A transgenic rotifer-based RNA interference approach for antiviral protection against Covert mortality nodavirus in shrimp aquaculture

Viral diseases inflict severe economic losses on global aquaculture, driving the urgent need for sustainable and effective disease management strategies. In this study, we developed a feeding-based RNA interference (RNAi) strategy, utilizing the rotifer Brachionus plicatilis as a live delivery vehicle for short hairpin RNA (shRNA) against Covert mortality nodavirus (CMNV) to control covert mortality disease (VCMD) in Penaeus vannamei . A piggyBac-mediated transgenic platform was established in B. plicatilis based on genomic and transcriptomic resource development. Stable and heritable expression of a reporter gene was first demonstrated, confirming the feasibility of genetic manipulation in this non-model organism. CMNV was subsequently detected in farmed P. vannamei , and CMNV-specific shRNA expression cassettes were introduced into rotifers using endogenous promoter elements. Feeding experiments revealed that shrimp fed CMNV-shRNA –expressing rotifers exhibited a significant reduction in viral load, as determined by quantitative PCR. Under artificial CMNV challenge conditions, shrimp receiving transgenic rotifers showed reduced cumulative mortality compared with control groups. Importantly, no adverse effects on shrimp growth performance were observed. In summary, transgenic rotifers can serve as an effective and scalable live-feed platform for antiviral RNA delivery. This study provides a new concept for integrating RNAi-based disease control strategies into shrimp hatchery systems and offers a promising approach for sustainable management of viral diseases in aquaculture.