CRISPR/Cas9‐mediated germline mutagenesis in the subsocial parasitoid wasp, <i>Sclerodermus guani</i>

The ectoparasitoid wasp Sclerodermus guani (Hymenoptera: Bethylidae), as a subsocial insect, is widely applied in biological control against beetle vectors of pine wood nematodes. Despite significant advances in behavioural research, functional genetics in S. guani remains underdeveloped due to the absence of efficient gene manipulation tools. In this study, we employed CRISPR-mediated mutagenesis to achieve germline gene knockout targeting the eye pigment-associated gene kynurenine 3-monooxygenase (KMO). Phylogenetic analysis revealed that S. guani KMO shares a close relationship with its homologue in Prorops nasuta (Hymenoptera: Bethylidae). Two single-guide RNAs (sgRNAs), coupled with Cas9 protein with and without nuclear localisation signal (NLS) were tested. Both sgRNAs induced specific in vitro DNA cleavage and in vivo heritable indels at the target genomic loci. Homozygous null mutant females and males exhibit a white-eye phenotype, which was identified during pupal stage. Optimal editing efficiency in vivo was achieved using the Cas9-NLS variant. Given the complication of germline gene editing in eusocial Hymenopterans, the application of CRISPR in the subsocial parasitoid wasp S. guani provides an accessible research platform for the molecular evolution of insect sociality.