Formation of the unusual semivolatile diterpene rhizathalene by the Arabidopsis class I terpene synthase TPS08 in the root stele is involved in defense against belowground herbivory.

Secondary metabolites are major constituents of plant defense against herbivore attack. Relatively little is known about the cell type-specific formation and antiherbivore activities of secondary compounds in roots despite the substantial impact of root herbivory on plant performance and fitness. Here, we describe the constitutive formation of semivolatile diterpenes called rhizathalenes by the class I terpene synthase (TPS) 08 in roots of Arabidopsis thaliana. The primary enzymatic product of TPS08, rhizathalene A, which is produced from the substrate all-trans geranylgeranyl diphosphate, represents a so far unidentified class of tricyclic diterpene carbon skeletons with an unusual tricyclic spiro-hydrindane structure. Protein targeting and administration of stable isotope precursors indicate that rhizathalenes are biosynthesized in root leucoplasts. TPS08 expression is largely localized to the root stele, suggesting a centric and gradual release of its diterpene products into the peripheral root cell layers. We demonstrate that roots of Arabidopsis tps08 mutant plants, grown aeroponically and in potting substrate, are more susceptible to herbivory by the opportunistic root herbivore fungus gnat (Bradysia spp) and suffer substantial removal of peripheral tissue at larval feeding sites. Our work provides evidence for the in vivo role of semivolatile diterpene metabolites as local antifeedants in belowground direct defense against root-feeding insects.

Unusual macrocyclic lactone sex pheromone of Parcoblatta lata, a primary food source of the endangered red-cockaded woodpecker.

Wood cockroaches in the genus Parcoblatta, comprising 12 species endemic to North America, are highly abundant in southeastern pine forests and represent an important prey of the endangered red-cockaded woodpecker, Picoides borealis. The broad wood cockroach, Parcoblatta lata, is among the largest and most abundant of the wood cockroaches, constituting >50% of the biomass of the woodpecker's diet. Because reproduction in red-cockaded woodpeckers is affected dramatically by seasonal and spatial changes in arthropod availability, monitoring P. lata populations could serve as a useful index of habitat suitability for woodpecker conservation and forest management efforts. Female P. lata emit a volatile, long-distance sex pheromone, which, once identified and synthesized, could be deployed for monitoring cockroach populations. We describe here the identification, synthesis, and confirmation of the chemical structure of this pheromone as (4Z,11Z)-oxacyclotrideca-4,11-dien-2-one [= (3Z,10Z)-dodecadienolide; herein referred to as "parcoblattalactone"]. This macrocyclic lactone is a previously unidentified natural product and a previously unknown pheromonal structure for cockroaches, highlighting the great chemical diversity that characterizes olfactory communication in cockroaches: Each long-range sex pheromone identified to date from different genera belongs to a different chemical class. Parcoblattalactone was biologically active in electrophysiological assays and attracted not only P. lata but also several other Parcoblatta species in pine forests, underscoring its utility in monitoring several endemic wood cockroach species in red-cockaded woodpecker habitats.

Pheromone trap for the eastern tent caterpillar moth.

The discovery that the eastern tent caterpillar Malacosoma americanum (F.) causes mare reproductive loss syndrome (MRLS), and thus has the potential to continue to result in major economic losses to the equine industry of Kentucky, has resulted in an intensive effort to identify practical means to monitor and control this defoliator, including these experiments to optimize a sex pheromone trap for this pest. A pheromone-baited delta trap with a large opening, such as InterceptST Delta, was more effective than other tested traps. Orange delta traps caught more moths than other tested colors. ETC males are caught at all tested heights within the tree canopy. For monitoring flights, setting traps at 1.5 m would allow easy counting of moths. A 9:1 blend of (E,Z)-5,7-dodecadienal (ETC-Ald) and (E,Z)-5,7-dodecadienol (ETC-OH) was most effective in capturing males. Increasing loading doses of a 3:1 blend (Ald:OH) resulted in the capture of increasing numbers of moths, but a 9:1 blend was more effective than 3:1 blend even at a nine-fold lower loading rate. Pheromone-impregnated white septa caught more moths than gray septa at the same loading dose. The advantages and limitations of using pheromone traps for monitoring M. americanum are discussed.

Chemical composition of some components of the arrestment pheromone of the black-legged tick, Ixodes scapularis (Acari: Ixodidae) and their use in tick control.

Chemical analysis (high-performance liquid chromatography) and bioassay demonstrated the presence of compounds that seem to be components of the Ixodes scapularis arrestment pheromone. Only two purines, guanine and xanthine, were found in acidified saline extracts made from cast skins after molting of fed nymphs, fed larvae, and fecal/excretory exudates deposited by unfed adults on substrates in their environment. The ratio of guanine to xanthine was 10.6:1 in an extract from the nymphal skins versus 0.95:1 in an extract from the larval skins. Guanine, xanthine, and traces of a third purine, tentatively identified as 8-azaguanine, were found in extracts made from filter paper strips or washings from glass vials contaminated with tick feces and excreta left by unfed adults. 8-azaguanine may be a product of microbial degradation of the other purines rather than a natural product from the ticks. Low concentrations of ammonia also were detected in saline extracts of excreta from feeding ticks. Hematin also was found in NH4OH extracts of the black fecal/excretory exudates deposited by the unfed ticks. Hematin was tentatively identified by comparison of spectra with that of the authentic standard. Bioassays demonstrated a strong positive arrestment response to cast skins found to contain a mixture of guanine and xanthine and to black fecal/excretory exudates containing guanine, xanthine, the putative 8-azaguanine, and hematin. A Noldus video tracking system using a CCD video camera and Ethovision Pro tracking software showed statistically significant increases in the frequency of visits to the treated zone versus the control. Ticks were significantly more likely to assemble in response to the tick exudates within as little as 3 h compared with the controls. Previous bioassay studies also showed strong positive responses to guanine, xanthine, other purines, and hematin. Comparisons with the arrestment pheromones of other tick species are described. The inclusion of the pheromone components in a permethrin-impregnated oily matrix, Last Call, increased the lethal activity of the product to 95% compared with only 65% in the formulation with permethrin alone. More detailed knowledge of I. scapularis arrestment pheromone may be useful for improving the efficacy of this tick-killing technology even further.

Synthesis of the female sex pheromone of the citrus mealybug, Planococcus citri.

The citrus mealybug, Planococcus citri (Risso) is a common pest in the Southern U. S. and the Mediterranean. Two alternative syntheses of the female sex pheromone, (1R)-(+)-cis-2,2-dimethyl-3-isopropenyl-cyclobutane methanol acetate, have been developed. Key transformations include an allylic oxidation of (1R)-(+)-alpha-pinene to (+)-R-verbenone, oxidative decarboxylation using RuCl(3)-NaIO(4), and methylenation with Zn/CH(2)Br(2)/TiCl(4).

Microparticle dispensers for the controlled release of insect pheromones.

The potential utility of micrometer-sized particles as controlled-release devices for the volatilization of insect pheromones for mating disruption applications is evaluated in this study for two pheromone/model compound systems (codlemone/1-dodecanol and disparlure/1,2-epoxyoctadecane). To expedite the measurement of release rates from these particle devices, two techniques based on thermogravimetric analysis (TGA) have been exploited: isothermal TGA (I-TGA) at elevated temperatures (40-80 degrees C) with N(2) convection and volatilization temperature (VT) by dynamic TGA. A correlation between these two methods has been established. Samples that exhibit a higher VT provide a lower release rate from a particle substrate. Using these techniques, it has been demonstrated that chemical interactions between adsorbed liquids and particle surfaces may play a small role in defining release characteristics under conditions of low surface area, whereas parameters associated with total surface area and micropore structure appear to be much more significant in retarding evaporation for uncoated particles containing an adsorbed liquid. Additional regulation of release rates was achieved by coating the particle systems with water-soluble or water-dispersible polymers. By careful selection of particle porosity and coating composition, it is envisioned that the evaporation rate of pheromones can be tailored to specific insect control applications.

Chemical and behavioral analysis of the cuticular hydrocarbons from Asian citrus psyllid, Diaphorina citri.

Huanglongbing (HLB) is the most destructive disease of citrus worldwide. The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the vector of the phloem-inhabiting bacterium, Candidatus Liberibacter asiaticus, which is presumed to cause HLB in Florida citrus. Laboratory and field studies were conducted to examine the behavioral responses of male and female D. citri to their cuticular extracts. In olfactometer assays, more male D. citri were attracted to one, five, or 10 female cuticular extract equivalent units than blank controls. The results were confirmed in field studies in which clear or yellow traps baited with 10 female cuticular extract equivalent units attracted proportionately more males than clear traps baited with male cuticular extract or unbaited traps. Analyses of cuticular constituents of male and female D. citri revealed differences between the sexes in chemical composition of their cuticular extracts. Laboratory bioassays with synthetic chemicals identified from cuticular extracts indicated that dodecanoic acid attracted more males than clean air. Traps baited with dodecanoic acid did not increase total catch of D. citri as compared with blank traps at the dosages tested; however, the sex ratio of psyllid catch was male biased on traps baited with the highest lure loading dosage tested (10.0 mg).

Nanogram-scale preparation and NMR analysis for mass-limited small volatile compounds.

Semiochemicals are often produced in infinitesimally small quantities, so their isolation requires large amounts of starting material, not only requiring significant effort in sample preparation, but also resulting in a complex mixture of compounds from which the bioactive compound needs to be purified and identified. Often, compounds cannot be unambiguously identified by their mass spectra alone, and NMR analysis is required for absolute chemical identification, further exacerbating the situation because NMR is relatively insensitive and requires large amounts of pure analyte, generally more than several micrograms. We developed an integrated approach for purification and NMR analysis of <1 µg of material. Collections from high performance preparative gas-chromatography are directly eluted with minimal NMR solvent into capillary NMR tubes. With this technique, (1)H-NMR spectra were obtained on 50 ng of geranyl acetate, which served as a model compound, and reasonable H-H COSY NMR spectra were obtained from 250 ng of geranyl acetate. This simple off-line integration of preparative GC and NMR will facilitate the purification and chemical identification of novel volatile compounds, such as insect pheromones and other semiochemicals, which occur in minute (sub-nanogram), and often limited, quantities.

Identification of the sex pheromone of the German cockroach, Blattella germanica.

The sex pheromone of the German cockroach, Blattella germanica, has been characterized as gentisyl quinone isovalerate. This cockroach is a major cause of allergic disease and serves as a mechanical vector of pathogens, making it one of the most important residential and food-associated pests worldwide. The sex pheromone-producing gland in adult females was identified in 1993, but thermal instability of the pheromone made characterization difficult. Now, using a new preparative gas chromatography approach coupled with electroantennographic detection, we have isolated and characterized the pheromone, which we term blattellaquinone, and confirmed the identification by chemical synthesis. The synthetic pheromone was active in behavioral assays and highly effective in field trapping tests, which suggest that it may provide a new tool in cockroach population detection, monitoring, and control.

Submicro scale NMR sample preparation for volatile chemicals.

A simple, inexpensive, and highly efficient NMR sample preparation technique for volatile chemicals has been devised using a micropreparative GC system. The recovery efficiency of a volatile chemical using this technique was >80% with sample sizes of 0.05 to 0.5 microg. The purity of the acquired NMR samples was sufficient for high sensitive NMR analyses including two dimensional experiments.