Identification and gene regulation of the desaturase enzymes involved in sex-pheromone biosynthesis of pest moths infesting grain.
Authors: Rafaeli Ada , Roelofs Wendell , Zada Byers Anat 
Published: January 2022

The original objectives of the approved proposal included: 1. Establishment of the biosynthetic pathways for pheromone production using labeled precursors and GC-MS. 2. The elucidation of a circadian regulation of key enzymes in the biosynthetic pathway. 3. The identification, characterization and confirmation of functional expression of the delta-desaturases. 4. The identification of gene regulatory processes involved in the expression of the key enzymes in the biosynthetic pathway. Background to the topic: Moths constitute one of the major groups of pest insects in agriculture and their reproductive behavior is dependent on chemical communication. Sex-pheromone blends are utilized by a variety of moth species to attract conspecific mates. The sex pheromones used are commonly composed of blends of aliphatic molecules that vary in chain length, geometry, degree and position of double bonds and functional groups. They are formed by various actions of specific delta-desaturases to which chain shortening, elongation, reduction, acetylation, and oxidation of a common fatty acyl precursor is coupled. In most of the moth species sex-pheromone biosynthesis is under circadian control by the neurohormone, PBAN (pheromone-biosynthesis-activating neuropeptide). The development of specific and safe insect control strategies utilizing pheromone systems depends on a clear knowledge of the molecular mechanisms involved. In this proposal we aimed at identifying and characterizing specific desaturases involved in the biosynthetic pathway of two moth pest-speciesof stored products, P. interpunctella and S. cerealella, and to elucidate the regulation of the enzymes involved in pheromone biosynthesis. Due to technical difficulties the second stored product pest was excluded from the study at an early phase of the research project. Major conclusions: Within the framework of the planned objectives we confirmed the pheromone biosynthetic pathway of P. interpunctella and H. armigera by using labeled precursor molecules. In addition, in conjunction with various inhibitors we determined the PBAN-stimulated rate-limiting step for these biosynthetic pathways. We thereby present conclusive evidence that the enzyme Acetyl Coenzyme A Carboxylase is activated as a result of PBAN stimulation. We also found that P. interpunctella produce the main pheromone component Z9, E12 Tetradecenyl acetate through the action of a D11 desaturase working on the 16:Acid precursor. This is evidenced by the high amount of incorporation of ²H-labeled 16:Acid into pheromone when compared to the incorporation of ²H-labeled 14:Acid. However, in contrast to reports on other moth species, P. interpunctella is also capable of utilizing the 14:Acid precursor, although to a much lesser extent than the 16:Acid precursor. Despite the discovery of nine different desaturase gene transcripts in this species, from the present study it is evident that although PCR detected all nine gene transcripts, specific to female pheromone glands, only two are highly expressed whereas the other 7 are expressed at levels of at least 10⁵ fold lower showing very low abundance. These two genes correspond to D11-like desaturases strengthening the hypothesis that the main biosynthetic pathway involves a D11 desaturase.