Pharmacological characterization and functional divergence of tyramine receptors PxTAR2 and PxTAR1A in the diamondback moth, Plutella xylostella.

Tyramine signaling orchestrates essential physiological processes in insects, yet the functional diversification of tyramine receptors (TARs) and the evolutionary significance of receptor gene duplication remain poorly understood. While most lepidopterans harbor a single TAR1, the diamondback moth Plutella xylostella uniquely possesses two TAR1 paralogs (PxTAR1 and PxTAR1A) alongside PxTAR2, providing an exceptional model to investigate sub-functionalization. However, functional characterization of PxTARs has been limited to TAR1. Here, we uncover striking functional differentiation of TARs through integrated pharmacological and genetical analyses. Despite high structural conservation with lepidopteran orthologs (∼80% identity), PxTAR2 exhibited no obvious plasma membrane localization and complete pharmacological silence in HEK293 cells-showing no measurable cAMP or Ca2+ responses to tyramine, suggesting obligate dependence on unavailable cellular cofactors. In sharp contrast, PxTAR1A was predominantly localized to the plasma membrane in HEK293 cells. It displayed canonical TAR1-like properties (tyramine EC50 = 51.36 nM; octopamine EC50 = 8.9 μM) yet distinct pharmacological fingerprinting, including unique antagonist susceptibility to phentolamine. CRISPR/Cas9-mediated gene knockout revealed drastic functional partitioning: while PxTAR2 deficiency caused only modest late-stage oviposition decline, PxTAR1A knockout severely compromised reproductive fitness, reducing fecundity by 24% (194 to 148 eggs/female) and, critically, collapsing embryonic hatchability from 81% to 47% due to late developmental arrest. qRT-PCR analyses further elucidated this divergence: PxTAR1A is predominantly expressed in embryos (14.6-fold and 9-fold higher than PxTAR2 and PxTAR1, respectively), while PxTAR2 displays the lowest expression among PxTARs throughout the entire life cycle. These findings advance our understanding of tyramine receptor diversification, establish PxTAR1A as a master regulator of insect embryogenesis, and identify a high-value target for next-generation pest management targeting reproduction.