1a. Objectives (from AD-416):
The main focus of the work proposed here will be to use an evolutionary bioassay-driven process to make lab and then field-ready pheromone lures by starting with highly purified (LC/HPLC) pheromone components and formulate them on rubber septa and other lab substrates that have been de-activated (cleaned up/extracted); ratios of emitted pheromone volatiles will be assessed regularly. The pheromone components will be stabilized with BHT and Sumisorb™ and these lures will be compared to “standard” filter-paper sources and a commercial lure formulation from ISCA Technologies.
1b. Approach (from AD-416):
The approach will be to be as rigorous as possible with chemical purity, lure dosing and assessment of pheromone release ratios and then add a graded or stepwise level of “stress” on the pheromone formulations. Lures made with rubber septa have been known to be unstable with some aldehyde-containing pheromone blends. To circumvent this problem, we will exhaustively extract rubber septa, plastic vials and tubes with a Soxhlet device or by boiling them in hot solvents to remove compounds that can catalyze aldehyde pheromone degradation. These prospective lures with then be loaded with the now known 4-component pheromone blend and their by quality and longevity testing will start in the laboratory by comparison with filter paper disks loaded with gland extract or the 4-component pheromone blend as positive controls.
3. Progress Report:
Progress was made on objective 2 to "develop a sex pheromone based program for use in the integrated management of navel orangeworm" in the parent project. The navel orangeworm (NOW), Amyelois transitella is well known as the primary lepidopteran pest of almonds as well as pistachios and is also a pest of walnuts, pomegranates and figs. Control of NOW is by removal of mummified nuts (orchard sanitation), insecticide sprays and some mating disruption with the primary sex pheromone component. Timing of insecticide sprays is determined by assessing NOW levels based on counts of NOW eggs on egg traps and by examination of nuts for NOW. Monitoring NOW presence with pheromone traps is expected to be a less labor intensive and a more accurate way to estimate NOW presence and numbers. Identification of the sex pheromone for NOW was completed recently. A four component sex pheromone blend was determined to be as attractive as females. Attractiveness of these four components was subsequently verified by a joint UC Riverside/ARS study. Preliminary field trials confirmed the 4-component blend could capture as many or more NOW males than females; however, lure effectiveness declined rapidly during the 4-day test. This year we worked further on both gland extracts and volatile collections (below) to determine which steps in the analytical process led to skewed isomer ratios and/or skewed ratios between pheromone components. Gland extracts and pheromone volatiles were collected during the females’ optimal time of pheromone release. Glands were extracted in hexane and volatiles were collected from manually everted sex pheromone glands, with volatile collection on an open capillary. Collection of pheromone components on these open glass capillaries is particularly efficient since all the adhered odor molecules can be stripped from the capillary wall using just a few microliters of solvent. Five microliters of hexane forms a solid “slug” of solvent that moves through the capillary collecting the odor molecules; at the end of the capillary this solvent slug is removed with a gas chromatography (GC) syringe for immediate chemical analysis. As we have noted on numerous occasions, the conjugated dienes (three of the four pheromone components for NOW), are extraordinarily labile during collection and handling periods. Even movements of the plunger in an older syringe would lead to isomerization of these compounds. Therefore, all of our methods were re-analyzed, and practiced carefully until we obtained consistent results with synthetic and natural pheromone components. We have analyzed the release ratios of NOW pheromone molecules from cleaned rubber septa and found consistent ratios of three components. Release rates also were constant over a 29 day sample period. We have developed handling methods for NOW pheromone release substrates and the volatiles collected from them that allow consistent release ratios and repeatable measurements of these volatiles.