Independent evolution of geraniol-8-hydroxylase activity involved in iridoid formation in the Argentine ant (Linepithema humile)

Iridoids are bicyclic monoterpenoids that function as defense and signaling compounds in both plants and insects. Although recent research suggested that iridoids evolved independently in these two kingdoms of life, it remained unclear whether independent evolution of iridoid biosynthesis also occurred across different insect lineages. In order to gain insight into the evolution of iridoids in insects, we examined the first committed step of iridoid biosynthesis, the hydroxylation of geraniol to 8-hydroxygeraniol, in the Argentine ant (Linepithema humile) of the order Hymenoptera. This transformation is typically catalyzed by cytochrome P450 monooxygenases in both plants and insects. By integrating transcriptomic and metabolomic analyses across various body parts, we identified candidate P450 genes potentially involved in this reaction. The candidate genes were heterologously expressed in yeast, and enzyme activity was assessed by supplying geraniol to the yeast cultures. One candidate P450 enzyme displayed geraniol 8-hydroxylase (G8H) activity and was designated LhG8H. Phylogenetic analysis showed that LhG8H is evolutionarily distinct from previously characterized G8H enzymes in the pea aphid (Acyrthosiphon pisum) of the order Hemiptera and the mustard leaf beetle (Phaedon cochleariae) of the order Coleoptera. These results support the hypothesis that geraniol 8-hydroxylase activity, a key step in iridoid biosynthesis, evolved independently within the insect orders Hymenoptera, Hemiptera, and Coleoptera.