Ommochrome pathway knockout via CRISPR/Cas9 reveals sex-linked eye pigmentation and establishes a heritable genome-editing platform in Rhynchophorus ferrugineus

The red palm weevil, Rhynchophorus ferrugineus, is the most economically destructive palm pest worldwide, threatening livelihoods, food security, and ecosystems across 49 countries. Weevil management currently relies predominantly on chemical insecticides, raising significant environmental and public health concerns. Despite its global agricultural importance, genetic approaches to pest management and the mechanistic basis of genome-editing strategies in Rhynchophorus remain largely unexplored. Here, we employed CRISPR/Cas9 genome editing to disrupt the R. ferrugineus ommochrome biosynthetic pathway -- a multi-enzymatic metabolic cascade that converts tryptophan into ommochrome pigments, including brown, yellow, and red pigments. We targeted two key pathway components: the ATP-binding cassette transporter white and the heme peroxidase cardinal. Both genes were ubiquitously expressed, with peak expression levels in the gut, fat body, and head. Elevated transcript levels were observed across early, mid, and late pupal stages and in 0-, 1-, and 2-day-old adult males and females, consistent with the progression of eye pigmentation throughout the R. ferrugineus life cycle. Embryonic microinjection of a single guide RNA (sgRNA)-Cas9 ribonucleoprotein complex targeting white produced in the Generation-0 (G0) adults with a distinct, white-eyed phenotype with a brownish outer margin, in contrast to the black eyes of wild-type adults. Genome-edited cardinal mutant adults displayed a translucent, brownish-white-eyed phenotype, with white streaks that gradually transitioned to a persistent translucent reddish-brown eye coloration. Mutations in both genes were confirmed in G0 adults by genomic DNA sequencing. Mutant adults were crossed to generate heterozygous G1 (+/-), G2 (-/-, -/+, and +/+), and G3 lines (-/-) with genotypes verified as carrying 2-, 3-, 9-, and 13-nucleotide deletions. A stable, heritable eye-color phenotype was established in homozygous knockout (-/-) G3 lines for both white and cardinal, confirmed by unambiguous indel (insertions/deletions) genotyping. Inheritance analysis revealed that both genes are X-linked, following a classical Mendelian sex-linked pattern: paternal alleles are transmitted exclusively to daughters, while maternal alleles are inherited equally by both daughters and sons. This study establishes the first fully homozygous knockout strain in R. ferrugineus and, by characterizing sex-linked inheritance in a coleopteran system, advances our understanding of how CRISPR/Cas9 can be efficiently applied to destructive palm weevil species. The present study represents the first report of CRISPR/Cas9 genome editing in any weevil (Curculionidae), using white and cardinal as marker genes. These findings provide a valuable platform for functional genomics and genome engineering in R. ferrugineus and offer a translational framework for genome editing in the invasive South American palm weevil, R. palmarum, laying a solid foundation for the development of gene-drive strategies aimed at sustainable palm weevil population control.