Double-stranded RNA injected into female Black Tiger shrimp (Penaeus monodon) prior to spawning does not transfer to progeny

Abstract

Injecting double-stranded (ds)RNA into abdominal muscle of female shrimp may provide a relatively simple means of reducing pre-existing virus infection loads and thus the propensity for infection to be transmitted vertically to seedstock used for aquaculture. However, to obtain regulatory approvals to use such RNA interference (RNAi) technology in hatcheries producing seedstock to be farmed for human consumption, there is a need to demonstrate that injected dsRNA will not transfer from broodstock or be maintained/amplified in their farmed progeny. To investigate this, at the time they were eyestalk ablated, female Black Tiger shrimp (Penaeus monodon) broodstock naturally impregnated with male spermatophores were injected with a 1:1 mix of dsRNAs synthesised to a 954 bp region of the gill-associated virus (GAV) genome and a 464 bp region of the firefly luciferase (Luc) gene. Eggs spawned 6 to 14 days later from each of 3 non-injected control females and 3 females injected at a dosage of 0.5 μg each dsRNA/g shrimp weight were reared separately and sampled at key developmental stages until they reached post-larval (PL) age. To detect residual dsRNA, cDNA synthesised to both native and heat-denatured (HD) total nucleic acid (TNA = RNA + DNA) extracted from the larval samples and pleopod, lymphoid organ and ovary tissues sampled from each female immediately after spawning was amplified using highly-sensitive TaqMan quantitative real-time (q)PCR tests targeting the dsGAV1 and dsLuc dsRNA sequences. Using HD TNA to strand-separate dsRNA and thus improve the detection sensitivity of the RT-qPCR tests, both dsRNAs were detected in all 3 tissues examined from injected females. Direct qPCR testing of the DNA component of each TNA confirmed that the detections were not due to residual template DNA in the purified dsRNA preparations. Similar RT-qPCR testing of both native and HD TNA extracted from pools of 50 embryos, nauplii, protozoea, and mysis and 32 individual PL reared separately for each female generated no unequivocal or consistent detections of dsGAV1 or dsLuc dsRNA. These findings provide confidence that purified dsRNA injected into female broodstock at the time an eyestalk is ablated and at the dosage tested was not effectively transferred to/amplified in progeny, and thus that the human health risks of using this RNAi strategy to reduce virus disease risks during shrimp grow-out will be negligible.