Determination of the Absolute Configuration of the Male-Produced Sex Pheromone of the Stink Bug Pellaea stictica, (2R,4R,8R)-2,4,8,13-Tetramethyltetradecan-1-ol by Stereoselective Synthesis Coupled with Enantiomeric Resolution.

In a previous study, we reported the identification and synthesis of a male-specific sex pheromone component of the stink bug, Pellaea stictica, as the alcohol 2,4,8,13-tetramethyltetradecan-1-ol (1). To establish the correlation between the stereochemistry of the pheromone and its bioactivity, it first was necessary to determine its absolute configuration. For this purpose, a series of syntheses were designed to: (a) furnish a mixture of all possible stereoisomers; (b) a narrowed down group of diastereomers, and (c) one specific enantiomer. A crucial step in the syntheses involved a coupling reaction between two key intermediates: a phosphonium salt and an aldehyde, through a Wittig olefination. Nuclear magnetic resonance data of a mixture of the synthetic pheromone diastereomers and further comparison of GC retention times with that of the natural product by gas chromatography suggested that the methyl branches at C2 and C4 were in a syn relationship, reducing the possibilities to only four of the eight possible stereoisomers. Employing GC analysis, chiral derivatization reagents and synthetic (8R)-2,4-syn-1 it was possible to confirm the configuration of the methyl branch at C8 as R, reducing the number of possible stereoisomers to two. After enantioselective synthesis of (2R,4R,8R)-1, the absolute configurations of all methyl branches of the natural compound were confirmed as R, fully identifying the male-produced sex pheromone of P. stictica as (2R,4R,8R)-2,4,8,13-tetramethyltetradecan-1-ol.

Identification of Zingiberenol and Murgantiol as Components of the Aggregation-Sex Pheromone of the Rice Stink Bug, Mormidea v-luteum (Heteroptera: Pentatomidae).

Mormidea v-luteum (Lichtenstein, 1796) feeds on commercial crops, such as rice and ryegrass, causing damage that slows growth and reduces productivity. With the aim of developing an eco-friendly control technique, we investigated the compounds involved in chemical communication in this species. The volatiles produced by a group of seven males or females allocated to different aerated glass chambers were collected for 24 h and analyzed by gas chromatograph/mass spectrometry and gas chromatography/Fourier transform infrared spectroscopy. The analyses showed six male-specific compounds, identified as two isomers of zingiberenol (compounds 1 and 2), three isomers of murgantiol (3, 5 and 6) and sesquipiperitol (4). Compounds 1 and 5 were the major components and were produced in a ratio of 6:4. The absolute stereochemistry of the two isomers of zingiberenol was established as (1S,4R,1'S)-1 and (1R,4R,1'S)-2 by chiral gas chromatography. Stereochemistry was not determined for all the other molecules. To confirm the attractiveness of these chemicals, bioassays were performed in a Y-tube olfactometer, first using crude extracts and, subsequently, synthetic compounds. Male volatiles were attractive to both sexes, demonstrating an aggregation pheromone. In bioassays with synthetic compounds, (1'S)-zingiberenol was highly attractive to both males and females. However, when (1'S)-murgantiol was tested, only females were attracted. Interestingly, when a mixture of zingiberenol and murgantiol isomers was tested, it was attractive to both sexes, with females more attracted to the mixture than to zingiberenol alone; males did not distinguish between treatments. Thus, the bioassay data suggest that the molecules have different functions in chemical communication of this species: zingiberenol acts primarily as an aggregation pheromone, while murgantiol plays a role as a sex pheromone.

Camphor and Borneol as the Male-Produced Sex Pheromone of the Shield Bug, Orsilochides leucoptera (Hemiptera: Scutelleridae).

The ability of stink bugs to release high amounts of strong-smelling and irritating defensive compounds is related to their metathoracic gland (MTG), which is an exocrine gland with defensive, sexual, alarm and aggregation signal functions. Orsilochides leucoptera (Scutelleridae) is a widespread species in the Neotropical region that feeds on plants of the families Malvaceae, Poaceae and Euphorbiaceae. A series of compounds (ketones, alcohols and esters) have been identified in the MTGs among the three species of Scutelleridae whose MTG secretions have been investigated thus far; however, no sex pheromone compounds have been described for any scutellerid species. The aim of this work was to study sex pheromone communication within this family of stink bugs, and identify the compounds present in the MTG of O. leucoptera. Analysis by gas chromatography/mass spectrometry (GC/MS) revealed two male specific compounds identified as (R)-camphor (1) and (R)-borneol (2), which were attractive to females in Y-tube olfactometer bioassays. Also, GC/MS analysis of secretions from MTG of males and females of O. leucoptera, identified eight additional compounds: 2-(E)-hexenal (3), (E)-4-oxo-2-hexenal (4), (E)-hex-2-enyl acetate (5), (R)-linalool (6), (R)-α-terpineol (7), dodecane (8), 1-tridecene (9) and n-tridecane (10). From these, (R)-α-terpineol was detected only in the gland of males, and is probably a biosynthetic intermediate of the pheromone components. Most of the MTG compounds identified in O. leucoptera have been identified in other heteropteran species. Camphor is often a toxic and repellent compound for insects. However, we report it, for the first time, as a sex pheromone component of an insect.

Kairomones from Euschistus heros egg masses and their potential use for Telenomus podisi parasitism improvement.

Telenomus podisi Ashmead (Hymenoptera: Scelionidae) is the most important egg parasitoid of Euschistus heros (Fabricius) (Hemiptera: Pentatomidae), and its successful parasitism is related to their searching ability to find suitable hosts under a complex chemical environment using host-reliable cues. Thus, the objective of this study was to elucidate chemical substances on the external layer of E. heros eggs and report its potential kairomonal activity on T. podisi. We tested female wasps in olfactometer system to synthetic compounds obtained from a chemical identification of E. heros egg masses. The synthetic blend was also evaluated in parasitism tests under laboratory and semi-field conditions. We identified 31 substances from egg surface extracts, including monoterpenes, aldehydes and alkanes. Among those compounds, a synthetic solution including camphene, ß-pinene, limonene and benzaldehyde-induced chemotaxic behaviour on the wasps and increased the parasitism on E. heros eggs, either in laboratory or semi-field test, suggesting its potential use to T. podisi manipulation and parasitism improvement.

Male-Produced Sex Pheromone of Tibraca limbativentris Revisited: Absolute Configurations of Zingiberenol Stereoisomers and their Influence on Chemotaxis Behavior of Conspecific Females.

The rice stalk stink bug, Tibraca limbativentris, is an important rice pest in Brazil with a high invasive potential for Mexico and the USA. The sex pheromone of this species was previously identified as a combination of two stereoisomers of 1,10-bisaboladien-3-ol (zingiberenol), but the absolute configurations of these sesquiterpenes were not determined, neither were their effect(s) on T. limbativentris behavior evaluated. In this study, using two chiral columns, we compared retention times of the two natural 1,10-bisaboladien-3-ol stereoisomers from air-entrainment samples of male T. limbativentris with those of synthetic stereoisomers of 1,10-bisaboladien-3-ol. The results showed that T. limbativentris males produce (3S,6S,7R)-1,10-bisaboladien-3-ol (1) and (3R,6S,7R)-1,10-bisaboladien-3-ol (5) as their sex pheromone. Two new minor, male-specific components were also identified as cis and trans isomers of 2,10-bisaboladien-1-ol (sesquipiperitol). Y-tube olfactometer bioassays showed that the major (3S,6S,7R) isomer 1 was essential for attraction of T. limbativentris females, but the minor (3R,6S,7R) isomer 2 was not, nor did it show synergistic/antagonistic effects when added to the major isomer. The (1S,6S,7R) and (1R,6S,7R) stereoisomers of sesquipepiritol also attracted T. limbativentris females.

Revision of Coptochilus (Heteroptera, Scutelleridae, Pachycorinae)

Coptochilus Amyot & Serville, 1843 is distinguishable from the other Neotropical Scutelleridae by the dorsally concave head, with lateral margins of mandibular plates elevated. Here we describe and revise the species of Coptochilus based on general and genital morphological traits. Coptochilus castaneus (Dallas, 1851), once synonymous with C. ferrugineus Amyot & Serville, 1843, is revalidated, and a new species, C. morrisi, is described from French Guiana. A key to the five species in the genus is provided.

Revision of Coptochilus (Heteroptera, Scutelleridae, Pachycorinae)

Coptochilus Amyot & Serville, 1843 is distinguishable from the other Neotropical Scutelleridae by the dorsally concave head, with lateral margins of mandibular plates elevated. Here we describe and revise the species of Coptochilus based on general and genital morphological traits. Coptochilus castaneus (Dallas, 1851), once synonymous with C. ferrugineus Amyot & Serville, 1843, is revalidated, and a new species, C. morrisi, is described from French Guiana. A key to the five species in the genus is provided.(AU)

Conditioning Native Telenomus and Trissolcus (Hymenoptera: Scelionidae) Egg Parasitoids to Recognize the Exotic Brown Marmorated Stink Bug (Heteroptera: Pentatomidae: Halyomorpha halys).

Halyomorpha halys (Stål) (Heteroptera: Pentatomidae), the brown marmorated stink bug (BMSB), is a polyphagous fruit and vegetable pest from eastern Asia and now invasive in the United States and Europe. Earlier research revealed that the main volatile from hexane egg extracts of a western North American pentatomid, Euschistus conspersus Uhler, to which two native scelionid egg parasitoids, Telenomus podisi Ashmead and Trissolcus erugatus Johnson are highly attracted, was methyl (2E,4Z)-2,4-decadienoate, the major component of the conspecific male-produced aggregation pheromone. Conversely, extracts of BMSB eggs lacked the male-produced sesquiterpenoid H. halys pheromone components but contained C16,18,20 aldehydes (hexadecanal, octadecanal, and eicosanal); both egg-surface extracts of BMSB eggs and the C16,18,20 synthetic aldehyde blend repelled the female parasitoids. The goal of the present research was to manipulate egg-surface volatiles to induce the scelionid egg parasitoids to adopt BMSB eggs as a host. Here it was demonstrated that individual females of both parasitoids could be conditioned to H. halys egg-surface aldehydes in Y-tube olfactometer experiments, and this positive memory lasted 3-4 d. Furthermore, conditioned wasps successfully parasitized fresh H. halys eggs in no choice tests, and their offspring continued to successfully parasitize fresh H. halys eggs for 2-3 more generations. Eventually, both the individual and generational memories faded. Unconditioned female parasitoids were not attracted to H. halys egg extract or the synthetic blend of egg-surface aldehydes, nor did they parasitize BMSB eggs. Theoretical and practical implications of the research are discussed in the contexts of Hopkins' host selection principle, biological control, and instinct evolution.

Volatiles Mediating Parasitism of Euschistus conspersus and Halyomorpha halys Eggs by Telenomus podisi and Trissolcus erugatus.

This study identified chemicals found on the eggs of two stink bug species, one native to western North America, Euschistus conspersus, and an invasive species from Asia, Halyomorpha halys. The responses of two scelionid egg parasitoids, Trissolcus erugatus and Telenomus podisi, toward natural stink bug egg volatiles, and synthetic reconstructions of egg volatiles, were tested in bioassays. A compound, methyl (2E,4Z)-2,4-decadienoate, previously identified as the major component of the male-produced aggregation pheromone of E. conspersus, was the major volatile identified from extracts of E. conspersus eggs. In contrast, for H. halys, the sesquiterpenoids that compose the male-produced aggregation pheromone of this species were not detected on eggs, whereas the presence of hexadecanal, octadecanal, and eicosanal was detected. In laboratory olfactometer tests, both Tr. erugatus and Te. podisi females were attracted to extracts of E. conspersus eggs, and to synthetic methyl (2E,4Z)-2,4-decadienoate. However, female Tr. erugatus and Te. podisi wasps were repelled, both by extracts of H. halys eggs and by a blend of the aldehydes identified from H. halys eggs. A follow-up field study, using hexane-washed and intact E. conspersus as sentinel eggs, showed that the parasitoids Trissolcus erugatus and Gryon obesum emerged from these eggs. Sentinel hexane-washed eggs treated with 3 ng of methyl (2E,4Z)-2,4-decadienoate were parasitized more by these two species than were hexane-washed or unwashed eggs, whereas hexane-washed eggs treated with a comparable dose of the C16,18,20 aldehyde mixture were avoided by these parasitoids. In a further field experiment, Trissolcus basalis was the primary parasitoid found in sticky traps baited with methyl (2E,4Z)-2,4-decadienoate, indicating that this species was attracted to, but either did not oviposit or develop in the E. conspersus sentinel eggs in the previous experiment.

The influence of volatile semiochemicals from stink bug eggs and oviposition-damaged plants on the foraging behaviour of the egg parasitoid Telenomus podisi.

During host selection, physical and chemical stimuli provide important cues that modify search behaviours of natural enemies. We evaluated the influence of volatiles released by eggs and egg extracts of the stink bug Euschistus heros and by soybean plants treated with the eggs and egg extracts on Telenomus podisi foraging behaviour. Responses to volatiles were evaluated in Y-tube olfactometers after exposure to (1) one egg cluster for 24 h; (2) plants with eggs laid by the stink bug, tested at 24, 48, and 72 h after treatment; (3) plants with eggs laid artificially, tested at 24, 48, and 72 h after treatment; and (4) plants treated with acetone or hexane extracts of eggs. Telenomus podisi was attracted to volatiles emitted by one egg cluster and to acetone extracts of one egg cluster, but not to air or acetone controls. There were no responses to odours of plants treated with eggs or egg extracts. Analysis of acetone extracts of egg clusters by gas chromatography revealed the major components were saturated and unsaturated fatty acids, including hexadecanoic acid, linoleic acid, and (Z)-9-octadecenoic acid. Our results suggest that one egg cluster and the acetone extract of one egg cluster contain volatile compounds that can modify T. podisi foraging behaviour, and that the amounts of these compounds, probably together with some minor compounds, are important for host recognition by T. podisi. Also, the oviposition damage or egg extracts on the plant did not elicit indirect defences that attracted Telenomus podisi.

The fungistatic and fungicidal effects of volatiles from metathoracic glands of soybean-attacking stink bugs (Heteroptera: Pentatomidae) on the entomopathogen Beauveria bassiana.

This study was initially designed to evaluate the differential susceptibility of three soybean-attacking pentatomids to the entomopathogenic fungus Beauveria bassiana in standardized bioassays. Euschistus heros (Eh) was shown to be significantly less susceptible than Chinavia ubica (Cu), whereas Dichelops melacanthus (Dm) adults were highly susceptible to fungal infections. A deeper look at the mechanisms involved in the possible role of volatiles from metathoracic glands on fungal infections was undertaken, and gland extracts from Nezara viridula (Nv), a species known for its resilience to fungal infections, were also included in the assays. Atmospheres with volatiles from pentatomids with very low-susceptibility to B. bassiana infections (Eh and Nv) had a significant effect on speed of germination as shown in counts performed up to 22h post-inoculation, by which time 0.1 (control), 0.6 (Dm), 17.9 (Cu), 32.6 (Eh), and 43.4% (Nv) of conidia had not germinated. The fungistatic (inhibitory) and fungicidal (lethal) effects of Eh and Nv volatile-rich atmospheres were subsequently quantified in Petri dishes with either PDA or PDA medium amended with carbendazim, which allowed germination rates to be determined at 18 and 48h post-inoculation, respectively. As opposed to control, Eh volatile-rich atmosphere had a clear fungistatic effect, since germination rate was only 27.4% within 18h, but reached 99.4% at 48h post-inoculation. For Nv volatile-rich atmospheres, only 15.1% of conidia germinated within 18h, and by 48h post-inoculation, approx. 18% of conidia were unviable (neither germ tubes nor intumescence), whereas in the control treatment rates were >99% at both reading times. Therefore, the gaseous phase of defensive secretions from fungus-resilient pentatomids possess a strong inhibitory effect and may display a less pronounced lethal effect on fungal germination, as was the case for Nv.

Identification and Expression Profile of Two Putative Odorant-Binding Proteins from the Neotropical Brown Stink Bug, Euschistus heros (Fabricius) (Hemiptera: Pentatomidae).

This work presents the first identification of putative odorant-binding proteins (OBPs) from a member of the Pentatomidae, i.e. the brown stink bug Euschistus heros (Fabricius), an important pest of soybean in Brazil. Antennae from both sexes of E. heros adults (12 days old and unmated) were used to construct a cDNA library, from which two transcripts encoding putative E. heros OBPs (EherOBPs) were identified. The expression levels of EherOBP1 and EherOBP2 were found to be higher in male antennae than in female and there was difference in expression in legs, wings, and abdomens of the two sexes. The histolocalization of EherOBP1 and EherOBP2 transcripts in antennae also showed a sexual dimorphism in the chemoreception system, with different expression sites in the antennal segments between males and females, occurring predominantly at the base of the sensillum. The implications of these findings for stink bug chemoreception are discussed.

Zingiberenol, (1R,4R,1'S)­4-(1',5'-Dimethylhex-4'-enyl)-1-methylcyclohex-2-en-1-ol, identified as the sex pheromone produced by males of the rice stink bug Oebalus poecilus (heteroptera: pentatomidae).

Bioassays using an olfactometer showed that Oebalus poecilus males produce the sexual pheromone, and the chemical analysis demonstrated that this compound is zingiberenol. Two groups of isomers, each containing four diastereoisomers, (1RS,4RS,1'S)- and (1RS,4RS,1'R)-zingiberenol, were prepared. These diastereoisomers were not separated on a chiral GC column. Therefore, to determine the absolute configuration of the carbon 1, 4, and 1' of zingiberenol produced by males, the following strategies were conducted. The extract containing males volatiles was submitted to dehydration microchemistry to produce zingiberene, in which the isomers are separated by chiral GC analysis, and by comparison with the natural zingiberene from ginger oil, the absolute stereochemistry of the carbons 4 and 1' was determined to be R and S, respectively, and the carbon 1 was determined as R from the (13)C NMR spectra of quercivorol. Finally, the bioassays showed that O. poecilus females responded to racemic mixture and to (1RS,4RS,1'S)-zingiberenol.

Secondary metabolites from the phloem of Piper solmsianum (Piperaceae) in the honeydew of Edessa meditabunda.

INTRODUCTION: The phytochemistry of species of the genus Piper has been studied extensively, including Piper solmsianum. However, no studies have addressed the phytochemistry of the sap content of Piper species. OBJECTIVE: To evaluate the transferring of secondary compounds from the saps of P. solmsianum to the honeydew of Edessa meditabunda. METHODOLOGY: The honeydew of E. meditabunda and saps of P. solmsianum were analysed by GC-MS, (1) H-NMR and LC-MS. RESULTS: The lignan (-)-grandisin and the phenylpropanoid (E)-isoelemicin were detected in both saps of P. solmsianum and honeydew of E. meditabunda. CONCLUSION: Analysis of honeydew secreted by the sap-sucking insect E. meditabunda indicated that (-)-grandisin and (E)-isoelemicin are absorbed from the phloem of Piper solmsianum.