Introduction
The exponential growth of aquaculture in recent years has led to an increase in diseases caused by opportunistic pathogens (viruses, bacteria, and fungi), resulting in significant economic losses. Among viral pathogens, one of the most relevant in the Mediterranean area is Betanodavirus (NNV), the causative agent of viral encephalopathy and retinopathy (VER) in fish. NNV can cause up to 100% mortality in juvenile stages of susceptible species, including the commercially important European sea bass (Dicentrarchus labrax) (Kibenge, 2019). Antimicrobial peptides (AMPs) are key components of the innate immune system and are found across a wide range of organisms. These small molecules regulate inflammation, promote leukocyte recruitment, and bridge the innate and adaptive immune responses. In addition, AMPs exhibit broad-spectrum activity, acting directly against bacteria, viruses, fungi, and parasites by disrupting their membranes. Due to their low likelihood of inducing resistance, AMPs are considered a promising alternative for enhancing disease resistance and improving overall fish health. Among them, beta-defensins (Bdef) have attracted attention for their antibacterial, antiviral, immunomodulatory, and anti-inflammatory properties. The use of AMPs as a preventive measure against NNV is increasingly supported by recent scientific evidence (Valero et al., 2020; Cervera et al., 2024). However, the in vivo role of orally administered Bdef in European sea bass has not yet been explored. Therefore, the aim of this study was to evaluate the involvement of European sea bass Bdef1 in NNV infection and its potential as an immunomodulatory agent in functional diets designed for sea bass prior to infection.
Material and methods
The synthetic mature peptide Bdef1 derived from European sea bass was encapsulated at 1 mg/mL in alginate-chitosan nanoparticles (NP) using a method that was previously described (Collado-González et al., 2020). Then, juvenile European sea bass were fed for 5 days with: Control (commercial diet), NP (diet with empty nanoparticles a), and NP+BD (nanoparticles with Bdef1) diets. After sampling for immune analysis, the NP and NP+BD groups were infected with a sublethal dose performed by intramuscular injection of 50 µL of the containing 104 TCID50 of NNV/fish. The control group was injected intramuscularly with 50 µL of PBS alone. At 2 and 24 days post-infection (dpi), samples of brain, head-kidney and hindgut of each fish were collected and processed for immune gene expression analysis by qPCR. Moreover, serum samples were collected to detect NNV specific antibodies using a ELISA assay and as well to evaluate the antibacterial activity agains Vibrio harveyi and Vibrio anguillarum using a method previously described (Oriol Sunyer & Tort, 1995). Data were expressed as the % of bacterial growth inhibition.
Results
Our results showed that sea bass fed with encapsulated Bdef1 during 5 days had not changed their immune parameters. In addition, the antiviral response elicited against NNV was again not altered. In fact, the results obtained from the ELISA assay performed on serum samples have shown that the production of specific antibodies was reduced by the intake of Bdef1.
Discussion and conclusions
This study suggests that 5 days dietary administration of encapsulated Bdef1 exerts a limited effect on the immune response of European sea bass as well as little response during NNV infection. Bdef1 appeared to modulate the immune response without inducing excessive activation. Additionally, a significant increase in antibacterial activity was observed against Vibrio harveyi, while no effect was detected for V. anguillarum. ELISA results showed lower virus-specific antibody levels in treated fish, indicating a potential modulation of the adaptive immune response. Overall, the promised benefits of Bdef1 as an immune-modulating agent are controversial, supporting further research to optimize its delivery and application in aquaculture.
References
Cervera, L., Arizcun, M., Mercado, L., Chaves-Pozo, E., & Cuesta, A. (2024). Hepcidin and dicentracin peptides show preventive antiviral applications against NNV infection in European sea bass through immunomodulatory roles. Aquaculture, 583, 740592. https://doi.org/10.1016/j.aquaculture.2024.740592
Collado-González, M., Ferreri, M. C., Freitas, A. R., Santos, A. C., Ferreira, N. R., Carissimi, G., Sequeira, J. A. D., Díaz Baños, F. G., Villora, G., Veiga, F., & Ribeiro, A. (2020). Complex Polysaccharide-Based Nanocomposites for Oral Insulin Delivery. Marine Drugs, 18(1), 55. https://doi.org/10.3390/md18010055
Kibenge, F. S. (2019). Emerging viruses in aquaculture. Current Opinion in Virology, 34, 97-103. https://doi.org/10.1016/j.coviro.2018.12.008
Oriol Sunyer, J., & Tort, L. (1995). Natural hemolytic and bactericidal activities of sea bream Sparus aurata serum are effected by the alternative complement pathway. Veterinary Immunology and Immunopathology, 45(3-4), 333-345. https://doi.org/10.1016/0165-2427(94)05430-Z
Valero, Y., Chaves-Pozo, E., & Cuesta, A. (2020). NK-lysin is highly conserved in European sea bass and gilthead seabream but differentially modulated during the immune response. Fish & Shellfish Immunology, 99, 435-441. https://doi.org/10.1016/j.fsi.2020.02.049
Acknowledgements
PID2022-139492NB-I00 funded by MICIU/AEI/10.13039/501100011033 and by ERDF/EU to AC, 22651/PI/24 funded by FSRM/10.13039/100007801, PRE2020-093771 to L.C., RYC2021-034184-I to MCG, IJC2020-042733-I from MICIU/AEI/10.13039/501100011033 and by the European Union NextGenerationEU/PRTR to YV. This study forms part of the ThinkInAzul programme and was supported by MCIU with funding from European Union NextGenerationEU (PRTR-C17.I1) and by Comunidad Autónoma de la Región de Murcia - Fundación Séneca to AC and EH.