Olfactory receptor neurons detecting plant odours and male volatiles in Anomala cuprea beetles (Coleoptera: Scarabaeidae)

We have identified several types of olfactory receptor neurons in male and female Anomala cuprea beetles. The receptor neurons were sensitive to female sex pheromone components, flower volatiles, green leaf volatiles and unknown volatiles from males. Olfactory sensilla were located on three lamellae forming the antennal club. There was a clear spatial separation between some types of sensilla on each lamella. Receptor neurons for the two sex pheromone components were situated in sensilla placodea covering a specific area on each lamella in both males and females. All sex pheromone receptor neurons were found in these sensilla. Most other receptor neurons were located in a longitudinal, heterogeneous streak formed by various types of sensilla. Receptor neurons for plant-derived compounds appeared to be specialists with a high sensitivity to their respective key compound. The most remarkable among these are the green leaf volatile-specific receptor neurons, which were both sensitive and selective, with the key compound being at least 1000 times as effective as any other compound. These green leaf volatile detectors are apparently homologous to detectors recently found in the scarab Phyllopertha diversa. Our results emphasize the role of single-sensillum recordings as a tool in the identification of biologically active odours.     

Conus venom peptides: correlating chemistry and behavior

Chemical communication in scarab beetles involves female-released long-distance sex pheromones. Electrophysiological recordings using tungsten microelectrodes demonstrated two types of olfactory receptor neurons in the scarab beetle Anomala cuprea, each specific for one of the two pheromone components (R)-buibuilactone and (R)-japonilure, respectively. No neurons were found that responded specifically to enantiomers of the pheromone compounds, i.e. (S)-buibuilactone and (S)-japonilure. Pheromone receptor neurons are present in high numbers on both the male and the female antenna, with a lower sensitivity in the females. As in bark beetles and moths, the pheromone receptor neurons in A. cuprea are very sensitive and selective. The difference in response thresholds between (R)- and (S)-enantiomers is almost three orders of magnitude. Pheromone receptor neurons are found in sensilla placodea located in a defined area on each lamella in the antennal club. (R)-buibuilactone and (R)-japonilure neurons are always found in different sensilla. Both types of sensilla contain two neurons, with the pheromone-sensitive neuron displaying a high spike amplitude and the second neuron, not responding to any of the tested compounds, always with a lower spike amplitude. Accepted: 19 December 1998     

A photoaffinity-labeled green leaf volatile compound 'tricks' highly selective and sensitive insect olfactory receptor neurons

The sex pheromone of the scarab beetle, Phyllopertha diversa, is emitted by females and specifically detected by olfactory receptor neurons in the male and female antennae. Single sensillum recordings showed that, in contrast to the less sensitive pheromone sensilla in females, olfactory receptor neurons in the male antennae had a low threshold (1 ng), which rivals those of moths. The male and female antennae also possessed olfactory receptor neurons specific for the detection of floral and green leaf volatile compounds. Detectors for the green leaf volatile (Z)-3-hexenyl acetate had a threshold (10 pg) far below the sensitivity of the pheromone-detecting machinery. In addition, these neurons showed a remarkable selectivity even when challenged with related compounds at 10000-fold higher concentrations. Surprisingly, a diazo analog, (Z)-3-hexenyl diazoacetate, elicited slightly higher nervous activity than the natural ligand in the neurons specific and selective for (Z)-3-hexenyl acetate. The inability of the green leaf volatile-detecting machinery to discriminate the photoaffinity-labeled compound from the natural product indicates that the synthetic ligand interacts with odorant-binding protein, odorant receptor and odorant-degrading enzyme as does the cognate ligand.     

草地贪夜蛾4个性信息素结合蛋白基因的克隆及表达模式分析

草地贪夜蛾是世界性的重大害虫,2019年1月入侵我国并迅速扩散到20多个省市。性诱剂是对草地贪夜蛾进行监测和诱杀的有效手段,但是其作用识别机制仍不清楚,限制了高效性诱剂的研发和应用。性信息素结合蛋白(PBPs)在鳞翅目昆虫包括草地贪夜蛾性信息素识别过程中起重要作用。本研究从草地贪夜蛾中克隆了4个编码PBPs的cDNA序列,命名为SfruPBP1-SfruPBP4。4个SfruPBPs均含有完整的开放阅读框,所编码的蛋白具有性信息素结合蛋白的典型特征:N-端有信号肽、具有保守的6个半胱氨酸残基。系统进化分析显示SfruPBPs与斜纹夜蛾和海灰翅夜蛾PBPs的亲缘关系最近,且4个SfruPBPs被聚在不同的进化分支。4个SfruPBPs基因均由3个外显子和2个内含子组成,内含子插入位点高度保守。此外,4个SfruPBPs在草地贪夜蛾基因组上呈串联排列。SfruPBP1、SfruPBP2和SfruPBP3在成虫触角中特异性表达,其中SfruPBP1和SfruPBP2在雄成虫触角中的表达水平显著高于雌虫,而SfruPBP3在雌雄触角中的表达水平无显著差异。SfruPBP4特异性表达于雄成虫腹部。本研究结果为揭示草地贪夜蛾性信息素识别机制奠定了基础。     

Pheromone binding proteins in the European and Asian corn borers: no protein change associated with pheromone differences.

Pheromone binding proteins (PBPs) are thought to play a role in the recognition of sex pheromone in male moth antennae. By binding selectively to different components of pheromone blends, these PBPs could play a role in differentiating between structurally related compounds. In this study we have characterized the pheromone binding proteins of two pheromone strains of the European corn borer (Ostrinia nubilalis) and also the closely related Asian corn borer (O. furnacalis). We have been able to detect only one PBP gene, which encodes a mature protein that is identical in amino acid sequence in individuals from different pheromone strains and different species. This result suggests that the PBP is not detecting differences between the two isomeric compounds of the European corn borer pheromone or the difference in double bond position between the pheromone molecules of the European and Asian corn borers.     

Do Pheromone Binding Proteins Converge in Amino Acid Sequence When Pheromones Converge?

Convergence in amino acid sequences between proteins can be strong evidence for selection. Here, I look for evidence of convergence in the amino acid sequences of pheromone binding protein (PBP) in response to convergence in pheromones. PBPs are involved in sex pheromone reception by the antennae of male moths. In this role PBPs may selectively bind pheromone components and experience convergent selection in response to convergence in pheromone components. However, examination of the PBPs of the taxa that have converged upon the use of (E)- or (Z)-11-tetradecenyl acetate as their major pheromone component reveals little evidence for convergence in the PBPs identified from these taxa. A few sites show a pattern consistent with convergence or parallelism; however, it cannot be ruled out that these sites share the ancestral state. Two of these sites fall within the proposed binding region of PBPs. These results suggest that PBPs either have not converged in sequence or have converged at very few sites in response to convergence on the same pheromone component. Received: 29 July 1999 / Accepted: 8 November 1999     

Characterization of olfactory receptor neurons for pheromone candidate and plant volatile compounds in the clover root weevil,Sitona lepidus

Antennal olfactory receptor neurons (ORNs) for pheromone and plant volatile compounds were identified and characterized in male and female clover root weevil, Sitona lepidus (Gyllenhal), using the single sensillum recording technique with five pheromone-related compounds, and 40 host and non-host plant volatile compounds. Overall, seven different types of olfactory sensilla containing specialized ORNs were identified in each sex of S. lepidus. Among them, three different types of sensilla in the males and two types in the females housed ORNs specialized for pheromone-related compounds. The ORNs in males were specialized for 4-methyl-3,5-heptanedione or one or more of four stereoisomers of 5-hydroxy-4-methyl-3-heptanone. In contrast, female sensilla did not contain ORNs sensitive to 4-methyl-3,5-heptanedione while they contained ORNs sensitive to and specialized for the stereoisomers of (4S,5S)-5-hydroxy-4-methyl-3-heptanone. In addition to the pheromone-related ORNs, four types of olfactory sensilla contained ORNs responsive to plant volatile compounds in male S. lepidus, and five types in females. Most of the ORNs identified in S. lepidus showed a high degree of specificity to specific volatile compounds although some of the active compounds showed overlapping response spectra in the ORNs across different types of sensilla. The most active plant volatile compounds were the four green leaf volatile compounds, (E)-2-hexenol, (Z)-2-hexenol, (Z)-3-hexenol and (E)-2-hexenal, and isomers of two monoterpenols, (±)-linalool and (±)-α-terpineol, all eliciting strong responses from relatively large numbers of ORNs in male and female S. lepidus. Our study indicates that S. lepidus has a set of highly sensitive and selective ORNs for pheromone and plant volatile compounds. Further work is needed to elucidate the behavioral implications of these findings.     

Smoke,pheromone and kairomone olfactory receptor neurons in males and females of the pine sawyer Monochamus galloprovincialis (Olivier) (Coleoptera: Cerambycidae)

The response of antennal olfactory receptor neurons (ORNs) of Monochamus galloprovincialis to several odourants was tested using single sensillum electrophysiology. Behaviourally active pheromone, and kairomone (host and sympatric bark beetle pheromone) odours were tested alongside smoke compounds released by burnt wood that are potentially attractive to the insect. The antennae bore several types of sensilla. Two plate areas in the proximal and distal ends of each antennal segment were covered with basiconic sensilla that responded to the odour stimuli. Sensilla basiconica contained one or two cells of different spike amplitude. The 32 male and 38 female ORNs tested responded with excitations or inhibitions to the different plant odours. In general the response of male and female receptors was very similar so they were pooled to perform a cluster analysis on ORN responses. Six ORNs were clearly specialised for pheromone reception. Responses to kairomone and smoke odours were less specific than those of pheromone, but a group of 9 cells was clearly excited by smoke compounds (mainly eugenol and 4-methyl 2-methoxyphenol), a group of 8 cells was very responsive to α-pinene, β-pinene and cis-verbenol, and a group of 14 cells responded to a wider range of compounds. The rest of the cells (47%) were either non-responsive or slightly inhibited by smoke compounds. Dose–response curves were obtained for several compounds. Different compounds induced significantly different latencies and these appeared to be unrelated to their boiling point.     

Pheromone binding proteins of Epiphyas postvittana (Lepidoptera: Tortricidae) are encoded at a single locus

The light brown apple moth, Epiphyas postvittana (Tortricidae: Lepidoptera) uses a blend of (E)-11-tetradecenyl acetate and (E,E)-9,11-tetradecadienyl acetate as its sex pheromone. Odorant binding proteins, abundant in the antennae of male and female E. postvittana, were separated by native PAGE to reveal four major proteins with distinct mobilities. Microsequencing of their N-terminal residues showed that two were general odorant binding proteins (GOBPs) while two were pheromone binding proteins (PBPs). Full length cDNAs encoding these proteins were amplified using a combination of PCR and RACE-PCR. Sequence of the GOBPs revealed two genes (EposGOBP1, EposGOBP2), similar to orthologues in other species of Lepidoptera. Eleven cDNAs of the PBP gene were amplified, cloned and sequenced revealing two major phylogenetic clusters of PBP sequences differing by six amino acid substitutions. The position of the six amino acid differences on the protein was predicted by mapping onto the three-dimensional structure of PBP of Bombyx mori. All six substitutions were predicted to fall on the outside of the protein away from the inner pheromone binding pocket. One substitution does fall close to the putative dimerisation region of the protein (Ser63Thr). Expression of three of the cDNAs in a baculovirus expression system revealed that one class encodes an electrophoretically slow form (EposPBP1-12) while the other encodes a fast form (EposPBP1-2, EposPBP1-3). A native Western of these expressed proteins compared with antennal protein extracts demonstrated that PBP is also expressed in female antennae and that PBP may be present as a dimer as well as a monomer in E. postvittana. The fast and slow forms of EposPBP1 are allelic. Westerns on single antennal pair protein extracts and allele-specific PCR from genomic DNA both show a segregating pattern of inheritance in laboratory and wild populations. Radio labelled (E)-11-tetradecenyl acetate binds to both fast and slow PBP forms in gel assays. Taken together, the genetic and biochemical data do not support the hypothesis that these PBPs are specific for each component of the E. postvittana pheromone. However, duplication of this PBP locus in the future might allow such diversification to evolve, as observed in the other species.     

Perception of volatiles associated with sex and food by different adult forms of the black bean aphid, Aphis fabae

Abstract. Electroantennograms (EAGs) were recorded from adult male and asexual forms (winged and wingless virginoparae and gynoparae) of the black bean aphid, Aphis fabae , during stimulation with two sex pheromone components, (+)-(4a S ,7 S ,7a R )-nepetalactone and (-)-(1 R ,4a S ,7 S ,7a R )-nepetalactol, as well as six plant volatiles, i.e. ( E )-2-hexenal, ( E )-2-hexenol-1, ( Z )-3-hexenol-1, ( Z )-3-hexenyl acetate, hexanal and allyl isothiocyanate. The male antennae are 1000-10,000 times more sensitive to nepetalactol and nepetalactone than to the plant compound ( E )-2-hexenal. Besides this marked difference of EAG peak responses in males, the EAG rise and decay are slower for the sex pheromone components. Males are also much more sensitive to the sex pheromone components than asexual females. This high sensitivity correlates with a predominance of antennal secondary rhinaria, the major sites of pheromone perception in the male. However, it is the primary rhinaria on the antennae of the wingless asexual females that are responsible for pheromone perception. Male antennae are as responsive as the asexual female antennae to the plant volatiles. The specialization of the male for mate location is discussed.     

Pheromone production, male abundance, body size, and the evolution of elaborate antennae in moths

The males of some species of moths possess elaborate feathery antennae. It is widely assumed that these striking morphological features have evolved through selection for males with greater sensitivity to the female sex pheromone, which is typically released in minute quantities. Accordingly, females of species in which males have elaborate (i.e., pectinate, bipectinate, or quadripectinate) antennae should produce the smallest quantities of pheromone. Alternatively, antennal morphology may be associated with the chemical properties of the pheromone components, with elaborate antennae being associated with pheromones that diffuse more quickly (i.e., have lower molecular weights). Finally, antennal morphology may reflect population structure, with low population abundance selecting for higher sensitivity and hence more elaborate antennae. We conducted a phylogenetic comparative analysis to test these explanations using pheromone chemical data and trapping data for 152 moth species. Elaborate antennae are associated with larger body size (longer forewing length), which suggests a biological cost that smaller moth species cannot bear. Body size is also positively correlated with pheromone titre and negatively correlated with population abundance (estimated by male abundance). Removing the effects of body size revealed no association between the shape of antennae and either pheromone titre, male abundance, or mean molecular weight of the pheromone components. However, among species with elaborate antennae, longer antennae were typically associated with lower male abundances and pheromone compounds with lower molecular weight, suggesting that male distribution and a more rapidly diffusing female sex pheromone may influence the size but not the general shape of male antennae.     

蚜虫性信息素

蚜虫属同翅目蚜总科,是重要的农作物害虫。蚜虫长年行孤雌生殖,在中、高纬度深秋时,产生性蚜,以卵越冬。雌性蚜分泌性信息素荆芥内酯(nepetalactone)和荆芥醇(nepetalactol)。该文综述了蚜虫性信息素的结构与组分、性腺的形态结构、性信息素释放及影响因素;性信息素对雌性母和天敌的引诱作用;性信息素在蚜虫种间隔离中的地位和作用以及蚜虫性信息素在蚜虫防治中的应用前景。     

Discrimination of pheromone enantiomers by two pheromone binding proteins from the gypsy moth Lymantria dispar

The gypsy moth, Lymantria dispar, uses (7R, 8S)-cis-2-methyl-7, 8-epoxyoctadecane, (+)-disparlure, as a sex pheromone. The (-) enantiomer of the pheromone is a strong behavioral antagonist. Specialized sensory hairs, sensillae, on the antennae of male moths detect the pheromone. Once the pheromone enters a sensillum, the very abundant pheromone binding protein (PBP) transports the odorant to the sensory neuron. We have expressed the two PBPs found in gypsy moth antennae, PBP1 and PBP2, and we have studied the affinity of these recombinant PBPs for the enantiomers of disparlure. To study pheromone binding under equilibrium conditions, we developed and validated a binding assay. We have addressed the two major problems with hydrophobic ligands in aqueous solution: (1) concentration-dependent adsorption of the ligand on vial surfaces and (2) separation of the protein-bound ligand from the material remaining free in solution. We used this assay to demonstrate for the first time that pheromone binding to PBP is reversible and that the two PBPs from L. dispar differ in their enantiomer binding preference. PBP1 has a higher affinity for the (-) enantiomer, while PBP2 has a higher affinity for the (+) enantiomer. The PBP from the wild silk moth, Antheraea polyphemus (Apol-3) bound the disparlure enantiomers more weakly than either of the L. dispar PBPs, but Apol-3 was also able to discriminate the enantiomers. We have observed extensive aggregation of both L. dispar PBPs and an increase in pheromone binding at high (>2 microM) PBP concentrations. We present a model of disparlure binding to the two PBPs.     

Effects of aromatic compounds on antennal responses and on the pheromone-binding proteins of the gypsy moth (Lymantria dispar)

Female gypsy moths emit a pheromone, (+)-disparlure, which the males follow until they locate the emitter. The male moths' antennae are covered with innervated sensory hairs, specialized in detection of the pheromone. The neurons in these sensory hairs are bathed by a solution rich in pheromone-binding protein (PBP). PBPs are soluble proteins that bind the pheromone and other odorants reversibly with variable thermodynamic and kinetic selectivity and are essential for olfactory responses. Here, we have studied the interaction between 2 gypsy moth PBPs with aromatic compounds that modulate the responses of male moth antennae to (+)-disparlure. The aromatic compounds do not elicit responses by themselves, but when administered together with pheromone, they inhibit, enhance, or prolong the electrophysiological response to the pheromone. Three interactions between the compounds and PBPs were studied: 1) the equilibrium binding of the compounds by themselves to the PBPs, 2) the equilibrium binding of the compounds in the presence of pheromone or a fluorescent reporter ligand, and 3) the effect of the compounds on the conformation of the pheromone-PBP complex. A subset of compounds causes a prolongation of the electroantennogram response, and from this study, we conclude that these compounds follow a structure-activity pattern and stabilize a particular conformer of the PBPs that appears to activate the olfactory response.     

Working range of stimulus flux transduction determines dendrite size and relative number of pheromone component receptor neurons in moths

We are proposing that the "relative" abundances of the differently tuned pheromone-component-responsive olfactory receptor neurons (ORNs) on insect antennae are not a result of natural selection working to maximize absolute sensitivity to individual pheromone components. Rather, relative abundances are a result of specifically tuned sensillum-plus-ORN units having been selected to accurately transduce and report to the antennal lobe the maximal ranges of molecular flux imparted by each pheromone component in every plume strand. To not reach saturating stimulus flux levels from the most concentrated plume strands of a pheromone blend, the dendritic surface area of the ORN type that is tuned to the most abundant component of a pheromone blend is increased in dendritic diameter in order to express a greater number of major pheromone component-specific odorant receptors. The increased ability of these enlarged dendrite, major component-tuned ORNs to accurately report very high flux of its component results in a larger working range of stimulus flux able to be accurately transduced by that type of ORN. However, the larger dendrite size and possibly other high-flux adjustments in titers of pheromone-binding proteins and degrading enzymes cause a decrease in absolute sensitivity to lower flux levels of the major component in lower concentration strands of the pheromone blend. In order to restore the ability of the whole-antenna major pheromone component-specific channel to accurately report to its glomerulus the abundance of the major component in lower concentration strands, the number of major component ORNs over the entire antenna is adjusted upward, creating a greater proportion of major component-tuned ORNs than those tuned to minor components. Pheromone blend balance reported by the whole-antennal major and minor component channels in low plume-flux strands is now restored, and the relative fluxes of the 2 components occurring in both low- and high-flux strands are thereby accurately reported to the component-specific glomeruli. Thus, we suggest that the 2 phenomena, dendrite size and relative numbers of differentially tuned ORNs are linked, and both are related to wide disparities in molecular flux ranges occurring for the more abundant and less abundant components in the pheromone blend plume strands.     

Analysis of odorant-binding proteins in antennae of a geometrid species, Ascotis selenaria cretacea, which produces lepidopteran Type II sex pheromone components

Information on the olfactory system in antennae of Geometridae moths is very limited, and odorant-binding proteins (OBPs) working as transporters of lipophilic odors have not been identified. In the first investigation on this family of insects, we examined antennal OBPs of the Japanese giant looper, Ascotis selenaria cretacea. RT-PCR experiments using several pairs of degenerate primers designed from known cDNA sequences encoding lepidopteran OBPs successfully amplified partial sequences of two pheromone-binding proteins (PBPs), named AscrPBP1 and AscrPBP2 in reference to their corresponding nucleotide sequence homologies with other PBPs. Using 5′- and 3′-rapid amplification of cDNA end strategies, a cDNA clone for AscrPBP1 encoding a protein of 141 amino acids was isolated. Western blotting with the antiserum against recombinant AscrPBP1 overexpressed in Escherichia coli showed that the AscrPBP1 gene was more strongly expressed in male antennae than in female antennae. Furthermore, natural AscrPBP1was isolated by immunoprecipitation with the antiserum, and its binding ability was evaluated by using synthetic sex pheromonal compounds with a C₁₉ chain. The result indicated that AscrPBP1 bound not only the pheromone components, 3,6,9-nonadecatriene and its 3,4-epoxy derivative, but also unnatural 6,7- and 9,10-epoxy derivatives. While no general odorant-binding proteins (GOBPs) were amplified in the RT-PCR experiments, two antisera prepared from GOBP1 and GOBP2 of Bombyx mori suggested the occurrence of at least two GOBPs in the A. s. cretacea antennae.     

Investigation of a sex pheromone for the oriental cockroach,Blatta orientalis

A sex pheromone for adult male oriental cockroaches Blatta orientalis was isolated from the faeces of adult virgin female oriental cockroaches. It elicited a sexual response at 10 pg and 1 ng with B. orientalis and Periplaneta americana adult males, respectively. The site of production appers to be the crop, oesophagus, and proventriculus. Electroantennogram responses of male antennae toward the isolated pheromone were greater than those of the female antennae. The adult male oriental cockroach also responded to the American cockroach sex pheromone. The isolated pheromone with a mol. wt of 232 may be similar to one of the components of the American cockroach sex pheromone.     

金龟甲性信息素的化学成分及其应用

金龟甲Scarabaeoidae性信息素研究主要集中在丽金龟亚科Rutelinae和鳃金龟亚科Melolonthinae。丽金龟亚科金龟甲的性腺由臀板和腹片顶端的上皮细胞组成,其性信息素成分主要是脂肪酸衍生物;而鳃金龟亚科金龟甲的性腺可以从腹部外翻,性信息素成分主要是氨基酸衍生物和萜烯类化合物。一些存在地理或季节隔离的物种具有结构相同的性信息素成分,但手性不同。在某些种类中,性信息素成分的手性对映体可能具有行为拮抗作用。本文综述了金龟甲性信息素的化学结构与应用的新进展。     

榆木蠹蛾性信息素通讯系统的超微结构观察

为了解榆木蠹蛾Holcocerus vicarius （Walker）雌蛾性信息素分泌腺（性信息素释放系统）位置、 表面形态和超微结构及雄蛾触角感受器（性信息素接收系统）的种类、 形态、 分布及功能, 利用扫描电镜和透射电镜对榆木蠹蛾雌蛾性信息素分泌腺和雄蛾的触角进行观察。结果表明： 榆木蠹蛾雌蛾性信息素分泌腺位于腹尖末端第8～9节节间膜上的背面中央区域, 腺体表面分布着许多饱满的锥状突起, 2日龄处女雌蛾腺体细胞间有明显的胞连接, 细胞基底膜基褶较多, 质膜上分布着微绒毛, 并与内表皮连接, 内表皮上含有多层几丁质, 胞质中含有脂质粒、 大量空泡、 光面内质网、 粗面内质网及线粒体; 雄蛾触角鞭节上有5种感受器, 为毛形感器、 刺形感器、 锥形感器、 腔锥形感器和曲毛形感器, 其中毛形感器数量最多, 曲毛形感器最少。柄节和梗节被大量鳞片覆盖, 未观察到感器。榆木蠹蛾性信息素通讯系统的研究为榆木蠹蛾性信息素的生物合成、 性信息素的提取、 鉴定及成虫生殖交配生物学行为提供了依据。