Major Sex Pheromone Component Research Articles

Interactive parallel sex pheromone circuits that promote and suppress courtship behaviors in the cockroach.

Many animals use multicomponent sex pheromones for mating, but the specific function and neural processing of each pheromone component remain unclear. The cockroach Periplaneta americana is a model for studying sex pheromone communication, and an adult female emits major and minor sex pheromone components, periplanone-B and -A (PB and PA), respectively. Attraction and courtship behaviors (wing-raising and abdominal extension) are strongly expressed when adult males are exposed to PB but weakly expressed when they are exposed to PA. When major PB is presented together with minor PA, behaviors elicited by PB were impaired, indicating that PA can both promote and suppress courtship behaviors depending on the pheromonal context. In this study, we identified the receptor genes for PA and PB and investigated the effects of knocking down each receptor gene on the activities of PA- and PB-responsive sensory neurons (PA- and PB-SNs), and their postsynaptic interneurons, and as well as effects on courtship behaviors in males. We found that PB strongly and PA weakly activate PB-SNs and their postsynaptic neurons, and activation of the PB-processing pathway is critical for the expression of courtship behaviors. PA also activates PA-SNs and the PA-processing pathway. When PA and PB are simultaneously presented, the PB-processing pathway undergoes inhibitory control by the PA-processing pathway, which weakens the expression of courtship behaviors. Our data indicate that physiological interactions between the PA- and PB-processing pathways positively and negatively mediate the attraction and courtship behaviors elicited by sex pheromones.

Identification of the female sex pheromone of Bastilla arctotaenia (Lepidoptera: Erebidae).

(3Z,6Z,9Z)-3,6,9-henicosatriene was identified as a major component of female sex pheromone of Bastilla arctotaenia (Lepidoptera: Erebidae), a pest of cultivated roses, by gas chromatograph-electroantennographic detector( GC-EAD) and gas chromatograph/mass spectrometry (GC/MS) analyses. The single (3Z,6Z,9Z)-3,6,9-henicosatriene (1.0 mg/lure) successfully attracted B. arctotaenia males in the field.

Molecular mechanism of sex pheromone perception in male Mythimna loreyi revealed by in vitro system.

Mythimna loreyi is an important agricultural pest with a sensitive sex pheromone communication system. To clarify the pheromone binding proteins (PBPs) and pheromone receptors (PRs) involved in sex pheromone perception is important for both understanding the molecular olfactory mechanism and developing a new pest control strategy in M. loreyi. First, the electroantennogram (EAG) assay showed that male M. loreyi displayed the highest response to the major sex pheromone component Z9-14:Ac, and higher responses to two minor components, Z7-12:Ac and Z11-16:Ac. Second, the fluorescence competition binding assay showed that PBP1 bound all three pheromones and other tested compounds with high or moderate affinity, while PBP2 and PBP3 each bound only one pheromone component and few other compounds. Third, functional study using the Xenopus oocyte system demonstrated that, of the six candidate PRs, PR2 was weakly sensitive to the major pheromone Z9-14:Ac, but was strongly sensitive to pheromone analog Z9-14:OH; PR3 was strongly and specifically sensitive to a minor component Z7-12:Ac; PR4 and OR33 were both weakly sensitive to another minor component, Z11-16:Ac. Finally, phylogenetic relationship and ligand profiles of PRs were compared among six species from two closely related genera Mythimna and Spodoptera, suggesting functional shifts of M. loreyi PRs toward Spodoptera PRs. Functional differentiations were revealed among three PBPs and six PRs in sex pheromone perception, laying an important basis for understanding the molecular mechanism of sex pheromone perception and for developing new control strategies in M. loreyi. © 2023 Society of Chemical Industry.

A shift in the paradigm? A male-specific lactone increases the response of both sexes of the olive fruit fly Bactrocera oleae to the food lure ammonium bicarbonate

The olive fruit fly Bactrocera oleae (Rossi) (Diptera: Tephritidae) is a key pest species of wild and cultivated olive trees worldwide. Contrarily to most tephritid flies, in which males release the sex pheromone, in B. oleae the female is the sex responsible of attracting the opposite sex. However, and even though vast research has been done during the last decades, we are still far from understanding the chemical signals involved in the sexual communication of this species, including those produced by males. Here, we report for the first time the presence of two male-specific volatile compounds, namely γ-hexalactone and δ-hexalactone, with the former exerting a significant attraction upon both sexes under laboratory and field conditions. Volatile collections conducted on laboratory-reared virgin individuals of both sexes revealed the presence of these two compounds only in males, regardless of their age. In double-choice behavioral assays, γ-hexalactone resulted to be attractive for virgin males and females (7–14 days old), with no attractiveness reported for δ-hexalactone. Finally, in field assays traps baited with the binary blend of ammonium bicarbonate and γ-hexalactone yielded significantly more catches per week of both sexes than those baited with ammonium bicarbonate and ammonium bicarbonate plus 1,7-dioxaspiro[5.5]undecane, the major sex pheromone component. Altogether, our results shed light on the chemical ecology of the species and represent a promising experimental basis for the development of more innovative and effective mass trapping tools based on the use of γ-hexalactone.

Functional Characterization of Pheromone Receptors in the Beet Webworm, Loxostege sticticalis (Lepidoptera: Pyralidae).

Lepidopteran insects mainly rely on sex pheromones to complete sexual communications. Pheromone receptors (PRs) are expressed on the olfactory receptor neurons (ORNs) of the sensilla trichodea and play an essential role in sexual communication. Despite extensive investigations into the mechanisms of peripheral recognition of sex pheromones in Lepidoptera, knowledge about these mechanisms in L. sticticalis remains limited. In this study, five candidate LstiPRs were analyzed in a phylogenetic tree with those of other Lepidopteran insects. Electroantennography (EAG) assays showed that the major sex pheromone component E11-14:OAc elicited a stronger antennal response than other compounds in male moths. Moreover, two types of neurons in sensilla trichodea were classified by single sensillum recordings, of which the "a" neuron specifically responded to E11-14:OAc. Five candidate PRs were functionally assayed by the heterologous expression system of Xenopus oocytes, and LstiPR2 responded to the major sex pheromone E11-14:OAc. Our findings suggest that LstiPR2 is a PR sensitive to L. sticticalis's major sex pheromone compound, E11-14:OAc. Furthermore, this study offers valuable insights into the sexual communication behavior of L. sticticalis, forming a foundation for further analysis of the species' central nervous system.

Roles of GOBP1 in perception of host plant volatiles revealed by CRISPR/Cas9‐mediated mutagenesis in Spodoptera litura

AbstractThe general odorant‐binding proteins (GOBPs) in Lepidoptera form a distinct OBP subclass, which exclusively has two members (GOBP1 and GOBP2) in a single species. GOBPs are long thought to function in perception of plant odorants and recently in sex pheromones, but the in vivo evidence is rarely provided. Here, by using CRISPR/Cas9 technique, we demonstrated that GOBP1 in a noctuid moth Spodoptera litura is involved in perception of host plant volatiles but not of the female sex pheromone, and GOBP1 in females is less important than GOBP2 in host plant perception. First, the GOBP1 knockout strain (GOBP1−/−) of S. litura was obtained by CRISPR/Cas9 technique. Second, in the electroantennography (EAG) tests, GOBP1−/− females displayed significant reduction in responses to plant volatile farnesene, and leaf discs of the host plant cabbage (males only to high leaf doses), but GOBP1−/− adults did not show significant EAG reduction to the major sex pheromone component and two pheromone analogues. Third, compared with GOBP2−/− females, GOBP1−/− females were significantly lower in reduction (in relation to WT moths) of EAG response to host plant leaves. Our results provide in vivo evidence that GOBP1 is involved in perception of host plant volatiles but is less important than GOBP2 in S. litura.

Functional Analysis of Pheromone Biosynthesis Activating Neuropeptide Receptor Isoforms in Maruca vitrata.

Insect sex pheromones are volatile chemicals that induce mating behavior between conspecific individuals. In moths, sex pheromone biosynthesis is initiated when pheromone biosynthesis-activating neuropeptide (PBAN) synthesized in the suboesophageal ganglion binds to its receptor on the epithelial cell membrane of the pheromone gland. To investigate the function of PBAN receptor (PBANR), we identified two PBANR isoforms, MviPBANR-B and MviPBANR-C, in the pheromone glands of Maruca vitrata. These two genes belong to G protein-coupled receptors (GPCRs) and have differences in the C-terminus but share a 7-transmembrane region and GPCR family 1 signature. These isoforms were expressed in all developmental stages and adult tissues. MviPBANR-C had the highest expression level in pheromone glands among the examined tissues. Through in vitro heterologous expression in HeLa cell lines, only MviPBANR-C-transfected cells responded to MviPBAN (≥5 µM MviPBAN), inducing Ca2+ influx. Sex pheromone production and mating behavior were investigated using gas chromatography and a bioassay after MviPBANR-C suppression by RNA interference, which resulted in the major sex pheromone component, E10E12-16:Ald, being quantitatively reduced compared to the control, thereby decreasing the mating rate. Our findings indicate that MviPBANR-C is involved in the signal transduction of sex pheromone biosynthesis in M. vitrata and that the C-terminal tail plays an important role in its function.

Development of a high-efficiency sex pheromone formula to control Euproctis pseudoconspersa

The tea tussock moth (Euproctis pseudoconspersa) is one of the most destructive chewing pests in tea plantations and causes a serious allergic reaction on the skin of tea plantation workers. The sex pheromone components of its Japanese population were first identified as 10,14-dimethylpentadecyl isobutyrate (10Me14Me-15:iBu) and 14-methylpentadecyl isobutyrate (14Me-15:iBu), with a stereogenic center. Only 10Me14Me-15:iBu has been identified in the Chinese E. pseudoconspersa population. However, field tests have shown that 10Me14Me-15:iBu cannot meet the demand of effective pest control in China. To develop a high-efficiency E. pseudoconspersa sex pheromone formula, electroantennogram (EAG) recordings of (S)- and (R)-enantiomers of 10Me14Me-15:iBu and 14Me-15:iBu were obtained in the present study. The results demonstrated that the EAG responses of male antennae to (R)-enantiomers were significantly higher than responses to the (S)-enantiomers, and 14Me-15:iBu also elicited EAG activity. Field tests showed that the catch numbers of male moths by (R)-enantiomers were significantly higher (P<0.05) than those of (S)-enantiomers. Addition of 14Me-15:iBu significantly increased the catch numbers of both the (S)- and (R)-enantiomers. The efficient pheromone formula containing 0.75 mg (R)-10Me14Me-15:iBu and 0.1 mg 14Me-15:iBu showed significantly higher attractiveness than commercial pheromone products. Our study demonstrated that (R)-10Me14Me-15:iBu was the major sex pheromone component of E. pseudoconspersa, and 14Me-15:iBu might be the minor sex pheromone component. Furthermore, a high-efficiency sex pheromone formula for E. pseudoconspersa control was defined in this study.

A closer look at sex pheromone autodetection in the Oriental fruit moth

Female moths emit sex pheromone to attracts males, and although they are not attracted to their own sex pheromone, they appear to detect it as it affects their behavior. In order to elucidate the mechanism of pheromone “autodetection” we compared responses of olfactory receptor neurons (ORNs) of male and female Grapholita molesta, a species with reported pheromone autodetection. Two concentrations of the major (Z8-12:Ac) and minor (E8-12:Ac) sex pheromone components, a plant-volatile blend containing methyl salicylate, terpinyl acetate and (E)-β-farnesene, and the male-produced hair-pencil (i.e., courtship) pheromone (ethyl trans-cinnamate) were tested in 45 male and 305 female ORNs. Hierarchical cluster analysis showed radically different peripheral olfactory systems between sexes that could be linked to their specific roles. In males 63% of the ORNs were tuned specifically to the major or minor female sex pheromone components, and 4% to the plant volatile blend, while the remaining 33% showed unspecific responses to the stimulus panel. In females 3% of the ORNs were specifically tuned to the male hair-pencil pheromone, 6% to the plant volatile blend, 91% were unspecific, and no ORN was tuned their own sex pheromone components. The lack of sex pheromone-specific ORNs in females suggests that they are not able to discriminate pheromone blends, and thus pheromone autodetection is unlikely in this species. We discuss our results in the context of the methodological limitations inherent to odor stimulation studies.

A female-biased odorant receptor tuned to the lepidopteran sex pheromone in parasitoid Microplitis mediator guiding habitat of host insects

IntroductionThe parasitoid wasp Microplitis mediator is an important natural enemy of the turnip moth Agrotis segetum and other Noctuidae pests. In our field observation, it was fortuitously discovered that sex pheromone traps used for A. segetum also attract female wasps, verified by a simulated field condition dual-choice laboratory assay. Therefore, it was hypothesized that olfactory recognition could be crucial in this process. In this regard, a female-biased odorant receptor of the wasp, MmedOR49, attracted our attention. ObjectivesTo unravel the significance of the female-biased MmedOR49 regulating host pheromone recognition. MethodsExpression analysis (fluorescence in situ hybridization; quantitative realtime PCR), in vitro (two-electrode voltage-clamp recordings) and in vivo (RNAi combined with behavioral assessments) functional studies, and bioinformatics (structural modeling and molecular docking) were carried out to investigate the characteristics of MmedOR49. ResultsMmedOR49 expression was detected in the antennae of females by FISH. Quantification indicated that the expression level of MmedOR49 increased significantly after adult emergence. In vitro functional study revealed that MmedOR49 was specifically tuned to cis-5-decenyl acetate (Z5-10:Ac), the major sex pheromone component of A. segetum. Molecular docking showed that Z5-10:Ac strongly bound to the key amino acid residues His 80, Ile 81, and Arg 84 of MmedOR49 through hydrogen bonding. Behavioral assays indicated that female wasps were significantly attracted by Z5-10:Ac in a three-cage olfactometer. RNAi targeting further confirmed that MmedOR49 was necessary to recognize Z5-10:Ac, as female wasps lost their original behavioral responses to Z5-10:Ac after down-regulation of the MmedOR49 transcript. ConclusionAlthough M. mediator is a larval endoparasitoid, female wasps have a behavioral preference for a sex pheromone component of lepidopteran hosts. In this behavior, for female M. mediator, MmedOR49 plays an important role in guiding the habitat of host insects. These data provide a potential target for enhancing natural enemy utilization and pest control.

Functional analysis of pheromone receptor repertoire in the fall armyworm, Spodoptera frugiperda.

The fall armyworm, Spodoptera frugiperda (J. E. Smith), is a polyphagous moth species that is spreading all around the globe. It uses (Z)-9-tetradecenyl acetate (Z9-14:Ac) and (Z)-7-dodecenyl acetate (Z7-12:Ac) (100:3.9) as essential sex pheromone components. However, our understanding of the molecular basis of pheromone detection of S. frugiperda is still incomplete. Herein, we identified six PRs, i.e. SfruOR6, 11, 13, 16, 56, and 62, by transcriptome sequencing. Subsequently, we heterologously expressed them in Drosophila OR67d neurons and determined their response spectra with a large panel of sex pheromones and analogs. Among them, SfruOR13-expressing neurons strongly respond to the major sex pheromone component Z9-14:Ac, but also comparably to (Z,E)-9,12-tetradecadienyl acetate (Z9,E12-14:Ac) and weakly to (Z)-9-dodecenyl acetate (Z9-12:Ac). Both SfruOR56 and SfruOR62 are specifically tuned to the minor sex pheromone component Z7-12:Ac with varying intensities and sensitivities. In addition, SfruOR6 is activated only by Z9,E12-14:Ac, and SfruOR16 by both (Z)-9-tetradecenol (Z9-14:OH) and (Z)-9-tetradecenal (Z9-14:Ald). However, the OR67d neurons expressing SfruOR11 remain silent to all compounds tested, a phenomenon commonly found in the OR11 clade of Noctuidae species. Next, using single sensillum recording, we characterized four sensilla types on the antennae of males, namely A, B, C and D types that are tuned to the ligands of PRs, thereby confirming that S. frugiperda uses both SfruOR56 and SfruOR62 to detect Z7-12:Ac. Finally, using wind tunnel assay, we demonstrate that both Z9,E12-14:Ac and Z9-14:OH act as antagonists to the sex pheromone. We have deorphanized five PRs and characterized four types of sensilla responsible for the detection of pheromone compounds, providing insights into the peripheral encoding of sex pheromones in S. frugiperda.

Suppression of pheromone biosynthesis and mating behavior by RNA interference of pheromone gland-specific fatty acyl reductase in Maruca vitrata.

In moths, various enzymes, such as fatty acid synthases, fatty acyl desaturases, and fatty acyl reductases (FARs), are involved in pheromone biosynthesis. In particular, pheromone gland-specific FAR (pgFAR) plays an important role in converting the functional group from carboxylic to alcohol during pheromone biosynthesis. A novel pgFAR of Maruca vitrata, Mvi-pgFAR, was identified through transcriptome sequencing of its pheromone gland. To investigate the involvement of Mvi-pgFAR in pheromone biosynthesis, Mvi-pgFAR was cloned from the pheromone gland and suppressed by RNA interference (RNAi). Mvi-pgFAR harbored several conserved motifs related to NAD(P)H-binding, N-glycosylation, and adenosine / guanosine triphosphate binding. Phylogenetic analysis revealed that Mvi-pgFAR with other lepidopteran pgFARs formed an independent clade. Mvi-pgFAR was specifically expressed only in the pheromone gland. Quantitative real-time polymerase chain reaction showed that the diurnal expression levels of Mvi-pgFAR in the pheromone gland were the highest at 2 h before the scotophase. After primarily confirming Mvi-pgFAR suppression by RNAi, (E,E)-10,12-hexadecadienal (E10E12-16:Ald), a major sex pheromone component, was quantified by gas chromatography. When Mvi-pgFAR was successfully suppressed, E10E12-16:Ald production was reduced by up to half of that of the control, and the mating rate was subsequently decreased. Our results demonstrate that Mvi-pgFAR downregulation can suppress mating behavior by changing the relative sex pheromone component ratio, suggesting that Mvi-pgFAR can be used as a novel control target.

(Z,E)‐α‐Farnesene – sex pheromone component of female click beetle Selatosomus aeripennis destructor with intra‐ and inter‐sexual communication function

AbstractSex pheromones are known for only a few major click beetle (Coleoptera: Elateridae) pests in North America. These pheromones could be used to monitor, as well as control, adult beetles. Our objective was to identify the sex pheromone of female Selatosomus aeripennis destructor (Brown), a predominant elaterid pest species in farmland in the Canadian Prairie provinces. Headspace volatiles from a group of S. a. destructor females were collected and analyzed by coupled gas chromatography‐electroantennographic detection (GC‐EAD) and gas chromatography‐mass spectrometry (GC‐MS). The GC‐EAD recordings revealed two candidate pheromone components that elicited weak (CPC‐1) or strong (CPC‐2) responses from adult male S. a. destructor antennae. The mass spectra of CPC‐1 and CPC‐2 indicated, and authentic standards confirmed, that they were the isomers (E)‐β‐farnesene and (Z,E)‐α‐farnesene, respectively. In three field experiments at various sites in Alberta, Canada, traps baited with synthetic (Z,E)‐α‐farnesene captured 7.1×, 6.8×, and 2.6× more males than unbaited control traps. In follow‐up experiments, traps baited with (Z,E)‐α‐farnesene captured significantly more males, but significantly fewer females, than unbaited control traps, indicating sex pheromone autodetection by females. (E)‐β‐Farnesene on its own was not attractive as a trap lure, and when added to (Z,E)‐α‐farnesene at a 20:1 ratio, it reduced the lure’s attractiveness. Our data support the conclusion that (Z,E)‐α‐farnesene is the major sex pheromone component of S. a. destructor females, with both an intra‐ and inter‐sexual communication function.

Involvement of GOBP2 in the perception of a sex pheromone component in both larval and adult Spodoptera litura revealed using CRISPR/Cas9 mutagenesis

General odorant-binding proteins (GOBPs) are long considered responsible for the perception of plant odorants. In this study with the important noctuid pest Spodoptera litura, we functionally characterized that GOBP2 is also involved in the perception of sex pheromone components using in vivo CRISPR/Cas9 technique. First, the GOBP2 sgRNA and Cas9 protein were injected into the newly laid insect eggs, resulting in a 35.6% target mutagenesis in G0 moths. Then, the homozygous GOBP2 knockout strain (GOBP2−/−) was obtained after the screening of three generations. The knockout male and female moths displayed a significant reduction in EAG responses to the sex pheromone components, and the knockout females also displayed a significant reduction to plant odorants. In the behavioral assay of food choice, GOBP2−/− larvae lost the preference to artificial diet added with the major sex pheromone component Z9, E11-tetradecadienyl acetate (Z9, E11-14:Ac), whereas the WT larvae highly preferred the pheromone diet. Y-tube olfactometer assay and direct pheromone stimulation assay showed that GOBP2−/− male adults reduced significantly than WT males in percentages of choice, hair pencil displaying and mating attempt to Z9, E11-14:Ac. In the oviposition test, GOBP2−/− females showed significantly reduced preference for the soybean plants compared to the WT females. Our study demonstrated that GOBP2 plays an important role in perceiving sex pheromones in adult and larval stages, providing new insight into sex pheromone perception and a potential target for sex pheromone-based behavioral regulation in the pest.

Bioproduction of (Z,E)-9,12-tetradecadienyl acetate (ZETA), the major pheromone component of Plodia, Ephestia, and Spodoptera species in yeast.

BACKGROUND(Z,E)‐9,12‐tetradecadienyl acetate (ZETA, Z9,E12‐14:OAc) is a major sex pheromone component for many stored‐product moth species. This pheromone is used worldwide for mating disruption, detection, monitoring, and mass trapping in raw and processed food storage facilities. In this study, we demonstrate the biological production of ZETA pheromone by engineered yeast Saccharomyces cerevisiae.RESULTSWe mined the pheromone gland transcriptome data of the almond moth, Ephestia (Cadra) cautella (Walker), to trace a novel E12 fatty acyl desaturase and expressed candidates heterologously in yeast and Sf9 systems. Furthermore, we demonstrated a tailor‐made ZETA pheromone bioproduction in yeast through metabolic engineering using this E12 desaturase, in combination with three genes from various sources coding for a Z9 desaturase, a fatty acyl reductase, and an acetyltransferase, respectively. Electrophysiological assays (gas chromatography coupled to an electroantennographic detector) proved that the transgenic yeast‐produced ZETA pheromone component elicits distinct antennal responses.CONCLUSIONThe reconstructed biosynthetic pathway in yeast efficiently produces ZETA pheromone, leaves an undetectable level of biosynthetic intermediates, and paves the way for the economically competitive high‐demand ZETA pheromone's bioproduction technology for high‐value storage pest control.

Functional Characterization of Sex Pheromone Neurons and Receptors in the Armyworm, Mythimna separata (Walker).

Pheromone receptors (PRs) of moths are expressed on the dendritic membrane of odorant receptor neurons (ORNs) housed in the long trichoid sensilla (TS) of antennae and are essential to sex pheromone reception. The function of peripheral neurons of Mythimna separata in recognizing sex pheromones is still unclear. In this study, electroantennogram recordings were performed from male and female antennae of M. separata, and showed that the major component of sex pheromones, (Z)-11-hexadecenal (Z11–16:Ald), evoked the strongest response of male antennae with significant differences between sexes. Single sensillum recording was used to record responses of neurons housed in TS of male M. separata. The results revealed four types of TS with three neurons housed in each type, based on profiles of responses to sex pheromone components and pheromone analogs. ORN-B of type-I TS was specifically tuned to the major sex pheromone component Z11–16:Ald; ORN-Bs in type-III and type-IV TSs were, respectively, activated by minor components (Z)-11-hexadecen-1-yl acetate (Z11–16:OAc) and hexadecenal (16:Ald); and ORNs in type-II TS were mainly activated by the sex pheromone analogs. We further cloned full-length sequences of six putative PR genes and an Orco gene. Functional characterization of PRs in the Xenopus oocyte system demonstrated that male antennae-biased MsepPR1 responded strongly to (Z)-9-tetradecenal (Z9-14:Ald), suggesting that MsepPR1 may be expressed in type-II TS. MsepPR6 was exclusively tuned to (Z)-9-tetradecen-1-yl acetate (Z9–14:OAc). MsepPR2 and MsepPR4 showed no responses to any tested components. Female antennae-biased MespPR5 was broadly tuned to Z9–14:Ald, Z9–14:OAc, Z11–16:Ald, and (Z)-11-hexadecen-1-ol (Z11–16:OH). Our results further enriched the sex pheromone recognition mechanism in the peripheral nervous system of moth M. separata.

Biosynthesis of the Sex Pheromone Component (E,Z)-7,9-Dodecadienyl Acetate in the European Grapevine Moth, Lobesia botrana, Involving \u220611 Desaturation and an Elusive \u22067 Desaturase

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.

Contribution of odorant binding proteins to olfactory detection of (Z)-11-hexadecenal in Helicoverpa armigera

Helicoverpa armigera utilizes (Z)-11-hexadecenal (Z11-16:Ald) as its major sex pheromone component. Three pheromone binding proteins (PBPs) and two general odorant binding proteins (GOBPs) are abundantly expressed in the male antennae of H. armigera. However, their precise roles in the olfactory detection of Z11-16:Ald remain enigmatic. To answer this question, we first synthesized the antibody against HarmOR13, an olfactory receptor (OR) primarily responding to Z11-16:Ald and mapped the local associations between PBPs/GOBPs and HarmOR13. Immunostaining showed that HarmPBPs and HarmGOBPs were localized in the supporting cells of trichoid sensilla and basiconic sensilla respectively. In particular, HarmPBP1 and HarmPBP2 were colocalized in the cells surrounding the olfactory receptor neurons (ORNs) expressing HarmOR13. Next, using two noninterfering binary expression tools, we heterologously expressed HarmPBP1, HarmPBP2 and HarmOR13 in Drosophila T1 sensilla to validate the functional interplay between PBPs and HarmOR13. We found that the addition of HarmPBP1 or HarmPBP2, not HarmPBP3, significantly increased HarmOR13's response to Z11-16:Ald. However, the presence of either HarmPBP1 or HarmPBP2 was ineffective to change the tuning breadth of HarmOR13 and modulate the response kinetics of this receptor. Taken together, this work demonstrates both HarmPBP1 and HarmPBP2 are involved in Z11-16:Ald detection. Our results support the idea that PBPs can contribute to the peripheral olfactory sensitivity but do little in modulating the selectivity and the response kinetics of corresponding ORs.

Functional Characterization of Sex Pheromone Receptors in the Fall Armyworm (Spodoptera frugiperda)

Pheromone receptors (PRs) found in the antennae of male moths play a vital role in the recognition of sex pheromones released by females. The fall armyworm (FAW), Spodoptera frugiperda, is a notorious invasive pest, but its PRs have not been reported. In this report, six candidate PRs (SfruOR6, 11, 13, 16, 56 and 62) suggested by phylogenetic analysis were cloned, and their tissue–sex expression profiles were determined by quantitative real-time PCR (qPCR). All six genes except for SfruOR6 were highly and specifically expressed in the antennae, with SfruOR6, 13 and 62 being male-specific, while the other three (SfruOR11, 16 and 56) were male biased, suggesting their roles in sex pheromone perception. A functional analysis by the Xenopus oocyte system further demonstrated that SfruOR13 was highly sensitive to the major sex pheromone component Z9-14:OAc and the pheromone analog Z9,E12-14:OAc, but less sensitive to the minor pheromone component Z9-12:OAc; SfruOR16 responded weakly to pheromone component Z9-14:OAc, but strongly to pheromone analog Z9-14:OH; the other four candidate PRs did not respond to any of the four pheromone components and four pheromone analogs. This study contributes to clarifying the pheromone perception in the FAW, and provides potential gene targets for developing OR-based pest control techniques.

Identification and field evaluation of the sex pheromone of Apolygus lucorum (Hemiptera: Miridae) in China.

The plant bug, Apolygus lucorum Meyer-Dür, has begun a resurgence and has become a key pest in cotton in northern China, with the wide-scale adoption of transgenic Bt cotton. We attempted to develop a new approach to the control of this plant bug by identifying and utilizing its sex pheromone. Extracts from A. lucorum adults and nymphs were analyzed using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-electroantennogram detection (GC-EAD) to identify the sex pheromone components. (E)-4-Oxo-2-hexenal and (E)-2-hexenyl butyrate were the major sex pheromone components from females and were also electrophysiology-active towards male antennae. For males, (E)-4-oxo-2-hexenal and hexyl butyrate were the major sex pheromone components. These three components, however, were not detected in nymphs. Field tests showed that binary blends of (E)-4-oxo-2-hexenal and (E)-2-hexenyl butyrate at a ratio of 3:2 resulted in trapping the greatest numbers of A. lucorum males. A long-term dispenser was developed by loading the pheromone components dissolved in sunflower oil into polyethylene vials. Further field evaluation showed that the polyethylene dispensers attracted significantly more bugs even when deployed in the field for 5 weeks. We identified the sex pheromone of A. lucorum and developed a type of high-efficiency and long-term lure. Our results could expand knowledge of the pheromones of plant bugs, and provide novel technologies to monitor and control this pest. © 2019 Society of Chemical Industry.