Introduction
Global aquaculture production is steadily increasing, alongside rising reports of pathogen infections, highlighting the urgent need for effective disease control measures. Scuticociliates are opportunistic parasites that infect a wide range of species, including fish, crustaceans, and mollusks. Scuticociliatosis, caused by scuticociliates, has been reported worldwide and causes systemic tissue damage in skeletal muscle, the brain, the liver, the spleen, and the blood. Clinical signs typically include systemic ulceration and hemorrhage, followed by body darkening and abnormal swimming behavior. Formalin and hydrogen peroxide are the most widely used treatments for scuticociliate control. However, both agents were effective only against external parasites and showed limited effect against internal parasites. Furthermore, continuous chemical exposure increases residual drug concentrations in fish tissues and aquatic environments, leading to immune suppression, pathogen resistance, and environmental contamination. Thus, there is an urgent need to develop safe alternative therapeutics to replace conventional chemical treatments. In this study, we evaluated the antiparasitic efficacy of a marine-derived biotoxin against scuticociliates, assessed its safety in host fish, and elucidated its underlying antiparasitic mechanisms through transcriptomic analysis.
Materials and Methods
Miamiensis avidus was aseptically isolated from the brain of infected fish at an aquaculture farm located in Jeju, South Korea. The isolated scuticociliates were cultured using the RTgill-W1 cell line. M. avidus was treated with biotoxin at various concentrations and exposure durations, and cellular safety was established through cytotoxicity assessment. Antiparasitic efficacy was evaluated in olive flounder (Paralichthys olivaceus) infected with scuticociliates following a 5 hour biotoxin treatment, with observations over 14 days. To assess residual biotoxin levels in fish tissues, muscle samples were collected immediately after treatment, and at 1 and 3 days post treatment. Host safety was further evaluated by monitoring mortality and biochemical parameters over a 28 day period. To elucidate the antiparasitic mechanism of biotoxin, transcriptome analysis was performed. Differentially expressed genes (DEGs) were identified using DESeq2, and gene ontology (GO) enrichment analysis was conducted using clusterProfiler package in R. We identified core co-expression modules through weighted gene co-expression network analysis (WGCNA). By integrating these modules with enriched GO terms, we prioritized candidate genes at the functional intersection to elucidate the key molecular drivers underlying antiparasitic efficacy.
Results
Biotoxin demonstrated 100% antiparasitic efficacy against M. avidus at 1,000 μg/L within 4 hours. No cytotoxicity was observed in cells treated with 100 μg/L biotoxin for 5 hours. At this concentration, biotoxin exhibited antiparasitic efficacy against M. avidus in infected olive flounder. Fish treated with biotoxin following M. avidus infection showed delayed mortality compared to the M. avidus only group, and co-treatment of M. avidus and biotoxin resulted in a greater than 50% increase in survival rate. No residual toxin was detected in the muscle tissue of biotoxin-treated flounder, and biochemical parameters showed no significant differences compared to the control group over 28 days, indicating no adverse effects on host physiology. Transcriptome analysis identified 6,186 differentially expressed genes (DEGs; p < 0.05, log2FC > 1), of which 3,146 were upregulated, and 3,040 were downregulated. Enrichment analysis revealed upregulation of protein synthesis machinery, lipid metabolism, and ER components, and downregulation of GPCR signaling, ion channel signaling, and antioxidant defenses. WGCNA identified seven modules, of which four showed significant ROS correlation. Hub gene analysis identified ion channel, cytoskeletal, and motility regulators, implicating sensory-motor disruption as the antiparasitic mechanism.
Discussion
This study shows biotoxin effectively treats scuticociliatosis, killing M. avidus rapidly without harming the fish or leaving residues. Infected flounder treated with biotoxin showed improved survival and normal biochemical parameters, confirming its safety. Transcriptome analysis revealed that biotoxin works by suppressing ion channels, GPCR signaling, and cytoskeletal genes. These changes likely disrupt sensory perception and motility. The upregulation of protein synthesis terms may represent a compensatory or stress-related response. These findings elucidate the antiparasitic mechanism of action and suggest that targeting sensory-motor pathways could be a promising strategy for developing novel antiparasitic drugs in aquaculture.
Acknowledgment
This work was supported by a grant from the Korea Institute of Ocean Science and Technology (KB0012), the Korean government.
References
Park J., Lee K.W., Park Y., Yeo J.H., Jung J.H., Kang H.M., (2025) Characteristics of different scuticociliates from an infected olive flounder (Pararlichthys olivaceus) farm in South Korea. Developmental and Comparative Immunology. 166 (105357) 1-10. https://doi.org/10.1016/j.dci.2025.105357