The Preferential Binding of a Sensory Organ Specific Odorant Binding Protein of the Alfalfa Plant Bug Adelphocoris lineolatus AlinOBP10 to Biologically Active Host Plant Volatiles

Semiochemicals such as sex pheromones and plant volatiles are crucial components of insect mating systems and host plant localization. In the olfactory signal transduction pathway, odorant-binding proteins (OBPs) are important elements that function in the first step of the pathway by carrying hydrophobic semiochemicals across the sensillum lymph to the olfactory receptors (ORs). In this study, we examined the binding affinities of semiochemicals to AlinOBP10, a putative OBP from the alfalfa plant bug, Adelphocoris lineolatus, that we demonstrate is expressed mainly in sensory organs. We then characterized the biological activities of the high affinity semiochemicals by measuring their electrophysiological activities in antennae and behavioral responses in the plant bug. AlinOBP10 displayed weak binding affinities to two major putative pheromone components, hexyl butyrate and (E)-2-hexenyl butyrate. In contrast, AlinOBP10 exhibited higher binding affinities to six host plant volatiles, namely myrcene, β-pinene, β-ionone, 3-hexanone, (E)-2-hexenal, and 1-hexanol. The biological activities of these six putative ligands were further studied in electroantennogram recordings and Y-tube olfactometer trials. The three compounds, (E)-2-hexenal, 1-hexanol, and 3-hexanone elicited strong electrophysiological responses, but elicited distinct behaviors. While 3-hexanone was attractive to female adults, (E)-2-hexenal and 1-hexanol were significant repellents. Although a weak electrophysiological response was elicited with β-pinene, it was a strong repellent. These results demonstrate that AlinOBP10 can interact with attractants, as well as repellents, with some specificity toward plant volatiles over sex pheromones.     

Identification of the Female-Produced Sex Pheromone of the Plant Bug Apolygus spinolae

Apolygus spinolae (Meyer-Dür) (Heteroptera: Miridae) is an important pest of fruit and tea trees in Korea and Japan. Analyses of extracts of metathoracic scent glands revealed that those of female bugs contained hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal in a ratio of 20:100:7. The glands of males contained the same three compounds, but the ratio of the components was quite different, with hexyl butyrate being the most abundant. Field trapping tests with various blends of the synthetic compounds dispensed from high-density polyethylene tubes showed that (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal were essential for attraction of male A. spinolae, and catches with a wide range of ratios of these two compounds did not differ significantly. However, adding hexyl butyrate at 50 % or more of the (E)-2-hexenyl butyrate to the binary blend strongly inhibited attraction of males. Trap catches increased with increasing amounts of a 10:1 blend of (E)-2-hexenyl butyrate and (E)-4-oxo-2-hexenal from 0.011 to 11 mg loaded into the tube. Catches of males in traps baited with lures containing 1.1 mg of the binary blend were not significantly different from catches in traps baited with live virgin females.     

Male-Produced Sex Attractant Pheromone of the Green Stink Bug, Acrosternum hilare (Say)

Sexually mature virgin adult males of the green stink bug, Acrosternum hilare attracted sexually mature virgin adult females in laboratory bioassays using a vertical Y-tube. There was no indication that males attracted other males, or that females attracted either sex. These results suggested that A. hilare males produce a sex pheromone. Extracts of odors collected from sexually mature males contained compounds that were not present in extracts from females or sexually immature males. (4S)-Cis-(Z)-bisabolene epoxide ((4S)-cis-Z-BAE) was the major sex-specific component of the extract. The crude extract was attractive to female A. hilare, but when separated into four fractions, only the portion containing (4S)-cis-Z-BAE and the minor component (4S)-trans-Z-BAE was attractive to females. This fraction was as attractive as the crude extract, suggesting that the former contained all the pheromone components. Neither synthetic (4S)-cis-Z-BAE nor (4S)-trans-Z-BAE alone was attractive to females, but a 95:5 cis:trans blend, mimicing the ratio naturally produced by males, was attractive to females in Y-tube bioassays. Bioassays in a field cage showed that significantly more A. hilare females were attracted to cotton string lures treated with 1 mg of a 95:5 blend of (4S)-cis-Z-BAE and (4S)-trans-Z-BAE placed inside a bouquet of alfalfa than to an alfalfa bouquet containing a pentane-treated control. In field cage studies, attraction of females was greatest during the late afternoon and evening hours, and female A. hilare approached the synthetic pheromone source almost exclusively by walking.     

Defensive Compounds and Male-Produced Sex Pheromone of the Stink Bug, Agroecus griseus

Agroecus griseus is a serious corn pest in Brazil. Contents of the dorsal abdominal glands (DAGs) in nymphs, and the metathoracic gland (MTG) in adults of this species were characterized and quantified. Compounds found were similar to those of other Pentatomidae species and included aliphatic hydrocarbons, aldehydes, oxo-alkenals, and esters. However, two compounds were found in the MTG that have not been described previously for this family. Mass spectrometry, infrared spectroscopy, and gas chromatographic analysis using coinjection with authentic standards confirmed the identities of the compounds as enantiopure (S)-2-methylbutyl acetate and 3-methyl-2-butenyl acetate. The five nymphal instars showed significantly different ratios of components, mainly between those of the first and later instars. No significant differences were detected in MTG contents between sexes. Gas chromatography (GC) analysis of aeration extracts of males and females showed the presence of a compound released exclusively by males. Gas chromatography – electroantennographic detection (GC-EAD) assays indicated that the male-specific compound is bioactive in females, suggesting the presence of an attractant pheromone. The mass spectrum and infrared data for this compound matched with methyl 2,6,10-trimethyltridecanoate, a sex pheromone component previously detected in the stink bugs, Euschistus heros and E. obscurus. The synthetic standard coeluted with the natural pheromone on two different GC stationary phases, confirming the proposed structure. Y-tube olfactometer assays showed that the synthetic standard was strongly attractive to females, and GC-EAD tests produced responses with antennae from females similar to those of the natural pheromone.     

Investigation of Long-Range Female Sex Pheromone of the European Tarnished Plant Bug, Lygus rugulipennis: Chemical, Electrophysiological, and Field Studies

The European tarnished plant bug, Lygus rugulipennis, is an important pest of agricultural and horticultural crops throughout Europe. Adult male L. rugulipennis were previously shown to be attracted to traps baited with live virgin females, which suggests the females produce a sex pheromone. Volatiles produced by virgin female L. rugulipennis were shown to contain three components, hexyl butyrate, (E)-2-hexenyl butyrate, and (E)-4-oxo-2-hexenal which elicited electroantennographic (EAG) responses from males in analyses by linked gas chromatography-electroantennography (GC-EAG). They were produced in 1.5:1:0.08 ratio, respectively, by single females. Collections from 1, 2, or 4 virgin females showed the proportions of hexyl butyrate and (E)-4-oxo-2-hexenal to increase relative to that of (E)-2-hexenyl butyrate with increasing number of females. Although these compounds were found in body extracts of both male and female L. rugulipennis, they were not detected in volatiles released by virgin males. EAG dose-response studies showed that both males and females responded to these chemicals with minimal differences in sensitivity between the sexes or to the three components, except that males were more responsive than females to (E)-4-oxo-2-hexenal at the two highest doses tested. Release rates of the compounds from rubber septa, polyethylene vials, and polyethylene sachets were measured under laboratory conditions. Four field tests were carried out using sticky traps baited with all possible binary and tertiary combinations of the three chemicals using different combinations of dispensing systems. Catches of male L. rugulipennis in baited traps were similar to those in unbaited traps. Significantly fewer females were caught on traps baited with blends containing hexyl butyrate than on traps without hexyl butyrate or unbaited traps in one test and overall. The roles of the three compounds and possible reasons for their failure to attract males are discussed.     

Identification of Female Sex Pheromone of the Rice Leaf Bug, Trigonotylus caelestialium

The female sex pheromone of the rice leaf bug, Trigonotylus caelestialium, was analyzed by GC-EAD and GC-MS. Ten EAD active compounds—n-hexyl n-hexanoate, (E)-2-hexenyl n-hexanoate, n-hexyl (E)-2-hexenoate, n-octyl n-butyrate, n-octyl n-hexanoate, (E)-2-octenyl n-hexanoate, n-pentyl n-hexanoate, n-hexyl n-butyrate, (E)-2-hexenyl n-butyrate, and n-hexyl (E)-2-butenoate—were detected in the ratio of 1000:414–491:trace–5:5–11:55–71:50–63:trace–3:225:90:32 from female body extracts, and in the ratio of 1000:271–342:10–43:1–3:58–78:14–19:trace:178:36:26 from male body extracts. Field trapping tests with these synthetic compounds indicated that n-hexyl n-hexanoate, (E)-2-hexenyl n-hexanoate, and n-octyl n-butyrate are pheromone components, and mixtures in ratios of 1000:400–500:10–100 were more attractive to males. Doses ranging from 4.29 g to 14.3 g of the three-component mixture in the ratio of 1000:400:30 loaded into glass capillary tubes were most attractive to males.     

The Male-produced Sex Pheromone of the True Bug, Phthia picta, is an Unusual Hydrocarbon

Phthia picta is part of a complex of true bugs (Heteroptera) in Brazil that attack tomatoes, being particularly damaging because nymphs and adults feed on both leaves and fruit. Gas chromatography (GC) of aeration extracts of adult males vs. females revealed the presence of a male-specific compound. GC-electroantennographic detector experiments indicated that the antennae of females are highly sensitive to this male-specific compound. GC-mass spectrometry and GC-FTIR analyses suggested a methyl branched hydrocarbon structure for this compound. After synthesis of three different proposed structures, the natural product was indentified as 5,9,17-trimethylhenicosane, which was strongly attractive to females in Y-tube olfactometer bioassays. Analysis of dissected body parts of adults revealed that the pheromone is produced in the lateral accessory glands of the metathoracic scent gland of males only.     

Hero Turned Villain: Identification of Components of the Sex Pheromone of the Tomato Bug,Nesidiocoris tenuis

Journal of Chemical Ecology - Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae) is a tropical mirid bug used as a biocontrol agent in protected crops, including tomatoes. Although N. tenuis...     

Sex Pheromone of the Plant Bug, <Emphasis Type="Italic">Phytocoris calli</Emphasis> Knight

Female Phytocoris calli Knight produce a sex pheromone from metathoracic scent glands. The pheromone consists of hexyl acetate (HA; present in both sexes), with the female-specific compounds (E)-2-hexenyl acetate (E2HA), octyl acetate (OA), and (E)-2-octenyl acetate (E2OA). HA and E2OA are key components of the pheromone, since deletion of either ester from the blend resulted in a total suppression of conspecific male trap catches. However, the binary blend of HA and E2OA was only slightly attractive to males, and was significantly less active than the four-component blend. The two ternary blends, HA/OA/E2OA and HA/E2HA/E2OA, were each as attractive as the full four-component blend. Evidence from previous research on the pheromones of Phytocoris species suggests that the apparent chemical redundancy in the pheromone of P. calli may actually be involved in maintaining reproductive isolation from other sympatric species. The patterns observed for pheromones of the five Phytocoris species whose pheromones have been directly (P. californicus, P. relativus, P. difficilis, and P. calli) or indirectly (P. breviusculus) studied are discussed vis-à-vis the pheromone intractable species of Lygus and Lygocoris plant bugs.     

Sex pheromone of the mullein bug,Campylomma verbasci (Meyer) (Heteroptera: Miridae)

The first known sex pheromone for the family Miridae is reported for the mullein bug,Campylomma verbasci (Meyer) (Heteroptera: Miridae). Extracts of and Porapak Q-trapped volatiles from females were attractive to males in field bioassays. Butyl butyrate and hexyl butyrate were the predominant compounds in the females' volatiles, but these compounds previously had been found to be inactive when released alone or in combination. Butyl butyrate in combination with an isolated trace chemical, (E)-crotyl butyrate (which was inactive alone), rivaled the attraction elicited by five live females. When synthetic butyl butyrate and (E)-crotyl butyrate in a 16: 1 ratio were released at 91 or 183 g/day, they comprised an effective attractant for male bugs.     

Repeatability of Pheromone Blend Composition in Individual Males of the Southern Green Stink Bug, Nezara viridula

The males of Nezara viridula (Heteroptera, Pentatomidae) produce a blend of four terpenoid compounds. The proportion of these four compounds varies among different populations. The terpenoid content of the volatile emission from individual males of a French population was monitored for several days with headspace solid-phase microextraction. The total amount of terpenoid compounds collected on an SPME fiber from one virgin male bug emitting over the course of 1 hr was very variable between samples. More than 50% of males released pheromone, at least during one part of the observation period. The relative percentages of bisabolene, nerolidol, trans-bisabolene epoxide, and cis-bisabolene epoxide were calculated and compared. The proportion of nerolidol in the blend showed a large dispersion between samples and a high coefficient of variation. The percentage values for bisabolene and its trans and cis epoxides were less dispersed, and the coefficients of variation for both epoxides were low. The proportion of trans epoxide relative to the two epoxides, which has been considered characteristic of pheromone strains of N. viridula, showed the smallest coefficient of variation. The repeatability index (RI) varied between 0.57 and 0.85 for the four compounds. Repeatability was particularly high for the two epoxide isomers. Stability in epoxide production was confirmed by statistical analysis (ANOVA), which revealed significant differences in the percentages of cis and trans epoxides between individuals. Correspondingly, the RI of the ratio of trans to cis + trans epoxides was over 0.8. Our study confirms the existence of an interindividual variation of the trans to cis epoxide ratio within populations of Nezara viridula but indicates that each individual reproducibly emits both isomers in the same proportion day after day.     

Effects of Age and Mating on Female Sex Attractant Pheromone Levels in the Sorghum Plant Bug, <Emphasis Type=Italic>Stenotus rubrovittatus</Emphasis> (Matsumura)

Previous work has shown that male sorghum plant bugs, Stenotus rubrovittatus (Matsumura) (Heteroptera: Miridae), are more attracted to young virgin females than to old virgin females and are not attracted to mated females. Therefore, we examined the effects of age and mating status on female sex pheromone levels. The pheromone components, hexyl butyrate, (E)-hex-2-en-1-yl butyrate, and (E)-4-oxohex-2-enal, were collected from females in two different ways. First, the compounds were extracted from whole bodies of each female and quantified by gas chromatography-mass spectrometry (GC-MS). Pheromone levels extracted from virgin female bodies decreased with age. In females that had just mated, pheromone levels did not differ from those of virgin females, and pheromone levels in mated females remained relatively constant up to 15 d after mating. This absence of change in pheromone levels extracted from whole bodies of mated females is not congruent with previous reports of lack of male attraction to these females. In a second method for pheromone recovery (adsorption on a PDMS-coated stir bar), GC-MS analysis showed that mated females released lower amounts of pheromone components compared to those emitted by virgin females of the same age (4 d). As was the case with whole body extracts, young virgin females (3 d) released higher amounts of pheromone components than did old virgin females (8 d). The results suggest that male response in S. rubrovittatus is dictated more by the quantities of pheromone components released into the volatile headspace by females than by the quantities present in the body.     

Geraniol, E-3,7-dimethyl-2,6-octadien-1-ol, as the Alarm Pheromone of the Sycamore Lace Bug Corythucha ciliata (Say)

Although adult sycamore lace bugs Corythucha ciliata (Say) show no sign of aggregation, nymphs at all developing stages are gregarious. When an individual nymph in the center of a colony was squashed with a needlepoint, proximate nymphs showed evasive behavior. The same evasive reaction was produced by exposing aggregated nymphs to nymph hexane extract. The active component, E-3,7-dimethyl-2,6-octadien-1-ol, geraniol, was responsible for the evasive behavior, and identified as the alarm pheromone. One nanogram of the alarm pheromone elicited activity similar to that in a third instar nymph. Presence of 2-acylcyclohexane-1,3-diones and their 4-hydroxy analogues are reconfirmed as nymph-specific components, though their biological significance remains unknown.     

The Sex Pheromone of the Pine Brown-Tail Moth,Euproctis terminalis (Lepidoptera: Erebidae)

Journal of Chemical Ecology - The pine brown tail moth, Euproctis terminalis (Walker 1855), is a periodic pest in pine plantations in South Africa. The larvae feed on pine needles and can cause...     

Identification of Female Sex Pheromone of the Legume Pod Borer, Maruca vitrata and Antagonistic Effects of Geometrical Isomers

An EAD-active component in a female abdominal tip extract of the legume pod borer, Maruca vitrata (Fabricius) (= M. testulalis), was identified as (E,E)-10,12-hexadecadienal (E10,E12–16:Ald). In laboratory bioassays, the purified synthetic E10,E12–16:Ald with 99% isomeric purity had attractancy to male moths equal to the crude extract. However, the unpurified chemical with 92% isomeric purity did not show any attractancy. Addition of 0.1 ng EZ isomer or 1 ng of EZ, ZE, or ZZ isomers of 10,12–16: Ald to 1 ng of the purified synthetic pheromone antagonized its attractancy as did addition of each isomer to 1 female equivalent of the crude extract. These results suggest that it is necessary to develop formulation and release techniques that maintain extremely high isomeric purity of the synthetic pheromone for male trapping in the field.     

Female Sex Pheromone of a Carpenter Moth, Cossus insularis (Lepidoptera: Cossidae)

This study describes the identification of a sex pheromone component of a cossid moth, Cossus insularis. Coupled gas chromatographic–electroantennographic detection (GC–EAD) analysis of solid-phase microextraction (SPME) collections of volatiles released by live female moths showed that two compounds elicited EAG responses from the antennae of male moths. These compounds were identified as (E)-3-tetradecenyl acetate (E3-14:Ac) and (Z)-3-tetradecenyl acetate (Z3-14:Ac) by mass spectral analysis and retention index comparisons with synthetic standards. The ratio of E3-14:Ac and Z3-14:Ac was 95:5 in the effluvia of a female. In field bioassays, sticky traps baited with blends of E3-14:Ac and Z3-14:Ac showed that E3-14:Ac is an essential component of the pheromone. However, the role of Z3-14:Ac is unclear, because E3-14:Ac as a single component was as attractive to male moths as blends of E3-14:Ac and Z3-14:Ac, including the 95:5 blend released by live female moths.     

Chemocommunication in Phyllonorycter ulmifoliella (HBN.) (Lepidoptera: Gracillariidae): Periodicity, Sex Pheromone, and Inhibitors

(Z)-10-Tetradecenyl acetate (Z10-14:OAc) from abdominal tip extracts of virgin females of the tentiform leafminer moth Phyllonorycter ulmifoliella (Lepidoptera: Gracillariidae) was identified by gas chromatography and mass spectrometry. The biological activity of the component was confirmed by field tests with synthetic compounds. As a sex pheromone component this ester is novel both in the family Gracillariidae and in the superfamily Gracillarioidea. Field trapping of P. ulmifoliella with synthetic Z10-14: OAc at dosages of 1 and 0.2 mg/dispenser led to catches of approximately 9000 and 3000 male moths, respectively. The attractivity of the Z10-14:OAc was strongly inhibited by a 10% admixture of either (Z)-9-tetradecenyl acetate (Z9-14:OAc), (E)-9-tetradecenyl acetate (E9-14:OAc), or (E)-11-tetradecenyl acetate (E11-14:OAc). Addition of 10% (E)-10-tetradecenyl acetate (E10-14:OAc) to the sex pheromone reduced attractivity, but significantly less than the inhibitors previously mentioned. The pheromone releasing (or “calling”) behavior of virgin P. ulmifoliella females was recorded under laboratory conditions. Calling activity started about half an hour before lightson and the maximum number of calling females was registered half an hour after the start of photophase. A high level of pheromone releasing activity lasted for about 2 hr and ceased about 5 hr after the start of photophase. Chemocommunication activity in the light period of day is assumed to be an adaptation which allows this phyllonoryctid to avoid inhibitors emitted as pheromones by many other species. A scheme of probable interactions by means of semiochemicals between P. ulmifoliella and other lepidopterans is presented and the appearance of Z10-14: OAc as a sex pheromone component in Lepidoptera during evolution of the order is discussed.     

Olfactory reception of potential pheromones and plant odors by tarnished plant bug,Lygus lineolaris (Hemiptera: Miridae)

Olfactory reception of potential pheromones and host-plant odors by male and female tarnished plant bugs (TPBs),Lygus lineolaris (Hemiptera: Miridae), was investigated by utilizing electroantennogram (EAG) techniques. In general, EAGs were similar between the sexes. Among 31 compounds of seven chemical groups tested, insect-produced butyrates and host-plant-containing green leaf volatiles (GLVs) were the most active. Hexyl butyrate and (E)-2-hexenyl butyrate elicited greater EAGs in males than in females. Females responded with significantly greater EAGs to alcohol and aldehyde GLVs than to their acetate derivatives. Among GLVs, sexual dimorphism was also observed in response to (E)-2-hexenol and (E)-2-hexenal. Females were more sensitive to the monoterpene geraniol than were males. While nonanal was the most stimulatory compound tested, no sexual differences in EAGs to this compound were observed. These studies reveal olfactory receptors on TPB antennae responsive to insect and host-plant volatiles that are likely to play a role in host finding and sexual attraction.     

Sex Pheromone of the Leaf-Miner Phyllonorycter Platani: (Z10)-Tetradecenyl Acetate

A female produced sex pheromone of the leaf-miner pest Phyllonorycter platani (Staudinger) (Lepidoptera: Gracillariidae) was identified as (Z10)-tetradecenyl acetate. Field trapping trials confirmed activity of this compound. The presence of the geometrical isomer (E10)-tetradecenyl acetate, a pheromone component of other Phyllonorycter species, reduced trap efficacy.