Function analysis of <i>flightin</i> gene in the global tortricid fruit borer <i>Grapholita molesta</i> using CRISPR/Cas9

Many tortricid moths are significant fruit borers characterized by limited flight capacity. However, some individuals within a population of tortricid species exhibit extended flight capabilities, facilitating gene flow between orchards and enabling host switching. To date, research on the proteins involved in flight among fruit borers is limited. Flightin is recognized as a flight muscle protein, yet its function remains unexplored in lepidopteran insects. In this study, quantitative polymerase chain reaction analysis revealed that the flightin gene is expressed at various developmental stages and tissues of Grapholita molesta, with the highest expression in adults and the thorax. Using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) gene editing technology, we successfully generated a homozygous flightin gene knockout strain of G. molesta. The knockout of the flightin gene resulted in contraction of indirect flight muscle fibers, irregularities in the Z-disc of the flight muscles, and a significant elongation of sarcomere length. Additionally, cumulative flight distance and flight time were significantly reduced. The larval period and preoviposition period were significantly prolonged, while larval weight, pupal weight, longevity, and fecundity were all significantly decreased. The results indicate that the flightin gene not only plays an important role in the flight capacity of G. molesta, but also has an effect on the growth and development, and reproduction of the insect, suggesting that flightin may be a potential target for pest management of G. molesta. This is the first investigation into the function of the flightin gene using CRISPR/Cas9.