Sex Pheromone Components Research Articles

Comparison of functions of pheromone receptor repertoires in Helicoverpa armigera and Helicoverpa assulta using a Drosophila expression system

Helicoverpa armigera and H. assulta are sympatric closely related species sharing two sex pheromone components, (Z)-11-hexadecenal (Z11-16:Ald) and (Z)-9-hexadecenal (Z9-16:Ald) but in opposite ratios, 97:3 and 3:97 respectively. This feature makes them a feasible model for studying the evolution of pheromone coding mechanisms of lepidopteran insects. Despite a decade-long study to deorphanize the pheromone receptor (PR) repertoires of the two species, the comparison of the function of all PR orthologs between the two species is incomplete. Moreover, the ligands of OR14 and OR15 have so far not been found, likely due to the missing of the active ligand(s) in the compound panel and/or incompatibility of heterologous expression systems used. In the present study, we expressed the PR repertoires of both Helicoverpa species in Drosophila T1 neurons to comparatively study the function of PRs. Among those PRs, OR13, OR6, and OR14 of both species are functionally conserved and narrowly tuned, and the T1 neurons expressing each of them respond to Z11-16:Ald, (Z)-9-hexadecenol (Z9-16:OH), and (Z)-11-hexadecenyl acetate (Z11-16:Ac), respectively. While HarmOR16-expressing neurons respond strongly to (Z)-9-tetradecenal (Z9-14:Ald) and (Z)-11-hexadecenol (Z11-16:OH), the neurons expressing HassOR16 mainly respond to Z9-14:Ald and also weakly respond to (Z)-9-tetradecenol (Z9-14:OH). Moreover, HarmOR14b-expressing neurons are activated by Z9-14:Ald, whereas HassOR14b-expressing neurons are sensitive to Z9-16:Ald, Z9-14:Ald, and (Z)-9-hexadecenol (Z9-16:OH). In addition, HarmOR15-expressing neurons are selectively responsive to Z9-14:Ald. However, the Drosophila T1 neurons expressing either HarmOR11 or HassOR11 are silent to all of the compounds tested. In summary, except for OR11, we have deorphanized all the PRs of these two Helicoverpa species using a Drosophila expression system and a large panel of pheromone compounds, thereby providing a valuable reference for parsing the code of peripheral coding of pheromones.

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.

CRISPR/Cas9 mutagenesis against sex pheromone biosynthesis leads to loss of female attractiveness in Spodoptera exigua, an insect pestt.

Virgin female moths are known to release sex pheromones to attract conspecific males. Accurate sex pheromones are required for their chemical communication. Sex pheromones of Spodoptera exigua, a lepidopteran insect, contain unsaturated fatty acid derivatives having a double bond at the 12th carbon position. A desaturase of S. exigua (SexiDES5) was proposed to have dual functions by forming double bonds at the 11th and 12th carbons to synthesize Z9,E12-tetradecedienoic acid, which could be acetylated to be a main sex pheromone component Z9,E12-tetradecenoic acetate (Z9E12-14:Ac). A deletion of SexiDES5 using CRISPR/Cas9 was generated and inbred to obtain homozygotes. Mutant females could not produce Z9E12-14:Ac along with Z9-14:Ac and Z11-14:Ac. Subsequently, pheromone extract of mutant females did not induce a sensory signal in male antennae. They failed to induce male mating behavior including hair pencil erection and orientation. In the field, these mutant females did not attract any males while control females attracted males. These results indicate that SexiDES5 can catalyze the desaturation at the 11th and 12th positions to produce sex pheromone components in S. exigua. This study also suggests an application of the genome editing technology to insect pest control by generating non-attractive female moths.

Detection of Volatile Organic Compounds by Antennal Lamellae of a Scarab Beetle

Chemoreceptive sensilla are abundantly distributed on antennal lamellae of scarab beetles. Olfactory reception by these sensory lamellae plays a major role in feeding behaviors and sexual communication of these beetles. A new electroantennogram (EAG) recording technique is here described for evaluation of electrophysiological responses of antennal lamellae of Pseudosymmachia flavescens to sex pheromones and host plant-related compounds. EAG responses were recorded simultaneously from each lamella and the closed antennal club. All test stimuli elicited similar EAG depolarization profiles in all the three lamellae and the closed club although EAG amplitudes from the same lamella or the club varied widely among different chemical stimuli. The mid lamella tended to produce significantly greater EAG responses. EAG responses evoked by a sex pheromone component, anisole, showed a significant correlation with the density of sensilla placodea subtype 1 (SP1). However, no general patterns were obtained for correlations between the density of any test sensilla type and EAG amplitudes evoked by all the six plant volatiles. Single sensillum recordings are needed to elucidate the specific roles of these sensilla in intraspecific sexual communication and perception of host plant volatiles.

A small number of male-biased candidate pheromone receptors are expressed in large subsets of the olfactory sensory neurons in the antennae of drones from the European honey bee Apis mellifera.

In the European honey bee (Apis mellifera), the olfactory system is essential for foraging and intraspecific communication via pheromones. Honey bees are equipped with a large repertoire of olfactory receptors belonging to the insect odorant receptor (OR) family. Previous studies have indicated that the transcription level of a few OR types including OR11, a receptor activated by the queen-released pheromone compound (2E)-9-oxodecenoic acid (9-ODA), is significantly higher in the antenna of males (drones) than in female workers. However, the number and distribution of antennal cells expressing male-biased ORs is elusive. Here, we analyzed antennal sections from bees by in situ hybridization for the expression of the male-biased receptors OR11, OR18, and OR170. Our results demonstrate that these receptors are expressed in only moderate numbers of cells in the antennae of females (workers and queens), whereas substantially higher cell numbers express these ORs in drones. Thus, the reported male-biased transcript levels are due to sex-specific differences in the number of antennal cells expressing these receptors. Detailed analyses for OR11 and OR18 in drone antennae revealed expression in two distinct subsets of olfactory sensory neurons (OSNs) that in total account for approximately 69% of the OR-positive cells. Such high percentages of OSNs expressing given receptors are reminiscent of male-biased ORs in moths that mediate the detection of female-released sex pheromone components. Collectively, our findings indicate remarkable similarities between male antennae of bees and moths and support the concept that male-biased ORs in bee drones serve the detection of female-emitted sex pheromones.

Functional Characterization of Two Antenna-Enriched Odorant-Binding Proteins From Bactrocera minax (Diptera: Tephritidae).

Olfaction is of great significance for insect mate-seeking and host-locating behaviors. Insect odorant-binding proteins (OBPs), especially those antenna-enriched OBPs, are thought to discriminate, capture and transport odorant molecules to olfactory receptors, but this has not been fully clarified in Bactrocera minax (Enderlein), an economically important pest of citrus crops. Our previous studies showed that seven OBP genes (BminOBP1-7) were identified from B. minax adults via a head transcriptome analysis, of which only BminOBP3 and 6 were highly expressed in antennae, suggesting an olfactory role. To confirm their functions, here, BminOBP3 and 6 were cloned, expressed in Escherichia coli cells. Binding properties of the recombinant BminOBPs with 13 volatiles, most of which can elicit a significant behavioral response from B. minax adults, were determined by fluorescent competitive binding assays. The results showed that Both BminOBP3 and 6 exhibited a remarkable selectivity towards the 13 ligands tested. BminOBP3 displayed strong binding affinity only with undecanol. BminOBP6 demonstrated strong binding affinity with undecanol and limonene among 13 ligands tested. Undecanol is believed to be main sex pheromone component of B. minax. Limonene is an important volatile compound enriched in citrus fruits. Taken together, we concluded that BminOBP3 and 6 may play a prominent role in the process of B. minax mate-seeking and host-locating behaviors through recognizing and transporting these volatiles. It is conceivable that this study will increase our molecular understanding of B. minax olfaction, facilitating the development of OBP-based behavioral interference that is potentially useful for the integrated management of B. minax.

Revisiting the sex pheromone of the fall armyworm Spodoptera frugiperda, a new invasive pest in South China.

The fall armyworm Spodoptera frugiperda is a worldwide serious agricultural pest, and recently invaded South China. Sex pheromone can be employed to monitor its population dynamics accurately in the field. However, the pheromone components previously reported by testing different geographic populations and strains are not consistent. On the basis of confirming that the S. frugiperda population from Yunnan Province belonged to the corn strain, we analyzed the potential sex pheromone components in the pheromone gland extracts of females using gas chromatography coupled with electroantennographic detection (GC-EAD), gas chromatography coupled with mass spectrometry (GC-MS) and electroantennography (EAG). The results show that (Z)-9-tetradecenal acetate (Z9-14:Ac), (Z)-11-hexadecenyl acetate (Z11-16:Ac), (Z)-7-dodecenyl acetate (Z7-12:Ac) or (E)-7-dodecenyl acetate (E7-12:Ac) with a ratio of 100 : 15.8 : 3.9 induced EAD responses to varying degrees: Z9-14:Ac elicited a strong EAD response, Z7-12:Ac or E7-12:Ac elicited a small but clear EAD response, while Z11-16:Ac elicited a weak EAD response. Further single sensillum recording (SSR) showed that Z9-14:Ac and Z7-12:Ac induced dose-dependent activities in two types (A and B) of sensilla in male antennae, respectively, while the sensilla in response to E7-12:Ac and Z11-16:Ac was not recorded. Finally, wind tunnel tests reveal that Z9-14:Ac and Z7-12:Ac are two principal sex pheromone components of the tested population.

Pheromone antagonism in Plu tella xylostella (Linnaeus) by sex pheromones of two sympatric noctuid moths.

Responses to sex pheromones are commonly antagonized by pheromone components of closely related species. Pheromone antagonism has not been widely explored for phylogenetically distant species that have completely different pheromone components. Yet, pheromone components of sympatrically occurring species may also interfere with each other even if these species are distantly related. Here, the effects of heterospecific pheromones on electrophysiology (electroantennogram, EAG) and behavioral responses were tested on the diamondback moth Plutella xyloslella (Plutellidae) and two sympatric noctuid moth species, Spodoptera litura and Spodoptera exigua, whose larvae also feed on Brassica crops. The sex pheromone blend of P. xyloslella, and its components, did not elicit EAG responses in males of the two noctuid species, while sex pheromone components of the noctuid moths elicited significant EAG responses in P. xyloslella males. In wind tunnel bioassays, both (Z, E)-9, 12-tetradecadienyl acetate (ZE-9,12-14:OAc) and (Z, E)-9, 11-tetradecadienyl acetate (ZE-9,11-14:OAc), sex pheromone components from the noctuid moths, inhibited the upwind flight behavior of P. xyloslella males toward an intraspecific pheromone odor source. In Brassica fields, sex pheromone lures of P. xyloslella did not influence trap catches of the noctuid moths, while P. xyloslella pheromone lures baited with either ZE-9,11-14:OAc or ZE-9,12-14:OAc decreased trap catches of P. xyloslella males in a dose-dependent manner. Trap catches of P. xylostella males were also affected by the proximity of ZE-9,11-14:OAc or ZE-9,12-14:OAc to P. xylostella lures. The uni-directional pheromone antagonism by ZE-9,11-14:OAc and ZE-9,12-14:OAc suggests innovative semiochemical-based strategies for the management of P. xyloslella and other economically important pests in Brassica fields. © 2021 Society of Chemical Industry.

Identification and Characterization of an Antennae-Specific Glutathione S-Transferase From the Indian Meal Moth.

Insect glutathione-S-transferases (GSTs) play essential roles in metabolizing endogenous and exogenous compounds. GSTs that are uniquely expressed in antennae are assumed to function as scavengers of pheromones and host volatiles in the odorant detection system. Based on this assumption, antennae-specific GSTs have been identified and functionally characterized in increasing number of insect species. In the present study, 17 putative GSTs were identified from the antennal transcriptomic dataset of the Indian meal moth, Plodia interpunctella, a severe stored-grain pest worldwide. Among the GSTs, only PiGSTd1 is antennae-specific according to both Fragments Per Kilobase Million (FPKM) and quantitative real-time PCR (qRT-PCR) analysis. Sequence analysis revealed that PiGSTd1 has a similar identity as many delta GSTs from other moths. Enzyme kinetic assays using 1-chloro-2,4-dinitrobenzene (CDNB) as substrates showed that the recombinant PiGSTd1 gave a Km of 0.2292 ± 0.01805 mM and a Vmax of 14.02 ± 0.2545 μmol·mg−1·min−1 under the optimal catalytic conditions (35°C and pH = 7.5). Further analysis revealed that the recombinant PiGSTd1 could efficiently degrade the sex pheromone component Z9-12:Ac (75.63 ± 5.52%), as well as aldehyde volatiles, including hexanal (89.10 ± 2.21%), heptanal (63.19 ± 5.36%), (E)-2-octenal (73.58 ± 3.92%), (E)-2-nonenal (75.81 ± 1.90%), and (E)-2-decenal (61.13 ± 5.24%). Taken together, our findings suggest that PiGSTd1 may play essential roles in degrading and inactivating a variety of odorants, especially sex pheromones and host volatiles of P. interpunctella.

Aphid Odorant-Binding Protein 9 Is Narrowly Tuned to Linear Alcohols and Aldehydes of Sixteen Carbon Atoms.

Simple SummaryOdorant-binding proteins (OBPs) mediate chemical communication in insects and can provide a shortcut to deciphering their olfactory code. Although being less specific than olfactory receptors, they are easy to express, purify and characterize. Aphids, in particular, are endowed with a limited repertoire of only 10 OBPs, which are exceptionally well conserved across species. Therefore, using OBPs to study olfaction in aphids appears to be a very attractive strategy. Within our project to functionally characterize all OBPs of the pea aphid, Acyrthosiphon pisum, we have expressed OBP9 and found it to be narrowly tuned to the linear alcohols and aldehydes of 16 carbon atoms. 1-Hexadecanol has been reported as a strong feeding repellant produced by toxic fungi. On the other hand, 16-carbon linear aldehydes are components of several lepidopteran pheromones and could represent warning signals to avoid potential feeding competition.Aphid odorant-binding protein 9 is almost exclusively expressed in antennae and is well conserved between different aphid species. In order to investigate its function, we have expressed this protein and measured ligand-binding affinities to a number of common natural compounds. The best ligands are long-chain aldehydes and alcohols, in particular Z9-hexadecenal and Z11-hexadecenal, as well as 1-hexadecanol and Z11-1-hexadecenol. A model of this protein indicated Lys37 as the residue that is likely to establish strong interactions with the ligands, probably a Schiff base with aldehydes and a hydrogen bond with alcohols. Indeed, when we replaced this lysine with a leucine, the mutated protein lost its affinity to both long aldehydes and alcohols, while the binding of other volatiles was unaffected. Long-chain linear alcohols are common products of molds and have been reported as aphid antifeedants. Corresponding aldehydes, instead, are major components of sex pheromones for several species of Lepidoptera. We speculate that aphids might use OBP9 to avoid mold-contaminated plants as well as competition with lepidopteran larvae.

Anatomical Comparison of Antennal Lobes in Two Sibling Ectropis Moths: Emphasis on the Macroglomerular Complex.

Ectropis obliqua and Ectropis grisescens are two sibling moth species of tea plantations in China. The male antennae of both species can detect shared and specific sex pheromone components. Thus, the primary olfactory center, i.e., the antennal lobe (AL), plays a vital role in distinguishing the sex pheromones. To provide evidence for the possible mechanism allowing this distinction, in this study, we compared the macroglomerular complex (MGC) of the AL between the males of the two species by immunostaining using presynaptic antibody and propidium iodide (PI) with antennal backfills, and confocal imaging and digital 3D-reconstruction. The results showed that MGC of both E. obliqua and E. grisescens contained five glomeruli at invariant positions between the species. However, the volumes of the anterior-lateral glomerulus (ALG) and posterior-ventral (PV) glomerulus differed between the species, possibly related to differences in sensing sex pheromone compounds and their ratios between E. obliqua and E. grisescens. Our results provide an important basis for the mechanism of mating isolation between these sibling moth species.

Limoniic Acid and Its Analog as Trap Lures for Pest Limonius Species (Coleoptera: Elateridae) in North America.

Four species of Limonius wireworms (Coleoptera: Elateridae), L. californicus, L. canus, L. infuscatus and L. agonus, are serious crop pests in North America. Limoniic acid, (E)-4-ethyloct-4-enoic acid, has been reported as a sex pheromone component of female L. californicus and L. canus, and a sex attractant for male L. infuscatus. In the same study, both limoniic acid and the analog (E)-5-ethyloct-4-enoic acid were highly attractive in field experiments. Moreover, six carboxylic acids in headspace volatiles of Limonius females elicited responses from male antennae but were not tested for behavioral activity. Here, we report trap catch data of Limonius spp. obtained in field experiments at 27 sites across North America. All four Limonius species were attracted to limoniic acid and to the analog but not to the carboxylic acids. Adding these carboxylic acids to limoniic acid, or to the analog, reduced its attractiveness. In dose-response studies, trap lures containing 0.4 mg or 4 mg of limoniic acid afforded large captures of L. californicus and L. infuscatus. Neither limoniic acid nor the analog were deterrent to other elaterid pest species. The broad attractiveness of limoniic acid to Limonius spp., and its non-deterrent effect on heterogeners, may facilitate the development of generic pheromone-based monitoring and management tools for multiple click beetle species.

Identification and functional characterization of sex pheromone receptors in mirid bugs (Heteroptera: Miridae)

Mirid bugs are a group of important insect pests that cause large annual losses in agricultural production. Many studies have focused on the isolation and identification of sex pheromones in mirid bugs, and the components and biological activity of the sex pheromones have also been studied as a way to control these pests. However, few studies have focused on the mechanisms of pheromone perception. In this study, we identified the odorant receptor repertoire in three mirid bug species, Apolygus lucorum, Adelphocoris lineolatus, and Adelphocoris suturalis using antennal transcriptome sequencing and bioinformatics analysis. The candidate pheromone receptor (PR) genes were then identified by comparative transcriptomic and expression pattern analysis. Importantly, in vitro functional studies have shown that the candidate PRs have robust responses to the main mirid bug sex pheromone components (E)-2-hexenyl butyrate (E2HB) and hexyl butyrate (HB). Our study uncovered the mechanism of pheromone peripheral coding in these three species and elucidated the mechanism by which mirid bugs can specifically recognize a mate. Moreover, the results of our study will provide a theoretical basis for screening effective sex attractants or mating disturbance agents at the molecular and neural levels for enhanced control of these destructive pests.

Exposure to (Z)-11-hexadecenal [(Z)-11-16:Ald] increases Brassica nigra susceptibility to subsequent herbivory

It is well established that plants emit, detect and respond to volatile organic compounds; however, knowledge on the ability of plants to detect and respond to volatiles emitted by non-plant organisms is limited. Recent studies indicated that plants detect insect-emitted volatiles that induce defence responses; however, the mechanisms underlying this detection and defence priming is unknown. Therefore, we explored if exposure to a main component of Plutella xylostella female sex pheromone namely (Z)-11-hexadecenal [(Z)-11-16:Ald] induced detectable early and late stage defence-related plant responses in Brassica nigra. Exposure to biologically relevant levels of vapourised (Z)-11-16:Ald released from a loaded septum induced a change in volatile emissions of receiver plants after herbivore attack and increased the leaf area consumed by P. xylostella larvae. Further experiments examining the effects of the (Z)-11-16:Ald on several stages of plant defence-related responses showed that exposure to 100 ppm of (Z)-11-16:Ald in liquid state induced depolarisation of the transmembrane potential (Vm), an increase in cytosolic calcium concentration [Ca2+]cyt, production of H2O2 and an increase in expression of reactive oxygen species (ROS)-mediated genes and ROS-scavenging enzyme activity. The results suggest that exposure to volatile (Z)-11-16:Ald increases the susceptibility of B. nigra to subsequent herbivory. This unexpected finding, suggest alternative ecological effects of detecting insect pheromone to those reported earlier. Experiments conducted in vitro showed that high doses of (Z)-11-16:Ald induced defence-related responses, but further experiments should assess how specific the response is to this particular aldehyde.

Functional Characterization of a Candidate Sex Pheromone Receptor AlinOR33 Involved in the Chemoreception of Adelphocoris lineolatus.

Sex pheromones are deemed to play a significant role in sexual communication of most insects. Although many sex pheromone components in mirid bugs have been identified, the roles of odorant receptors in sex pheromone perception in Adelphocoris spp. (Hemiptera: Miridae) remain unknown so far. Here, AlinOR33, a candidate sex pheromone receptor in Adelphocoris lineolatus was functionally characterized. Phylogenetic analysis showed that AlinOR33 clustered with the sex pheromone receptor AlucOR4 fromApolygus lucorum. Quantitative real-time PCR measurement revealed that the expression of AlinOR33 increased gradually from nymph to adult stage and reached its peak in the antennae of 3-day-old mated male bugs. The subsequent in situ hybridization demonstrated that AlinOR33 was mainly expressed in sensilla trichoid on the antennae of A. lineolatus. In the two-electrode voltage clamp recordings, AlinOR33/AlinOrco was specifically tuned to four sex pheromone components including butyl butyrate, hexyl hexanoate, trans-2-hexenyl butyrate and hexyl butyrate, and especially most sensitive to the major component trans-2-hexenyl butyrate. After dsAlinOR33 injection, the electroantennogram responses of males to four sex pheromone components were reduced significantly (∼50%). Compared to control bugs, dsAlinOR33-injected male bugs almost lost behavioral preference for trans-2-hexenyl butyrate. Furthermore, the wingbeat frequency of dsAlinOR33-injected male bugs notably declined. Therefore, we conclude that as a candidate sex pheromone receptor, AlinOR33 plays essential roles in the sexual behavior of A. lineolatus.

Identification and behavioral assays of sex pheromone components in Smerinthus tokyonis (Lepidoptera: Sphingidae)

Hawk moths are classified into the subfamilies Sphinginae, Macroglossinae and Smerinthinae. The sex pheromones of hawk moths have been intensively investigated recently. However, these reports were mainly on Sphinginae and Macroglossinae and there are only a few reports on Smerinthinae. Here, we identified sex pheromone components from the Smerinthinae, Smerinthus tokyonis Matsumura (Lepidoptera: Sphingidae). Observation of female calling behavior showed that the behavior started immediately after the photo-phase started. Gas chromatography (GC) coupled with electroantennography detection analysis indicated that male antenna responded to three components in the pheromone gland extract. GC–MS and GC analyses demonstrated that the three components were (10Z,12E)–, (10E,12Z)–, and (10Z,12Z)–hexadecadienyl acetates in a 6:7:87 ratio. We subsequently performed behavioral assays in cages. We observed the orientation and contact behavior of males in response to different odor sources, including a solvent control, calling female, pheromone gland extract, and synthetic blend. Males did not respond to the solvent control, but did respond to the other sources. Since males responded more to the calling female than to the synthetic blend, additional cues seem to be required for complete mating behavior. Nevertheless, the pheromone components determined in this first study of a Smerinthinae species are important chemicals in mating communication.

Effects of Multi-Component Backgrounds of Volatile Plant Compounds on Moth Pheromone Perception.

Simple SummaryIt is well acknowledged that some of the volatile plant compounds (VPC) naturally present in insect natural habitats alter the perception of their own pheromone when presented individually as a background to pheromone. However, the effects of mixing VPCs as they appear to insects in natural olfactory landscapes are poorly understood. We measured the activity of brain neurons and neurons that detect a sex pheromone component in a moth antenna, while exposed to simple or composite backgrounds of VPCs representative of the odorant variety encountered by this moth. Maps of activities were built using calcium imaging to visualize which brain areas were most affected by VPCs. In the antenna, we observed differences in VPC capacity to elicit firing response that cannot be explained by differences in stimulus intensities because we adjusted concentrations according to volatility. The neuronal network, which reformats the input from antenna neurons in the brain, did not improve pheromone salience. We postulate that moth olfactory system evolved to increase sensitivity and encode fast changes of concentration at some cost for signal extraction. Comparing blends to single compounds indicated that a blend shows the activity of its most active component, VPC salience seems more important than background complexity.The volatile plant compounds (VPC) alter pheromone perception by insects but mixture effects inside insect olfactory landscapes are poorly understood. We measured the activity of receptor neurons tuned to Z7-12Ac (Z7-ORN), a pheromone component, in the antenna and central neurons in male Agrotis ipsilon while exposed to simple or composite backgrounds of a panel of VPCs representative of the odorant variety encountered by a moth. Maps of activities were built using calcium imaging to visualize which areas in antennal lobes (AL) were affected by VPCs. We compared the VPC activity and their impact as backgrounds at antenna and AL levels, individually or in blends. At periphery, VPCs showed differences in their capacity to elicit Z7-ORN firing response that cannot be explained by differences in stimulus intensities because we adjusted concentrations according to vapor pressures. The AL neuronal network, which reformats the ORN input, did not improve pheromone salience. We postulate that the AL network evolved to increase sensitivity and to encode for fast changes of pheromone at some cost for signal extraction. Comparing blends to single compounds indicated that a blend shows the activity of its most active component. VPC salience seems to be more important than background complexity.

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.

Effect of pheromone blends, trap type and color on the capture of male clearwing moths, Synanthedon bicingulata (Lepidoptera: Sesiidae)

Two components of the Synanthedon bicingulata sex pheromone, (E,Z)-3,13-octadecadienyl acetate (E3,Z13-18:OAc) and (Z,Z)-3,13-octadecadienyl acetate (Z3,Z13-18:OAc), were synthesized to investigate the effect of pheromone blends, trap type and trap color on the capture of S. bicingulata males. The optimal sex pheromone ratio for E3,Z13-18:OAc and Z3,Z13-18:OAc was approximately 4.3:5.7 based on the purity of the two pheromone components in all test areas. A significant difference was observed in the number of S. bicingulata adult males caught in bucket and delta traps. The mean numbers of males caught in bucket and delta traps were 13.2 ± 2.2 and 7.6 ± 2.0, respectively. Trap color affected the number of adult males caught in bucket traps. More adult males were attracted to a yellow bucket trap than to green, white, blue, black and red traps. An analysis of the relationship between trap capture and trap surface-color values (L*a*b*) revealed a positive relationship between trap capture and b* value.

The Adelphocoris lineolatus OBP4: Support for evolutionary and functional divergence of a mirid pheromone-binding protein from that found in lepidopteran moths.

Pheromone-binding proteins (PBPs) have been extensively investigated in lepidopteran moths, but their evolution and function in hemipteran species remain unclear. Our previous study demonstrated that an odorant-binding protein, OBP4, of the mirid bug Adelphocoris lineolatus functions as a candidate hemipteran PBP but clustered with lepidopteran antennae-binding proteins (ABPs) rather than in the PBP/general odorant-binding protein (GOBP) clade. In this study, we hypothesized that origin and function of PBPs in hemipteran bugs may differ from those of lepidopteran moths. To test this hypothesis, we first constructed a phylogenetic tree using insect OBPs from sister hemipteran and holometabolous lineages, and the results indicated that neither OBP4 nor other types of candidate PBPs of mirid bugs clustered with the lepidopteran PBP/GOBP clade. Then, a fluorescence competitive binding assay was employed to determine binding affinities of recombinant OBP4 protein to host plant volatiles, with functional groups different from A. lineolatus sex pheromone components. The results revealed that OBP4 highly bound the female adult attractant 3-hexanone and 15 other mirid bug biologically active plant volatiles. Finally, we examined cellular expression profiles of OBP4 in putative antennal sensilla that are related to female A. lineolatus host plant location. The fluorescence in situ hybridization and immunocytochemical labeling assay showed that the OBP4 gene was highly expressed in the multiporous olfactory sensilla medium-long sensilla basiconica rather than in the short sensilla basiconica or uniporous sensilla chaetica. These results, together with those of our previous studies, indicate that OBP4 not only functions in recognition of bug-produced sex pheromones in males, but is probably involved in detection of host plant volatiles in both A. lineolatus sexes. Our findings support the hypothesis that the origin and function of PBPs in hemipteran bugs differ from those of well-known PBPs in lepidopteran moths, which provides a novel perspective on evolutionary mechanisms of sex pheromone communication across insect orders.