Knockout of odourant receptor co-receptor HvarOrco compromises reproductive fitness in the ladybird Hippodamia variegata

Abstract The predatory ladybird, Hippodamia variegata , relies critically on olfaction for host location, oviposition site selection, and predator avoidance. Here, we employed CRISPR/Cas9-mediated genome editing to generate a stable, heritable odourant receptor co-receptor ( Orco ) knockout line ( HvarOrco − /− ). Electrophysiological assessment using electroantennogram (EAG) recordings revealed that homozygous mutants exhibited profound anosmia, with significantly attenuated responses to six key semiochemicals, including the aphid alarm pheromone E -β-farnesene. Consistent with these physiological deficits, Y-tube assays demonstrated a complete loss of behavioural attraction towards host-associated cues in both sexes. Crucially, we uncovered a severe fitness cost associated with Orco deficiency. Mating assays revealed a sexual dimorphism in reproductive behaviour: while mutant females mated successfully, HvarOrco −/− males exhibited a significant failure to initiate copulation. Furthermore, post-copulatory reproductive output was drastically compromised; mutant pairs showed a 68.4% reduction in fecundity (10.3 ± 0.51 vs. 32.6 ± 0.96 eggs/30d) and a 90.4% decline in egg hatchability (8% ± 2% vs. 83% ± 1%). These findings establish HvarOrco as a dual determinant of both ecological adaptation and reproductive success. Collectively, our study validates a robust CRISPR/Cas9-based functional genomics platform for this ecologically important family of natural enemies and highlights the need for further investigation into potential non-canonical roles of this conserved co-receptor in reproductive physiology.