Targeted mutagenesis of the ryanodine receptor by Platinum TALENs causes slow swimming behaviour in Pacific bluefin tuna (Thunnus orientalis)

Abstract

In bluefin tuna aquaculture, high mortalities of hatchery-reared juveniles occur in sea cages owing to wall collisions that are caused by high-speed swimming in panic due to changes in illuminance. Here, we report that targeted gene mutagenesis of the ryanodine receptor (RyR1b), which allows the sarcoplasmic reticulum to release Ca2+ in fast skeletal muscle, using highly active Platinum TALENs caused slow swimming behaviour in response to external stimuli in Pacific bluefin tuna (PBT) larvae. This characteristic would be a useful trait to prevent wall collisions in aquaculture production. A pair of Platinum TALENs targeting exons 2 and 43 of the PBT ryr1b gene induced deletions in each TALEN target site of the injected embryos with extremely high efficiency. In addition, ryr1b expression was significantly decreased in the mutated G0 larvae at 7 days after hatching (DAH). A touch-evoked escape behaviour assay revealed that the ryr1b-mutated PBT larvae swam away much less efficiently in response to mechanosensory stimulation at 7 DAH than did the wild-type larvae. Our results demonstrate that genome editing technologies are effective tools for determining the functional characterization of genes in a comparatively short period, and create avenues for facilitating genetic studies and breeding of bluefin tuna species.

Prediction of the Sex-Associated Genomic Region in Tunas (Thunnus Fishes)

“…Our hypothesis may be tested by further genetic experiments, although the handling of tuna in tanks is not easy. Recently, genome-editing technology has been applied for the mutagenesis of tuna [ 48 ]. For example, it may be possible to check whether the sex ratio of larvae is heavily biased by the knockout of sult1st6y in fertilized eggs.…”

TALEN-Mediated Gene Editing of slc24a5 (Solute Carrier Family 24, Member 5) in Kawakawa, Euthynnus affinis

“…In this study, it was difficult to rear the kawakawa to adulthood for reasons other than the effects of the TALEN injection, which did not last beyond early stages of development. Although mortality of artificially reared tuna juveniles in sea cage or tanks is typically high due to injuries from collisions with the walls as the panicked fish attempt to escape by swimming at high speeds, platinum TALEN-induced mutants of the Pacific bluefin tuna embryos were relatively calm and swam slowly, resulting in comparatively higher survival [57]. In case of kawakawa, a small-scale rearing system for experiments has been established in our laboratory where the tanks are larger in size and the fish are moved to net cages when the fish reach a certain size.…”

Prediction of the sex-determination gene in tunas (Thunnus fishes)

“…Our hypothesis may be tested by further genetic experiments, although the handling of tuna in tanks is not easy. Recently, genome-editing technology has been applied to the mutagenesis of tuna (Higuchi et al, 2019). For example, it may be possible to check whether the sex ratio of larvae is heavily biased by the knockout of sult1st6y in fertilized eggs.…”