Female-Produced Sex Pheromone of Tetrastichus planipennisi, a Parasitoid Introduced for Biological Control of the Invasive Emerald Ash Borer, Agrilus planipennis.

The Asian eulophid wasp Tetrastichus planipennisi is being released in North America as a biocontrol agent for the emerald ash borer (Agrilus planipennis), a very destructive invasive buprestid beetle that is devastating ash trees (Fraxinus spp.). We identified, synthesized, and tested a female-produced sex pheromone for the wasp. The key component eliciting behavioral responses from male wasps in flight tunnel bioassays was identified as (6S,10S)-(2E,4E,8E)-4,6,8,10-tetramethyltrideca-2,4,8-triene. Female specificity was demonstrated by gas chromatographic (GC) comparison of male and female volatile emissions and whole body extracts. The identification was aided by coupled gas chromatography/mass spectrometry analysis, microchemical reactions, NMR, GC analyses with a chiral stationary phase column, and matching GC retention times and mass spectra with those of synthetic standards. The tetramethyl-triene hydrocarbon was synthesized as a mixture of two enantiomeric pairs of diastereomers, and as the pure insect-produced stereoisomer. In flight-tunnel bioassays, males responded to both the natural pheromone and the chiral synthetic material by upwind flight and landing on the source. In contrast, the mixture of four stereoisomers was not attractive, indicating that one or more of the "unnatural" stereoisomers antagonized attraction. Field trials, using yellow pan traps baited with natural pheromone, captured significantly more male wasps than control traps over a four week trial. The identified pheromone could increase the efficiency and specificity of the current detection methods for Tetrastichus planipennisi and aid in the determination of parasitoid establishment at release sites.

Pheromones of three ambrosia beetles in the Euwallacea fornicatus species complex: ratios and preferences.

Three cryptic species in the Euwallacea fornicatus species complex were reared in laboratory colonies and investigated for the presence of pheromones. Collections of volatiles from combinations of diet, fungus, beetles, and galleries from polyphagous shot hole borer (Euwallacea sp. #1) revealed the presence of 2-heneicosanone and 2-tricosanone only in the presence of beetles, regardless of sex. Subsequent examination of volatiles from the other two species, tea shot hole borer (Euwallacea sp. #2) and Kuroshio shot hole borer (Euwallacea sp. #5), revealed these two ketones were present in all three species but in different ratios. In dual choice olfactometer behavioral bioassays, mature mated females were strongly attracted to a synthetic binary blend of ketones matching their own natural ratios. However, females in each species were repelled by ketone blends in ratios corresponding to the other two species. Males of each species responded similarly to females when presented with ratios matching their own or the other two species. The presence of these compounds in the three beetle species, in ratios unique to each species, and their strong species-specific attraction and repellency, suggests they are pheromones. The ecological function of these pheromones is discussed. In addition to the pheromones, the previously known attractant (1S,4R)-p-menth-2-en-1-ol (also known as quercivorol) was discovered in the presence of the fungal symbionts, but not in association with the beetles. Quercivorol was tested in a dual-choice olfactometer and was strongly attractive to all three species. This evidence suggests quercivorol functions as a kairomone for members of the E. fornicatus species complex, likely produced by the symbiotic fungi.

Polyols, not sugars, determine the structural diversity of anti-streptococcal liamocins produced by Aureobasidium pullulans strain NRRL 50380.

Liamocins are polyol lipids produced by the fungus Aureobasidium pullulans, and have selective antibacterial activity against Streptococcus species. Liamocins produced by A. pullulans strain NRRL 50380 on sucrose medium have a d-mannitol head group ester-linked to 3,5-dihydroxydecanoate acyl chains, three or four of which are joined together by 1,5-polyester bonds (liamocins Man-A1 and Man-B1), and similar 3'-O-acetylated analogs (Man-A2 and Man-B2). However, other types of liamocins are produced depending on the choice of strain and growth conditions. In the current study, growth on different polyols, but not sugars, resulted in considerable structural variation, including liamocins with d-galactitol (dulcitol), d-sorbitol (glucitol), d- and l-arabitol, d-xylitol, l-threitol and glycerol head groups. The head groups of liamocins produced on arabitol were shown to be entirely composed of d-arabitol. These liamocin variants were structurally characterized by NMR and MS, and tested for antibacterial activity. The new liamocin variants also had selective activity against Streptococcus. Liamocin structural variants are novel antibacterials against Streptococcus sp. that merit further investigation.

A Culture Method for Darkling Beetles, Blapstinus spp. (Coleoptera:Tenebrionidae).

Darkling beetles, Blapstinus spp., have become a serious pest of Cucurbitaceae crops, especially in California. A culture method was sought to provide large numbers (>500) of adult beetles of known age and sex that could be used for laboratory testing when needed. A method previously developed for Alphitobius diaperinus (Panzer) using a diet of ground chick feed, with apple slices as a moisture source, was modified for use with Blapstinus spp. and then compared with the same method substituting apple slices with zucchini as the moisture source. Rearing boxes set up with apple slices produced significantly more pupae and adults than boxes containing zucchini slices. However, using either zucchini or apples as a moisture source yielded over the target of 500 adults per rearing box. A previous method designed to sex A. diaperinus based on the presence (♀) or absence (♂) of second valvifers in the pupal stage also proved to be effective for sexing the Blapstinus spp.

Can Coffee Chemical Compounds and Insecticidal Plants Be Harnessed for Control of Major Coffee Pests?

Pests and pathogens threaten coffee production worldwide and are difficult to control using conventional methods, such as insecticides. We review the literature on the chemistry of coffee, concentrating on compounds most commonly reported from Coffea arabica and Coffea canephora. Differences in chemistry can distinguish coffee species and varieties, and plants grown under different biogeographic conditions exhibit different chemotypes. A number of chemical groups, such as alkaloids and caffeoylquinic acids, are known to be insecticidal, but most studies have investigated their effects on coffee quality and flavor. More research is required to bridge this gap in knowledge, so that coffee can be bred to be more resistant to pests. Furthermore, we report on some pesticidal plants that have been used for control of coffee pests. Locally sourced pesticidal plants have been underutilized and offer a sustainable alternative to conventional insecticides and could be used to augment breeding for resilience of coffee plants.

Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism.

The mutualism between xyleborine beetles in the genus Euwallacea (Coleoptera: Curculionidae: Scolytinae) and members of the Ambrosia Fusarium Clade (AFC) represents one of 11 known evolutionary origins of fungiculture by ambrosia beetles. Female Euwallacea beetles transport fusarial symbionts in paired mandibular mycangia from their natal gallery to woody hosts where they are cultivated in galleries as a source of food. Native to Asia, several exotic Euwallacea species were introduced into the United States and Israel within the past two decades and they now threaten urban landscapes, forests and avocado production. To assess species limits and to date the evolutionary diversification of the mutualists, we reconstructed the evolutionary histories of key representatives of the Fusarium and Euwallacea clades using maximum parsimony and maximum likelihood methods. Twelve species-level lineages, termed AF 1-12, were identified within the monophyletic AFC and seven among the Fusarium-farming Euwallacea. Bayesian diversification-time estimates placed the origin of the Euwallacea-Fusarium mutualism near the Oligocene-Miocene boundary ∼19-24 Mya. Most Euwallacea spp. appear to be associated with one species of Fusarium, but two species farmed two closely related fusaria. Euwallacea sp. #2 in Miami-Dade County, Florida cultivated Fusarium spp. AF-6 and AF-8 on avocado, and Euwallacea sp. #4 farmed Fusarium ambrosium AF-1 and Fusarium sp. AF-11 on Chinese tea in Sri Lanka. Cophylogenetic analyses indicated that the Euwallacea and Fusarium phylogenies were largely incongruent, apparently due to the beetles switching fusarial symbionts (i.e., host shifts) at least five times during the evolution of this mutualism. Three cospeciation events between Euwallacea and their AFC symbionts were detected, but randomization tests failed to reject the null hypothesis that the putative parallel cladogenesis is a stochastic pattern. Lastly, two collections of Euwallacea sp. #2 from Miami-Dade County, Florida shared an identical cytochrome oxidase subunit 1 (CO1) allele with Euwallacea validus, suggesting introgressive hybridization between these species and/or pseudogenous nature of this marker. Results of the present study highlight the importance of understanding the potential for and frequency of host-switching between Euwallacea and members of the AFC, and that these shifts may bring together more aggressive and virulent combinations of these invasive mutualists.

Attraction of Agrilus planipennis (Coleoptera: Buprestidae) to a volatile pheromone: effects of release rate, host volatile, and trap placement.

Attraction of emerald ash borer, Agrilus planipennis Fairmaire, to a volatile pheromone was demonstrated in three field experiments using baited green sticky traps. A dose-response curve was generated for male A. planipennis to increasing release rates of (3Z)-dodecen-12-olide ((3Z)-lactone) in combination with the green leaf volatile, (3Z)-hexenol. Only the lowest release rate (<2.50 µg/d) of (3Z)-lactone significantly increased captures of male A. planipennis, as compared with traps baited with (3Z)-hexenol alone. Effect of trap height, (3Z)-lactone, and (3Z)-hexenol and their interactions on the trap capture of A. planipennis was determined in a factorial experiment. Number of males per trap was significantly and positively affected by (3Z)-lactone, (3Z)-hexenol, and trap height whereas number of females per trap was affected by trap height only; none of the interactions were significant. As predicted, the greatest mean catch of males was in traps baited with (3Z)-lactone and (3Z)-hexenol placed high in the canopy. Electroantennogram tests on the bark volatile, 7-epi-sesquithujene, demonstrated the ability of male and female A. planipennis antennae to detect and respond to this compound, particularly the (+)-7-epi-sesquithujene isomer. Results from an olfactometer bioassay and field testing did not demonstrate attraction of either males or females to (+)-7-epi-sesquithujene. These data increase our understanding of the pheromone ecology of the invasive emerald ash borer, provide further confirmation of the behavioral activity of the female-produced lactone pheromone, and should increase the ability to detect A. planipennis infestations where they are present.

Male-produced pheromone of Spathius agrili, a parasitoid introduced for the biological control of the invasive emerald ash borer, Agrilus planipennis.

The braconid wasp, Spathius agrili, has been released in the U.S. as a biocontrol agent for the invasive emerald ash borer (Coleoptera: Buprestidae: Agrilus planipennis), a destructive pest of ash trees (Fraxinus spp.). We identified and synthesized seven male-specific volatile compounds. Three of these, dodecanal, (4R,11E)-tetradecen-4-olide, and (Z)-10-heptadecen-2-one, were the key behaviorally active components in flight tunnel bioassays. Male specificity was demonstrated by gas chromatographic comparison of male and female volatile emissions and whole body extracts. Identifications were aided by coupled gas chromatographic-mass spectrometric (GC-MS) analysis, microchemical reactions, NMR, chiral GC analysis, and GC and MS comparison with authentic standards. Both the racemic and chiral forms of the γ-lactone, as well as both E- and Z-isomers were synthesized. Flight tunnel behavioral tests showed positive male and female S. agrili responses to both natural pheromone and synthetic blends, with upwind flight and landing on the source. Large field-cage tests, using yellow sticky traps baited with pheromone, captured approximately 50% of the released male and female wasps in 24-h periods. The use of pheromone-baited traps in the field could simplify the current detection method for determining parasitoid establishment (i.e., laboriously felling and peeling ash trees for recovery of S. agrili from infested EAB larvae).

Two-carbon homologation of aldehydes and ketones to α,ß-unsaturated aldehydes.

Phosphonate reagents were developed for the two-carbon homologation of aldehydes or ketones to unbranched- or methyl-branched α,ß-unsaturated aldehydes. The phosphonate reagents, diethyl methylformyl-2-phosphonate dimethylhydrazone and diethyl ethylformyl-2-phosphonate dimethylhydrazone, contained a protected aldehyde group instead of the usual ester group. A homologation cycle entailed condensation of the reagent with the starting aldehyde, followed by removal of the dimethylhydrazone protective group with a biphasic mixture of 1 M HCl and petroleum ether. This robust two-step process worked with a variety of aldehydes and ketones. Overall isolated yields of unsaturated aldehyde products ranged from 71% to 86% after the condensation and deprotection steps.

Absolute configuration of 7-epi-sesquithujene.

7-epi-sesquithujene (1) is a bicyclic sesquiterpene isolated from phoebe oil, an essential oil of the Brazilian walnut tree, Phoebe porosa. It is also produced by stressed ash trees and has been shown to elicit strong electrophysiological responses on emerald ash borer, Agrilus planipennis, antennae. In the course of the development of a synthetic 7-epi-sesquithujene lure for field testing against the emerald ash borer, we found that the absolute configuration of this compound had not been determined. We isolated >95% pure 7-epi-sesquithujene from phoebe oil via successive fractionation and conventional and argentation (HPLC) chromatographies. The specific optical rotation of this compound matched that of a synthetic product of known configuration. We also synthesized two other stereoisomers of sesquithujene and developed a chiral GC method to separate all four. Based on the specific rotation, stereoselective syntheses, and chiral GC analyses, 7-epi-sesquithujene present in phoebe oil and white ash was found to have the 2S,6S,7R absolute configuration.

Male-specific sesquiterpenes from Phyllotreta flea beetles.

Flea beetles in several genera are known to possess male-specific sesquiterpenes, at least some of which serve as aggregation pheromones that attract both sexes. In continuing research on the chemical ecology of Phyllotreta flea beetles, six new male-specific sesquiterpenes were identified, one from P. striolata (hydroxyketone 9) and five from P. pusilla (aldehydes 10-12 and 14 and alcohol 13); both species are crop pests. The minute amounts from beetles provided mass spectra and chromatographic data but were insufficient for complete structure determination. However, it was discovered that the new compounds could all be produced by applying organic reactions to previously identified flea beetle sesquiterpenes, and the resulting, larger amounts of material permitted definitive structure analysis by NMR. Molecular modeling was used in conjunction with NMR to define relative configurations of several newly created stereogenic centers. The absolute configurations of natural 9-14 were established by chiral gas chromatography/mass spectrometry. In electrophysiological tests (GC-EAD) conducted with P. striolata, compound 9 was detected with high sensitivity by the beetle antennae, which is consistent with a pheromonal function. The research opens new possibilities for using behavioral chemicals to monitor or manage these pest species.

Methyl 6-methylsalicylate: a female-produced pheromone component of the parasitoid wasp Spalangia endius.

Sex-pheromone-related behavior and chemistry were studied in the wasp Spalangia endius Walker (Hymenoptera: Pteromalidae), a pupal parasitoid of the house fly, Musca domestica L. (Diptera: Muscidae). Males responded behaviorally to female extracts by arrestment, whereas females did not arrest to male extracts. In a comparison of male and female extracts by gas chromatography-mass spectrometry (GC-MS), two female-specific compounds were found. One was identified as methyl 6-methylsalicylate (gas chromatographic retention time and mass spectrum versus an authentic standard), but the chemical structure of the second compound is still unknown. Male antennae were sensitive to both compounds in electrophysiological tests (GC-EAD). Males responded behaviorally to methyl 6-methylsalicylate by arrestment, but did not arrest to the second compound. Methyl 6-methylsalicylate has been reported previously from some ant and beetle species, but never from the Pteromalidae. Chemical analysis of the extracts and the male behavioral results are consistent with the hypothesis that methyl 6-methylsalicylate functions as a female-emitted pheromone component at short range, but the exact role of both compounds in intersexual interactions in S. endius remains to be determined.

Male-produced aggregation pheromone of the lesser mealworm beetle, Alphitobius diaperinus.

The lesser mealworm beetle, Alphitobius diaperinus (Panzer), is a widespread serious pest in poultry production facilities and is difficult to control by conventional means. Although pheromone-based tools have become useful in the management of other beetle pests, no pheromone was known for A. diaperinus, and this study sought to develop basic pheromone information. Volatiles were collected in the laboratory from groups of male and female A. diaperinus maintained on poultry food (chick starter mash). Gas chromatographic-mass spectrometric analysis of volatiles collected from feeding males and females revealed five male-specific compounds that were identified as (R)-(+)-limonene, (E)-beta-ocimene, (S)-(+)-linalool, (R)-(+)-daucene, and 2-nonanone. Emission of these began 1-2 weeks after adult emergence and could continue for at least 1 year, ceasing and resuming in response to changes in food availability and quality and other factors. No female-specific compounds were discovered. A synthetic blend of the five male compounds was attractive to both sexes in poultry production facilities in Illinois and Arkansas, indicating that the blend functions as an aggregation pheromone, but it is not yet known whether all five compounds are required for activity. A new pitfall trap is described for field use.

Sex pheromone of the pine false webworm Acantholyda erythrocephala.

Females of the pine false webworm Acantholyda erythrocephala (L) produce the sex pheromone (Z)-6, 14-pentadecadienal, which attracts flying males in the field. By using gas chromatography coupled with electroantennographic detection (GC-EAD) and mass spectrometry (GC-MS), we detected (Z)-6,14-pentadecadienal in volatile collections and in whole body extracts of female A. erythrocephala. Females, but not males, also exhibited a 25-carbon cuticular hydrocarbon, (Z,Z)-1,9,15-pentacosatriene, which can oxidize to (Z)-6,14-pentadecadienal upon exposure to air and sunlight. (Z,Z)-1,9,15-Pentacosatriene and (Z)-6,14-pentadecadienal identifications were corroborated by comparison with synthetic standards. (Z)-6, 14-Pentadecadienal is the second pheromone identified for pamphilliid sawflies, and the first to elicit strong field attraction, and thus offer potential as a pheromone lure to aid in control of this forest pest.

Antennally active macrolide from the emerald ash borer Agrilus planipennis emitted predominantly by females.

The macrocyclic lactone (3Z)-dodecen-12-olide was identified from the emissions of the emerald ash borer, Agrilus planipennis, feeding on ash foliage. The compound was detected from both sexes but was ca. 10 times more abundant from females. It was readily sensed by antennae of both males and females. Identification was confirmed by synthesis. The behavioral effects of the lactone remain unstudied in A. planipennis, but a verified pheromonal function could lead to improved monitoring for this invasive pest. The lactone is part of the pheromone of Cryptolestes pusillus, an unrelated beetle species.

Behaviorally active green leaf volatiles for monitoring the leaf beetle, Diorhabda elongata, a biocontrol agent of saltcedar, Tamarix spp.

Biological activity and chemistry of host plant volatiles were investigated for Diorhabda elongata, Brullé (Coleoptera: Chrysomelidae), a biological control agent for the invasive tree, saltcedar (Tamarix spp., Tamaricaceae). Gas chromatographic-electroantennographic detection (GC-EAD) analysis of volatiles collected from adult D. elongata feeding on saltcedar foliage or from saltcedar foliage alone showed 15 antennally active compounds. These compounds were more abundant in collections from beetle-infested foliage. Antennally active compounds were identified by GC-mass spectrometry (MS) and confirmed with authentic standards. The emissions of the most abundant GC-EAD-active compounds, green leaf volatiles (GLV), were quantitated by GC-MS. A blend of four GLV compounds, mimicking the natural blend ratio, was highly attractive to male and female D. elongata in the field, and a combination of GLV and male-produced aggregation pheromone attracted significantly greater numbers of D. elongata than did either bait alone. A preliminary experiment with a blend of seven additional GC-EAD-active saltcedar volatiles did not show any behavioral activity. The combination of the pheromone and the green leaf odor blend could be a useful attractant in detecting the presence of the biocontrol agent, D. elongata, in stands of saltcedar newly colonized by the beetle.

Male-produced aggregation pheromone compounds from the eggplant flea beetle (Epitrix fuscula): identification, synthesis, and field biossays.

Volatiles from the eggplant flea beetle, Epitrix fuscula Crotch (Coleoptera: Chrysomelidae), feeding on host foliage, were investigated. Six male-specific compounds were detected and were identified through the use of mass spectrometry, nuclear magnetic resonance (NMR) spectrometry, chiral and achiral gas chromatography, high-performance liquid chromatography, electrophysiology (gas chromatography-electroantennography, GC-EAD), and microchemical tests. The two most abundant of the six compounds were (2E,4E,6Z)-2,4,6-nonatrienal (1) and (2E,4E,6E)-2,4,6-nonatrienal (2). The other four compounds, present in minor amounts, were identified as himachalene sesquiterpenes; two of these, 3 and 4, were hydrocarbons and two, 5 and 6, were alcohols. All four sesquiterpenes were previously encountered from male flea beetles of Aphthona spp. and Phyllotreta cruciferae. Synthetic 1 and 2 matched the natural products by GC retention times, mass spectra, and NMR spectra. Sesquiterpenes 3-6 similarly matched synthetic standards and natural samples from the previously studied species in all ways, including chirality. Both natural and synthetic 1 and 2 gave positive GC-EAD responses, as did sesquiterpenes 3, 5, and 6. Field trials were conducted with a mixture of 1 and 2, and the baited traps were significantly more attractive than control traps to both male and female E. fuscula. The E. fuscula pheromone has potential for monitoring or controlling these pests in eggplants.

Dimethylfuran-lactone pheromone from males of Galerucella calmariensis and Galerucella pusilla.

Male Galerucella calmariensis and Galerucella pusilla (Coleoptera: Chrysomelidae) emit an aggregation pheromone while feeding on host foliage. Isolation of the compound from collected volatiles was guided by comparisons of gas chromatograms of extracts from males and females and by gas chromatography-electroantennographic detection. The compound was identified by a combination of spectrometric methods and microchemical tests as the novel dimethylfuran lactone, 12,13-dimethyl-5,14-dioxabicyclo[9.2.1]tetradeca-1(13),11-dien-4-one. The structure was confirmed by synthesis, and the synthetic compound attracted males and females of both species in field bioassays. These beetles were previously introduced into North America as biological control agents for the invasive wetland weed, purple loosestrife Lythrum salicaria, and the pheromone could become a tool for monitoring populations. A new method is described for distinguishing the two species based on the tibial spurs of the males.

Identification, synthesis, and field evaluation of the sex pheromone from the citrus leafminer, Phyllocnistis citrella.

Using male antenna as the sensing element, three electroantennographic detection (EAD)-active peaks were detected from pheromone gland extracts of the citrus leafminer, Phyllocnistis citrella. Based on gas chromatography (GC)-mass spectrometry and GC-infrared data, the semiochemicals were tentatively identified as a novel pheromone, (Z,Z,E)-7,11,13-hexadecatrienal, a previously identified attractant, (Z,Z)-7,11-hexadecadienal, and (Z)-7-hexadecenal in a ratio of 30:10:1, respectively. Identification was confirmed with synthetic compounds, which gave retention times identical to those of the natural products on three capillary columns with polar and nonpolar phases. While traps baited only with the previously identified attractant alone did not catch any males in Brazil, binary and tertiary mixtures with the major constituents caught significantly more male moths than traps baited with five virgin females.

Pheromonal activity of compounds identified from male Phyllotreta cruciferae: field tests of racemic mixtures, pure enantiomers, and combinations with allyl isothiocyanate.

Four himachalene sesquiterpenes and (+)-gamma-cadinene, previously identified as possible pheromone components from males of a North American population of Phyllotreta cruciferae Goeze (Coleoptera, Chrysomelidae), were tested for attractiveness in field trapping experiments in Hungary. A mixture of the four synthetic racemic himachalene derivatives and (+)-gamma-cadinene from a botanical source was slightly attractive to beetles, but much more attractive when blended with the known host-plant-derived attractant allyl isothiocyanate. This result was consistent with a previous study in North America. In tests with optically pure synthetic compounds, a blend of the same himachalene enantiomers found from male beetles was equivalent to the corresponding blend of racemic compounds, whereas a blend of the opposite enantiomers was not active. Through subtraction tests, it was found that the single compound, (6R,7S)-2,2,6,10-tetramethylbicyclo[5.4.0.]undeca-9,11-diene [compound (+)-A in this study], was as active as the whole mixture, suggesting that this compound is the key pheromone component of the European population of P. cruciferae. During field trials, several congeneric species, including P. vittula, P. nemorum, P. nodicornis, and P. ochripes, also were caught, suggesting that the same compound(s) may be relatively widespread as pheromone components in this genus.