Nezya Pramudya Wardani, Uun Yanuhar, Muhammad Musa, Heru Suryanto, Nico Rahman Caesar, Defa Rizqi Machfuda, Nafal Naqi Ramadhan
Viral Nervous Necrosis (VNN), caused by Nervous Necrosis Virus (NNV), is a major constraint in hybrid grouper (Epinephelus spp.) aquaculture, with larval and juvenile mortality reaching nearly 100%. Effective prophylactic strategies are urgently required to ensure sustainable production. This study aimed to test the hypothesis that recombinant Chlorella vulgaris protein, delivered using ZnO-chitosan nanoparticles, enhances immune response, improves growth, and mitigates VNN-induced tissue damage in hybrid grouper. An in vivo challenge experiment was conducted with juvenile fish assigned to 5 groups: Negative control (K−), positive control (K+), and 3 nanovaccine dosages (P1 = 33 µL, P2 = 66 µL and P3 = 112 µL). Vaccination was administered twice via oral sonde, with a 14-day interval between the primary and booster doses prior to viral challenge. The 66 µL dose (P2) yielded the most consistent protective effects, including significant improvements in body length and weight (p < 0.05), as well as elevated antioxidant enzyme activities (SOD = 3.997 U/mL; CAT = 109.7 U/mL), indicating enhanced oxidative defense. Histological analyses further confirmed reduced tissue damage in vaccinated groups, with P2 exhibiting attenuated vacuolization in the gills. Interestingly, although P2 was superior in growth and antioxidant responses, the 33 µL dose (P1) demonstrated the lowest tissue damage, suggesting a more favorable safety profile. These findings highlight a dose-dependent trade-off, where P2 maximizes immune and growth benefits, while P1 minimizes histopathological alterations. While ZnO-chitosan nanoparticles are generally considered safe at sub-toxic concentrations reported in fish toxicology studies (< 100 mg/L waterborne exposure), further ecotoxicological and bioaccumulation assessments are required to substantiate their long-term environmental safety. Within the scope of this study, the 33 µL dosage represents the most balanced option between efficacy and safety, supporting its potential as a nanovaccine platform for VNN control in hybrid grouper aquaculture. © 2026, Walailak University. All rights reserved.
Master Program of Aquaculture, Fisheries and Marine Science Faculty, Universitas Brawijaya, East Java, 65145, Indonesia; Study Program of Aquatic Resources Management, Faculty of Fisheries and Marine Sciences, Universitas Brawijaya, East Java, 65145, Indonesia; Center Excellent for Cellulose Composite, Department of Mechanical and Industrial Engineering, Faculty of Engineering, Universitas Negeri Malang, East Java, 65145, Indonesia
