Calcineurin is required for male sex pheromone biosynthesis and female acceptance

Lepidoptera sex pheromone biosynthesis is regulated by pheromone biosynthesis activating neuropeptide (PBAN). PBAN regulates not only female sex pheromone biosynthesis but also male sex pheromone biosynthesis. Previous research has confirmed that PBAN regulates sex pheromone biosynthesis using Ca²⁺ as a secondary messenger in all examined species to date. However, the downstream signal of Ca²⁺ has remained elusive. In the present study, calcineurin A (CNA), a downstream signal of Ca²⁺ , was discovered in Helicoverpa armigera male hairpencil and named HaCNA. Sequence analysis demonstrated that the open reading frame of HaCNA contains 1488 nucleotides encoding 495 amino acid residues. A homology search revealed that HaCNA shares a high amino acid identity with the CNA of other insects. Developmental and spatial expression analyses revealed that the mRNA levels of HaCNA peaked at 24 h after emergence and that HaCNA expression was ubiquitous in all examined tissues. Activity analysis revealed that PBAN activates HaCNA, and a Ca²⁺ inhibitor, Lacl₃ , attenuated the effect of PBAN by decreasing HaCNA activity. Pharmacological inhibitor and RNA interference-mediated knockdown assays revealed that both activity inhibition and decreased mRNA levels of HaCNA led to a significant decrease in the production of the male sex pheromone components [octadecanol and (Z)-11 hexadecanol)] and in the efficacy of female mating acceptance. Our results demonstrate that HaCNA acts as downstream signal of PBAN/Ca²⁺ and plays an important role in PBAN-induced male sex pheromone biosynthesis and female mating acceptance.