The Origins of Terpene Infochemicals in Insects: Identification and Evolutionary Analysis of Terpene Synthases in Diverse Lineages

Specialized metabolites have important roles as infochemicals in inter- and intraspecific interactions of insects. A particularly abundant class of specialized metabolites are terpenes, which are released by many members of taxonomically diverse insect lineages as pheromone and defense compounds. Despite the broad occurrence of terpenes in insects, knowledge of their biosynthesis remains limited compared to that in other forms of life. Terpenes are biosynthetically produced by the action of terpene synthase (TPS) enzymes. While insects lack TPS enzymes found in plants and microbes, there is growing evidence that insect TPS proteins have evolved independently from isoprenyl diphosphate synthase (IDS) enzymes in core terpene metabolism. To gain deeper insight into the transition from IDS to TPS function, I have explored the genomic and functional evolution of TPS enzymes in representatives of major insect lineages. First, I investigated evolutionary and functional relationships of TPS enzymes with roles in pheromone biosynthesis in pentatomids (stink bugs) including the invasive and economically critical pests Nezara viridula (Southern green stink bug) and Halyomorpha halys (brown marmorated stink bug). I also performed a comprehensive phylogenetic analysis of TPS genes in species across the broader order of piercing-sucking insects (Hemiptera), which provided evidence for an ancient emergence of TPS function in this group of insects. To gain a better understanding of core structural determinants of insect TPS evolution, we next defined distinct IDS catalytic motifs that are consistently substituted in enzymes with TPS function. These sequence characteristics were used to make predictions of TPS functionality in a large dataset of insect proteins. I determined the evolutionary dynamics of predicted and known TPS and IDS enzymes through extensive phylogenetic analysis to make top-level inferences about the distribution and evolution of TPS function in insects. Using this knowledge, I further explored functional transitions and subfunctionalization of TPS genes in the large order of beetles (Coleoptera), and more specifically, in species of the lady beetle family (Cocinellidae) including the globally invasive pest, Harmonia axyridis. Comparative genome analyses and IDS/TPS gene functional characterizations revealed gene duplication patterns and enzyme transitions that suggest TPS function evolved in part through processes of subfunctionalization and bifunctional enzymatic states. Additionally, this study provided the first experimental evidence for the mitochondrial localization of terpene metabolism in insects. Lastly, I identified putative TPS enzymes in the American cockroach, Periplaneta americana, and conducted an investigation into their catalytic activity. I found first evidence for TPS enzymatic activity in Blattodea as the most anciently diverging order of terpene-emitting insects and made inferences on the relationship of these enzymes to characterized IDS and TPS proteins in other insects. Our findings in the American cockroach point to the potential independent evolution of TPS function in blattodean cockroaches and termites in types of IDS ancestors. This work significantly advances our understanding of the evolution, functional diversity, and biochemical properties of TPS enzymes in insects, highlighting their recurring pattern of parallel evolution from IDS ancestors and its significance as a model for the emergence of novel specialized functions in core metabolic enzymes.