In vitro assay for sex pheromone biosynthesis by the female yellow mealworm beetle and identification of a regulated step

Several pheromones are known to be involved in mediating the mating behavior of the yellow mealworm beetle, Tenebrio molitor (Coleoptera: Tenebrionidae). The only sex pheromone that has been identified to date in this species is the female-produced male attractant, 4-methylnonanol (MNol). The objective of this study was to develop an in vitro assay for the last biosynthetic step involved in MNol biosynthesis, the reduction of 4-methylnonanoic acid (MNA) to the corresponding pheromone product. In vitro assays were conducted by adding appropriate cofactors and radioactively labelled precursor. Radioactive pheromone product was separated from the radioactive precursor by column chromatography, and quantified through scintillation counting. The conversion of MNA to MNol was observed, and was comparable to that observed in vivo using the same radiolabelled substrate. In both the in vivo and in vitro assays, the 3H-MNA was also converted to a nonpolar product, tentatively identified as a triacylglyerol (TAG) derivative. The reduction of 3H-MNA to pheromone proceeded through the coenzyme A intermediate. Mature females reduced significantly more precursor to pheromone than the immature females and the males, both in vivo and in vitro, indicating that the reduction of MNA to MNol is a regulated step involved in pheromone production. Using the in vitro assay reported herein, studies are ongoing in our lab to purify and characterize the enzyme catalyzing the reduction.