salmon marine production, where it results in large-scale mortalities and an increased frequency of antibiotic treatments. T. maritimum has been characterized at the genomic level in New Zealand, generating over 40 complete genomes, but the scale of that research has not occurred in other countries. The purpose of this work was to sequence 30 Canadian T. maritimum isolates using hybrid approaches and characterize population diversity, putative components (e.g., antimicrobial resistance genes, virulence factors, secretion systems) and location (e.g., cytoplasmic, outer-membrane), spatial-temporal relations, and plasmid inference. Isolates cultured from Atlantic salmon with mouthrot were collected in BC waters from 2016 to 2024. Isolates were cultured, and extracted genomic DNA was sequenced using 2^nd (i.e., Illumina MiSeq) and 3^rd (Oxford Nanopore MinION) generation sequencing platforms. Resultant sequences were assembled using Hybracter and subjected to several, publicly available, bioinformatic tools. Of the 30 isolates, 25 generated complete circular chromosomes, ranging from 3.3-3.8 Mbp and 30-32 % GC content, and both multi-locus sequence typing (MLST) and core-genome MLST generated two clades (i.e., TmarCan1 and TmarCan2) in agreement with prior research. Several antimicrobial resistance genes were identified; however, none were identified for florfenicol which is used extensively against mouthrot in BC. Putative virulence factors described in the type-strain were found in all isolates, with several exceptions. Of ~3000 proteins identified, 4-7 were predicted to be in the cell wall and surface, encoding gliding motility, transposase, T9SS type B, and Ig-like domain proteins. No associations were identified among isolates between time and space, suggesting little heterogeneity or dramatic changes among the two clades over time, though several isolates contained several small circular extra-chromosomal elements. This research establishes a baseline of the diversity and abilities of Atlantic salmon derived T. maritimum in BC marine waters and advances the development of disease mitigation and management practices such as targeted vaccine strategies in BC Atlantic salmon production.

1. Nowlan, Joseph, University of Guelph/Vancouver Island University, Presenter
2. Heese, Brianna, University of Guelph/Vancouver Island University, Author
3. Nguyen, Hai, Vancouver Island University, Author
4. Russell, Spencer, Vancouver Island University, Author

Koi Herpes Virus Whole Genome Sequencing Using Nanopore Long Read Sequencing

Introduction

Cyvirus cyprinidallo3, formerly Cyprinid herpesvirus 3 (CyHV-3) and also known as koi herpesvirus (KHV), has the largest genome (295 kbp) among all known herpesviruses. KHV is the aetiological agent of a serious disease that emerged in the 1990s, affecting common carp (Cyprinus carpio) and common carp hybrids. Since its emergence, KHV has spread globally and is listed by the World Organization for Animal Health (WOAH). The common carp is a highly valuable species in aquaculture and the angling industry. Despite KHV’s significance, genomic resources are limited, with only 23 KHV genomes published. This study had the following aims: Develop a long read whole genome sequencing protocol and bioinformatics pipeline for de novo assembly; assess the genomic diversity of KHV isolates from England and Wales between 2003 and 2024; identify polymorphic regions to develop improved markers for molecular epidemiology studies and investigate the association between genotypes and clinical signs.

Methodology

Approx. 60 KHV isolates from historical outbreaks between 2003 and 2024 were chosen based on host species, clinical signs and collection date. Isolates were cultured in common carp brain cells, clarified then concentrated 20x using a VivaSpin^®. Three different DNA extraction methods were trialed to maximise read length during Nanopore sequencing and extracted nucleic acids were assessed for quality, quantity and purity. Libraries were prepared with V14 native barcoding kits and sequenced using R10.4.1 flow cells and a MinION MK1D.

Results and Conclusions

Of the three nucleic acid extraction kits tested, the Quick-DNA HMW MagBead Kit (Zymo, USA, CA) showed the highest DNA titre, best integrity and purest extracts. Median read length (2,514 nt), N50 (20,545 nt) and read quality (Q15.6) were the greatest for the HMW kit. Following sequencing of the outbreak-related isolates modal Q-scores were ≈Q20. KHV genomes were assembled de novo giving ≈272 kb contigs and ≈295 kb complete genomes following curation of the terminal repeats.

Phylogenetic analysis identified isolates from the European, Asian and intermediate lineages. Five highly polymorphic regions were identified from previously published genomes with 4.7 to 10x higher nucleotide diversity. This study makes a significant contribution to the available KHV genomic resources and provides insights into the diversity of outbreak-related KHV isolates in England and Wales.

Keywords: KHV, whole genome sequencing, Nanopore

Funding: We acknowledge the funding from Defra and UKRI for the project SE0574 (GAPDC2)

1. Scott, George, Cefas, Presenter
2. Ryder, David, Cefas, Author
3. Wood, Gareth, Cefas, Author
4. Savage, Jacqueline, Cefas, Author
5. Paley, Richard, Cefas, Author
6. Batista, Frederico, Cefas, Author

Metagenomic analysis of environmental samples for virus surveillance in Galicia (northwest of Spain)

Aquaculture is one of the most important production sectors in Galicia (the northwest of Spain), headed by bivalve aquaculture. However, this sector is currently facing significant losses that may be attributed to the emergence of previously undescribed viruses and changes in virus virulence driven by climate change. Identifying novel viruses will be crucial for implementing outbreak prevention strategies and reducing the impact of pathologies. These factors underscore the need for a method of detection and characterization of viruses present in the environment. Also, an effective concentration method is needed, because of the low concentration of viral particles often found in environmental samples, which compromises the quality of viral material. Five different sample sites in Galician coastal waters were selected based on areas of significant fishery and aquaculture activities. At each site, samples were collected from two distinct zones: Zone 1 and zone 2, corresponding to areas of high and low shellfish productivity, respectively. Here, we implemented an optimized protocol for concentrating viruses from sediments and large volumes of sea water (20 L) by a concentration method using a REXEED-25A column. The concentrate was then ultracentrifuged. Viral concentrates have been successfully obtained. The applied concentration protocol yielded enough viral material from both water and sediment samples. All prepared samples had enough quality and quantity for next generation sequencing (NGS) confirming its suitability for metagenomic analysis. This approach provides a useful tool for identifying the viruses present in these areas and monitoring their evolution. In addition, this method allows the discovery of novel viruses and the evaluation of their potential risk and impact in the aquaculture industry over time.

This study has been financed by project CALIMAR, from the network REDEMAR, Consellería do Mar, Xunta de Galicia, through the European Maritime, Fisheries and Aquaculture Fund (EMFAF)

1. González-Rosales, Emma, ARCUS (Universidade de Santiago de Compostela), Presenter
2. Souto, Sandra, ARCUS (Universidade de Santiago de Compostela), Author
3. Olveira, José G., ARCUS (Universidade de Santiago de Compostela), Author
4. López-Vázquez, Carmen, ARCUS (Universidade de Santiago de Compostela), Author
5. P. Dopazo, Carlos, ARCUS (Universidade de Santiago de Compostela), Author