Author: marina02

Characterization of viperin in Haliotis discus hannai using transcriptomic analysis after poly(I:C) stimulation

Introduction: Viperin is an interferon-inducible antiviral protein known for its role in innate immune responses in vertebrates. However, its function and expression dynamics in mollusks, particularly in Haliotis discus hannai (Pacific abalone), remain poorly understood. This study aimed to characterize the viperin gene in H. discus hannai and investigate its immune-related expression patterns through transcriptomic analysis following immune stimulation with Poly(I:C), a synthetic analog of viral double-stranded RNA.

Methodology: Abalones were injected with poly(I:C) and sampled on days 2 and 7 post-injection. Hemocyte-derived RNA was extracted and subjected to Illumina-based de novo transcriptome sequencing. Differentially expressed genes (DEGs) were identified using Cuffdiff, and functional annotation was performed via GO and KEGG pathway analyses. The viperin gene was isolated from transcriptome data, cloned using RT-PCR and TA cloning techniques, and its sequence was analyzed. Tissue-specific expression patterns of viperin were evaluated using quantitative real-time PCR across multiple tissues and time points.

Results: Transcriptomic analysis revealed the up-regulation of multiple immune-related genes, including viperin, in response to Poly(I:C). GO and KEGG analyses indicated activation of TLR3-mediated signaling pathways, leading to the induction of antiviral and inflammatory responses. The full-length open reading frame (ORF) of viperin (1,059 bp) was successfully cloned and sequenced. Expression profiling showed that viperin was differentially expressed across tissues, with the highest induction observed in the gill tissue on day 7 post-Poly(I:C) stimulation. This suggests the gill as a key immune-responsive organ in abalone.

Conclusion: This study provides the first molecular characterization of viperin in H. discus hannai and demonstrates its inducible expression in response to viral mimic stimulation. These findings contribute to understanding the innate immune system of abalone and highlight viperin as a potential molecular marker for immune activation and antiviral defense in mollusks.

1. Yoo, Seung Hyun, Pukyong National University, Presenter
2. Park, Yong Jun, Pukyong National University, Author
3. Kim, Jeong Gyu, Pukyong National University, Author
4. Kim, Gun-Do, Pukyong National University, Author
5. Kim, Jong-Oh, Pukyong National University, Author

The secreted novel AID/APOBEC-like deaminase 1 (SNAD1) could play an important role in fish immunology

Introduction: AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a transition from cytidine to uridine. Secreted novel AID/APOBEC-like deaminases (SNADs) are unique among all intracellular classical AID/APOBECs due to their characteristic signal peptide. SNADs are central to antibody diversity and antiviral defence. Currently, no information is available on SNADs, including their protein characterisation, biochemical properties and catalytic activity.

Methodology: We used several in silico and in vivo approaches to better define the biological function of SNAD1. We analysed the phylogeny of SNADs, their common structural features and potential structural variations in fish species. Additionally, we searched published fish data and identified previously ‘uncharacterised proteins’ and transcripts as SNAD1 sequences. To validate the in silico predictions, we performed RT-qPCR-based expression studies of several SNAD1 gene variants in carp during temperature adaptation, as well as during infections with Aeromonas salmonicida, carp edema virus, and koi herpes virus.

Results: Our analysis provides strong evidence of the universal presence of multiple SNAD1 proteins/transcripts in fish, which are expressed after hatching and predominantly in anatomical organs associated with the immune system. A review of immunological research suggests that SNAD1 plays a role in the immune response to infection or immunization, as well as in interactions with the gut microbiota. We also found that SNAD1 is associated with temperature acclimation, environmental pollution and sex-based expression differences, with females exhibiting higher levels. During experimental infections, we observed that SNAD1 gene variants in carp exhibited different response patterns to temperature adaptation and bacterial and viral infections.

Conclusions: This dual sensitivity to environmental and pathogenic stressors highlights the importance of SNAD1 for fish, as it may improve thermotolerance and immune defence. Elucidating the biological role of SNADs is an exciting new area of research relating to the function of DNA and/or RNA editing in fish biology.

1. ADAMEK, MIKOLAJ, UNIVERSITY OF VETERINARY MEDICINE HANNOVER, Presenter
2. Majewska, Anna, Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Author
3. Dietrich, Mariola, Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Author
4. Budzko, Lucyna, Department of Molecular and Systems Biology, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland, Author
5. Figlerowicz, Marek, Department of Molecular and Systems Biology, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland, Author
6. Ciereszko, Andrzej, Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Author

Evolutionary and functional characterization of TNFRSF25 as a putative receptor for TNFSF15 in Rock bream (Oplegnathus fasciatus)

Tumor necrosis factor ligand superfamily member 15 (TNFSF15) and its receptor, TNFRSF25, are integral to immune regulation, inflammation, and apoptosis in mammals. This study aims to identify and characterize the putative TNFSF15 receptor, TNFRSF25, in the rock bream (Oplegnathus fasciatus). Although often labeled as “TNFRSF1A-like” in various databases, phylogenetic, syntenic, and homology analyses have confirmed that this gene is indeed TNFRSF25, which is distinctly separate from TNFRSF1A and clusters with orthologous TNFRSF25 genes found in other teleost species. Despite exhibiting limited sequence identity and weak syntenic conservation with mammalian TNFRSF25, the teleost homolog retains conserved features of the cysteine-rich domain (CRD), including residues essential for ligand binding. Notably, the teleost TNFRSF25 demonstrates a closer evolutionary relationship to vertebrate TNFRSF1A than to its mammalian TNFRSF25 counterpart, indicating a lineage-specific divergence. Expression profiling indicates that TNFRSF25 is highly expressed in blood and spleen, aligning with its relevance to immune functions. Additionally, in vitro stimulation with recombinant rock bream TNFSF15 (RB-rTNFSF15) resulted in a significant upregulation of TNFRSF25 and several downstream immune-related genes (TRAF3, TAB3, NEMO and Caspase 8) in head kidney and spleen cells. These findings contribute to a deeper understanding of the evolutionary and functional dynamics of TNFRSF25 in teleosts and underscore the necessity of characterizing species-specific TNF receptor-ligand interactions to enhance our comprehension of the complexity and diversification of TNF signaling in non-mammalian vertebrates.

1. Ko, Sungjae, Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Presenter
2. Hong, Suhee, Department of Aquatic Life Medicine, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea, Author

TESTICULAR BETA-DEFENSIN TRANSCRIPTIONAL PROFILE IN EUROPEAN SEA BASS

TESTICULAR BETA-DEFENSIN TRANSCRIPTIONAL PROFILE IN EUROPEAN SEA BASS

Laura Cervera^1,2, Yulema Valero^1,2, Alberto Cuesta², Marta Arizcun¹, Elena Chaves-Pozo^1*

¹ Centro Oceanográfico de Murcia (COMU-IEO), CSIC, Spain *elena.chaves@ieo.csic.es

² University of Murcia, Spain

Introduction

Antimicrobial peptides (AMPs) are small peptides with immunomodulatory properties and direct lytic effects on a wide range of pathogens. Among AMPs, beta-defensins (Bdef) are characterized by the presence of conserved cysteines and three disulfide bonds. In certain species mammals or teleost species, the presence of Bdef in the reproductive tract have been reported especially in males. Their presence is key for fighting against certain pathogens which affects gonadal tissues such as betanodavirus (NNV). NNV can reach the gonad and cause a persistence infection which can be transmitted to the highly susceptible progeny. In this context, our project focuses on the role of Bdef in testes via molecular characterization.

Methodology

Testes and ovaries from broodstock of European sea bass were collected at the gametogenesis stage. Testes of healthy specimens were also sampled monthly to study Bdefs coding gene expression during the male reproductive cycle An in vivo infection was performed by injecting male specimens with culture medium alone or containing 106 TCID50 of RGNNV/fish. Testes were removed after 1-, 7- and 15-days post-infection (dpi). All samples were processed for gene expression analysis using qPCR.

Results

The expression levels of bdef1 were similar in testis and ovary while bdef2 expression was higher in testis. Besides, the transcriptional levels of bdef2 were higher than bdef1 in testis. During the male reproductive cycle, the highest expression of bdef1 occurred during spermatogenesis stage while bdef2 was more expressed in spawning stage. During NNV in vivo infection bdef1 was up-regulated at 7 dpi while bdef2 remained unaltered.

Conclusions

Bdef2 seems to be preferentially expressed in the male reproductive tract. Otherwise, bdef1 and bdef2 might have different functionalities in testes since their expression reach their peak at different stages. The pattern of expression of bdef1 is similar to other AMPs in testis upon NNV infection pointing to the antiviral role of bdef1. More efforts are needed to clarify differences in regulation of both transcripts.

Funding

This research was funded by the European Union NextGenerationEU/PRTR, MCIN/AEI10.13039/501100011033 and Fundación Séneca (ThinkInAzulprogramme: PRTR-C17.I1), by MCIN/AEI/10.13039/501100011033 grant PRE2020-093771 to L.C. and by MICIU/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR to Y.V.

Acknowledgements

We would like to thank to the technical staff of the Aquaculture facilities of the COMU-IEO, CSIC.

1. CHAVES-POZO, ELENA, COMU-IEO, CSIC, Presenter
2. Cervera, Laura, University of Murcia, Author
3. Yulema, Valero, University of Murcia, Author
4. Cuesta, Alberto, University of Murcia, Author
5. Arizcun, Marta, COMU-IEO, CSIC, Author

Decrease in abundance ratio of gill-epithelial antigen sampling (GAS) cells along with sexual maturation

During immersion vaccination, gill-epithelial antigen sampling (GAS) cells uptake bacterins, degrade them in the cytoplasm and present antigens to T cells for induction of local immune responses in the gills of rainbow trout. In previous studies, we observed highly varied proportions of GAS cells in the gill-epithelium in each experiment and individuals because we used fish of different ages and strains in various timing. In this study, we investigated the changes in the abundance ratio of GAS cells, serum IgM titer and serum cortisol level in the same strain of rainbow trout throughout two years. The gonad somatic index (GSI) was calculated as an indicator of sexual maturity of fish.

Rainbow trout produced in November 2021 were reared with ground water around 17℃ under natural daylight. Fish were sampled in September, October, November and December 2023, January, March, August, October and December 2024 and January 2025. The gonad was weighed to calculate the GSI as an indicator of sexual maturity from March 2024 onwards. Gill epithelial cells were dispersed using 10 mM EDTA and incubated with the anti-GAS cell monoclonal antibody 2B4-1 and the lectin, Ulex europaeus agglutinin-1 (UEA-1). The proportions of GAS cells (2B4-1⁺/UEA-1⁺) in the epithelial cells were investigated by flow cytometry. Serum IgM titer and cortisol level were measured by ELISA.

There was no remarkable change in the abundance ratios of GAS cells during the fish age were between 1+ year old and 2+ years old. The other parameters also showed no significant change in the period. A significant decrease in the abundance ratio of GAS cells was observed in December 2024 when the fish age reached 3+ years old. Serum cortisol levels significantly declined at the same time, while no remarkable change was recorded in IgM titer throughout the entire experiment period. GSIs were gradually increased and reached its highest value in December 2024 (21.99 in females; 3.5 in males) and then decreased in January 2025. These data suggest that the abundance ratio of GAS cells in the gill-epithelium decreases along with sexual maturation in rainbow trout.

This result contributes to optimizing the timing of immersion vaccine administration and improving reproducibility of experiments about GAS cells.

1. Numata, Junsaku, Tokyo University of Marine Science and Technology, Presenter
2. Guruge, Aika, Tokyo University of Marine Science and Technology, Author
3. Matsumoto, Megumi, Tokyo University of Marine Science and Technology, Author
4. Sano, Motohiko, Tokyo University of Marine Science and Technology, Author
5. Kato, Goshi, Tokyo University of Marine Science and Technology, Author

Farm-level biosecurity and health management in floating net cage fish farms in Croatia

Introduction

Floating net cage farming is currently the only fish farming technology used in the Adriatic Sea. Its main advantages are simplicity and lower costs compared to other farming systems. However, it also presents significant challenges, particularly the inability to control environmental factors and the difficulty of implementing effective biosecurity measures. The risk of pathogen introduction into open farming systems, such as those using floating net cages, is challenging to manage. Despite advancements in identifying disease agents, the mariculture sector faces an ineffective approach to disease control, resulting in both economic and environmental impacts.

Methodology

The implementation of biosecurity measures on farms in the Republic of Croatia was analyzed, including steps to reduce the risk of introducing and spreading infectious diseases among farm animals, minimize the likelihood that infected animals or infectious agents will leave the facility and infect other locations and susceptible species, and enhance the effectiveness of responses to emerging diseases.

Results and Conclusions

The analysis identified key gaps in current biosecurity practices. Measures designed to prevent the entry and spread of pathogens within the farm are implemented in accordance with international standards regarding the health certification of live aquatic organisms. However, fish are farmed in open water, and the risk of introducing pathogens through water is high, often greater than the risk associated with introducing new fish from other areas. As long as cultured aquatic organisms maintain direct or indirect contact with wild organisms, new diseases will continue to emerge, and previously recognized diseases will reappear. The effort to control losses due to disease in aquaculture establishments is not rapid enough, and disinfecting net cages after removing dead fish is impractical. A risk communication mechanism has not been established to ensure transparent and auditable implementation of biosecurity for all stakeholders in the aquaculture sector. The use of veterinary medicinal products (VMP) for disease prevention and/or treatment is limited and does not address the occurrence of emerging diseases. This underscores the urgent need to strengthen stakeholder education and awareness regarding biosecurity practices.

1. ČOLAK, SLAVICA, UNIVERSITY OF ZADAR, Presenter
2. MEJDANDŽIĆ, DANIJEL, Cromaris d.d., Author
3. Župan, Ivan, University of Zadar, Author
4. Mustać, Bosiljka, University of Zadar, Author
5. Petani, Bruna, University of Zadar, Author
6. Bavčević, Lav, University of Zadar, Author
7. Šarić, Tomislav, University of Zadar, Author
8. Kolega, Matko, Cromaris d.d., Author

Use of transcriptomics for detection of novel viruses in molluscs

Detecting the presence of virus in an animal to diagnose or to predict a disease was dependent, most frequently, on the use of target techniques. Even cell culture isolation, a well-known non-target viral detection method, ultimately depends on serological and/or molecular techniques for identifying the isolate. As a result, in many cases, even when the presence of a virus is suspected, none is detected. Currently, the use of metagenomics and next generation sequencing to screen the microbiome or the virome of environmental samples is becoming increasingly common. However, sometimes the biological meaning of the presence of certain sequences is, particularly in the case of viruses, difficult to explain or even questionable.

In this study, we employed transcriptomics to investigate the presence of viruses actively expressed in the tissues of certain molluscs (queen scallop, Aequipecten opercularis; king scallop, Pecten maximus; Mediterranean mussel, Mytilus galloprovincialis). All sequences were compared, in a first stage (with E≤10-6), with the Reference Viral Database (RVDB), as well as with vFAM and RefSeqViral, obtaining close to 40,000 putative sequences (19841, 9436 and 9940, respectively). In a second filtration (with higher restrictive parameters), the number of sequences were drastically reduced to 176 (98, 39 and 39, respectively). A third comparison, against the NCBI nucleotide database using the blastN algorithm, reduced the total number of viral sequences to 53 (46, 7 and none, respectively). After removing the repeated sequences, 25 from queen scallop and 7 from king scallops were marked as putative sequences of viruses, mostly picorna-like viruses, whose presence should be demonstrated. To elucidate the biological significance of the presence of these viral sequences, we designed a large number of primers sets, and PCR was applied to the original tissue samples. The preliminary results suggest that some of these sequences are not NGS artifacts, and that certain viruses –unknown to date in our area– could actually be present in those species.

Financing: This study has been financed i) by project CALIMAR, from the network REDEMAR, Consellería do Mar, Xunta de Galicia, through the European Maritime, Fisheries and Aquaculture Fund (EMFAF), ii) by Red EpiMar (RED2022-134796-T), through MICIU/AEI/10.13039/501100011033, and European Regional Development Fund (ERDF), and iii) by project EpiVir (PID2022-14200OB-10), through MICIU/AEI/10.13039/501100011033, and ERDF.

1. González-Rosales, Emma, ARCUS,UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
2. Olveira, José G., ARCUS,UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
3. Pazos, Antonio, ARCUS,UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
4. Pérez-Parallé, Luz, ARCUS, UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
5. Souto, sandra, ARCUS, UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Author
6. DOPAZO, Carlos P., ARCUS, UNIVERSIDADE DE SANTIAGO DE COMPOSTELA, Presenter

Hatchery personnel’s perceptions on risk factors for fish mortality after sea transfer

Introduction: The transition from hatchery to sea site is a critical phase in the life cycle of Atlantic salmon, often associated with significant mortality rates. Understanding the perceptions of hatchery personnel regarding the risk factors contributing to these mortalities is essential for developing effective mitigation strategies. This study aims to identify what hatchery managers and fish health personnel perceive to be important pre-transfer risk factors for post-transfer mortality and reduced welfare in Atlantic salmon in Norway.

Methodology: A qualitative approach was employed, involving 15 semi-structured interviews with hatchery managers and fish health personnel. The interviews focused on their observations and experiences related to fish health and mortality during the sea transfer process. Data were analysed using systematic text condensation, a thematic cross-case analysis to identify common themes and risk factors.

Results: Participants identified several key factors contributing to fish mortality post-sea transfer. They emphasized that the highly intensive production environment, coupled with new protocols for smolt production, introduces new challenges within an already complex system. Poor smoltification, attributed to certain husbandry practices, was frequently mentioned as a significant cause of high post-transfer mortality. Participants also stressed the importance of prioritizing fish health and welfare within organizations to achieve low mortality rates. They noted that many risk factors stem from decisions made at various levels within salmon farming companies, suggesting that these risks could be mitigated if they are well described, understood, and prioritized.

Conclusion: The findings highlight the intricate nature of fish mortality post-sea transfer, underscoring the need for a holistic approach to address these challenges. The intensive production environment and evolving smolt production protocols necessitate continuous adaptation and improvement in husbandry practices. Ensuring proper smoltification is crucial, as it directly impacts post-transfer survival rates. The emphasis on fish health and welfare within organizations is important, as it fosters a proactive approach to identifying and mitigating risk factors. The study reveals that many of these risks originate from internal decisions within salmon farming companies, indicating that effective communication and prioritization of these issues can lead to significant improvements. By understanding and addressing the underlying causes of mortality, companies can implement targeted interventions to improve fish welfare and reduce mortalities.

Further research is needed to quantify these factors’ impact. Insights from hatchery personnel can guide a quantitative study on post-sea transfer mortality.

1. Sollien Gåsnes, Siri, Norwegian Veterinary Institute, Presenter
2. Bang Jensen, Britt, Technical University of Denmark, Author
3. Størkersen, Kristine, SINTEF Ocean, Author
4. Stomoen, Marit, Norwegian University of Life Sciences, Author
5. Gismervik, Kristine, Norwegian Veterinary Institute, Author

comparison of mortality cause assignments between fish health personnel and farmer recordings

introduction

high mortality is one of the biggest issues in norwegian salmon farming. understanding why and when salmon die during production is crucial for improving fish health and reducing mortality. mortalities are categorized daily by salmon farmers in norway, but the quality of these recordings is unknown. this study aimed to describe the agreement of mortality assignments between fish health personnel and farmers.

methods

two net pens with atlantic salmon were followed in 17 different facilities over 5-15 months. during monthly health visits, fish health personnel recorded mortality causes in necropsied fish according to the standard for cause-specific mortality and loss (1). macroscopic pathology was recorded for the necropsied fish. mortality assignments by fish health personnel were compared to daily assignments by farmers over the following weeks to assess the level of agreement.

preliminary results

a total of 2,445 dead fish were necropsied by fish health personnel during health visits and compared to approximately 375,000 dead fish categorized by farmers. varying degrees of agreement in mortality cause assignment were observed. the study also found differences in the occurrence of macroscopic pathological changes during the sea phase. associations between certain mortality causes and macroscopic pathological changes were found.

conclusion

the preliminary results indicate varying levels of agreement in mortality cause assignments by fish health personnel and farmers. these findings highlight the need for improved recording practices to ensure that the recorded mortality causes reflect the underlying cause. this can help farmers implement preventative measures at the correct time. associations between mortality causes and macroscopic changes should be further assessed and used to improve the recording practices.

references

1) aunsmo, a., persson, d., stormoen, m., romstad, s., jamtøy, o., & midtlyng, p. (2023). real-time monitoring of cause-specific mortality- and losses in industrial salmon farming. aquaculture, 563, 738969. doi:10.1016/j.aquaculture.2022.738969

1. RINGSTAD, NANNA KRISTINE, NMBU, Presenter
2. PERSSON, DAVID, NMBU, Author
3. STORMOEN, MARIT, NMBU, Author

Nodular Gill Disease in Swiss Aquaculture – Characterization of histological lesions in gills of rainbow trout (Oncorhynchus mykiss) and an outlook on diagnosis of involved amoeba species

Nodular gill disease (NGD) is an infectious disease associated with an infestation of the gills with amoebae, leading to proliferative gill lesions and consequently respiratory difficulties and mortality in freshwater salmonids. In Switzerland, NGD prevalence in rainbow trout (Oncorhynchus mykiss) has increased in recent years, with mortality rates reaching up to 50%.

A previous study by Vannetti et al. analyzed gill samples from 333 rainbow trout across six Swiss fish farms using a gross Gill Score (GS), wet mount microscopy and histology of few selected specimens. By morphological and molecular methods, six amoebic strains were identified. However, a broader histopathological assessment has not yet been performed.

The study presented here expands upon that work by histologically evaluating 121 of the original samples using a semi-quantitative scoring system to characterize NGD-related tissue changes in Swiss rainbow trout. The observed lesions and distribution patterns aligned with previous reports on the histopathology of NGD. Amoeba abundance was positively correlated with the severity of gill tissue alterations, including the proportion of affected filament tips and the degree of epithelial cell hypertrophy and hyperplasia. While these results support the hypothesis that amoebae play a central role in the development of NGD-associated lesions, the lesions observed are non-specific and are rather common responses of gills to injury. Thus, morphological evidence alone is insufficient to confirm amoebae as etiological agent of NGD.

Comparative analysis of diagnostic methods showed good agreement between gross and histological assessments, but notable discrepancies with wet mount microscopy, underscoring the need for integrated diagnostic strategies to detect amoebic gill infestation. Moreover, the advanced stage of disease at the time of visible gill alteration often limits treatment success, emphasizing the need for other diagnostic tools that enable early detection.

Recent metagenomic studies suggest that Vannella sp. may be the primary pathogen of NGD in Swiss rainbow trout and a qPCR method for its detection has been developed. Building on this, an ongoing project aims to validate this qPCR method for early detection of Vannella sp. and further investigate its role in NGD. Integration of qPCR, histological, and clinical findings may enhance diagnostic accuracy and support more effective NGD monitoring and management in aquaculture settings.

1. Seibold, Soyomi, Institute for Fish and Wildlife Health, University of Bern, Switzerland, Presenter
2. Vannetti, Stefania, Institute for Fish and Wildlife Health, University of Bern, Switzerland, Author
3. Delalay, Gary, Institute for Fish and Wildlife Health, University of Bern, Switzerland, Author
4. Nicolas, Diserens, Institute for Fish and Wildlife Health, University of Bern, Switzerland, Author
5. Schmidt-Posthaus, Heike, Institute for Fish and Wildlife Health, University of Bern, Switzerland, Author