2012 Annual Report
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.
Progress was made on objective 2a to "develop a stable formulation for the recently identified female sex pheromone" of the navel orangeworm in the parent project. The navel orangeworm (NOW), Amyelois transitella, is well known as the primary lepidopteran pest of pistachios and almonds and is also a pest of walnuts. Control of NOW is critical for these industries to maintain export markets as well as domestic consumption. Monitoring NOW presence with pheromone traps will enhance this control process. The pheromone components emitted by NOW trap lures are unstable under California summer field conditions due to their chemical structure.
This year’s progress on bioassays was greatly restricted by unknown issues with our insect colonies. Male NOW from our lab colony, used since 2000, largely failed to respond to female pheromone gland extract (which was taken from our research unit’s base culture, originated in 1966). We obtained NOW males (for flight assays) and female NOW (for pheromone extracts) from a separate NOW colony established in 2005 and maintained in our research unit. After cross checking male responsiveness and pheromone extracts, we determined that female pheromone gland extract from all three colonies elicited full male responsiveness as long as males came from our unit’s base strain or the 2005 strain. Thus, we concluded that an unknown change occurred in our 2000 strain, likely due to severe overheating of the growth chamber during a holiday weekend (more than 50% of individuals in the colony had died and our max/min thermometer indicated a high of 119 F). We subsequently started a colony of NOW derived from eggs from the 2005 strain, which we now use for our bioassays.
Most publications refer to rubber septa (used for synthetic pheromone lures) by description (size and color) plus a catalog number – finding a reference to most of these septa is no longer possible since the company and/or product are no longer available. We obtained rubber septa from West Co. made of natural rubber (isoprene) and synthetic rubber – butyl and chloro-butyl rubber. Batches of each were exhaustively extracted with solvent and stored in clean hexane until ready for use.
In previously examined pheromone collections we noted different ratios of compounds and isomers among individuals and these ratios also were affected by steps in the analytical approach. This year we worked further on both gland extracts and volatile collections 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 tube. The conjugated dienes (three of the four pheromone components for NOW), are extraordinarily labile during collection and handling periods (even plunger movements in an older GC syringe would lead to isomerization of these compounds). Therefore, all of our methods were re-analyzed until we obtained consistent results with synthetic and natural pheromone components. Collection of pheromone components on open glass capillaries is efficient since all the adhered odor molecules can be stripped from the capillary wall by just a few microliters of solvent. Ten microliters of hexane forms a solid “slug” of solvent that moves through the capillary collecting the odor molecules. This solvent slug can then be removed from the end of the capillary with a GC syringe for immediate analysis by GC/FID or GC/MS. We have analyzed the release ratios of NOW pheromone molecules.
Bioassays used during the pheromone elucidation/identification processes were conducted with prospective pheromone component blends loaded onto filter paper disks. We collected volatiles from similarly loaded disks and found that although the ZZ-aldehyde and ZZ-alcohol components were loaded in equal amounts (for optimal flight responsiveness by male NOW), the alcohol was only recovered in quantities approximately 10% of the aldehyde. When collected from Teflon disks loaded and handled in the same way, the alcohol was recovered at ca. 16% of the aldehyde. Since Teflon is putatively an inert substrate, we conclude that the aldehyde has a higher vapor pressure and also the alcohol adheres to the filter paper more strongly than the aldehyde since the alcohol is more polar.
New septa were exhaustively extracted with solvent systems different from last year, based on recommendations of experts, and as last year, these septa were/are held in clean hexane prior to use. We loaded a cleaned gray butyl rubber septum with 100ul of a mixture of (11Z,13Z)-hexadecadienal, (11Z,13Z)-hexadecadien-1-ol, and (3Z,6Z,9Z,12Z,15Z)-tricosapentaene (C23 pentaene); 1mg each. Release ratios were determined using a modified, single side-arm volatile collection device. The septum was held at room temperature (21-25 C) in a fume hood between measurements.
We have developed greatly improved handling methods for pheromone release substrates and the volatiles collected from them allow consistent and repeatable measurements of the release ratios. We should be able to repeat at least one week’s female equivalence in the field and likely extend that with altered loading of substrates and additional stabilization methods. Along with a repeat of field assays we will conduct laboratory wind tunnel assays using (lower dosage) rubber septa to correlate degree of responsiveness with volatile ratios and rough measures of release rates.