Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth,Amyelois transitella

Background

The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Methodology

By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.

Conclusion

We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.     

Molecular elements of pheromone detection in the female moth,Heliothis virescens

Pheromones play pivotal roles in the reproductive behavior of moths, most prominently for the mate finding of male moths. Accordingly, the molecular basis for the detection of female‐released pheromones by male moths has been studied in great detail. In contrast, little is known about how females can detect pheromone components released by themselves or by conspecifics. In this study, we assessed the antenna of female Heliothis virescens for elements of pheromone detection. In accordance with previous findings that female antennae respond to the sex pheromone component (Z)‐9‐tetradecenal, we identified olfactory sensory neurons that express its cognate receptor, the receptor type HR6. All HR6 cells coexpressed the “sensory neuron membrane protein 1” (SNMP1) and were associated with supporting cells expressing the pheromone‐binding proteins PBP1 and PBP2. These features are reminiscent to male antennae and point to congruent mechanisms for pheromone detection in the two sexes. Further analysis of the SNMP1‐expressing cells revealed a higher number in females compared to males. Moreover, in females, the SNMP1 neurons were arranged in clusters, which project their dendrites into a common sensillum, whereas in males there were only solitary SNMP1‐neurons and only 1 per sensillum. Not all SNMP1 positive cells in female antennae expressed HR6 but instead the putative pheromone receptors HR11 and HR18, respectively. Neurons expressing 1 of the 3 receptor types were assigned to different sensilla. Together the data indicate that on the antenna of females, sensory neurons in a subset of sensilla trichodea are equipped with molecular elements, which render them responsive to pheromones.     

Processing of sting pheromone and its components in the antennal lobe of the worker honeybee

In the honeybee Apis mellifera, a sting pheromone produced by sting glands plays an important role in coordinating defensive behavior. This pheromone is a blend constituted by several components. Little is known about the neural substrates underlying sting pheromone processing in the bee brain. Here, we investigated the neural activity elicited by eight components (five acetates and three alcohols) of the sting pheromone, and by real bee stings at the level of the antennal lobe (AL) of worker honeybees. We used in vivo calcium imaging to record odor-induced neural activity of 22 identified glomeruli in the AL. We found that acetates mainly activated medial glomeruli while alcohols mainly activated lateral dorsal glomeruli. The sting preparation evoked a glomerular pattern that was clearly distinct from those of individual pheromone components. No particular region of the imaged AL was found to process sting pheromone or any of its components. Further analyses in a putative honeybee olfactory space showed that the neural activity elicited by sting preparation cannot be linearly predicted by those of pheromone components and that such components are not clearly separated from non-sting pheromone odors. We conclude that sting pheromone is processed in the worker honeybee AL following the same principles of general odors so that the chemical structure of odorants is the main determinant of glomerular activation, rather than their pheromonal values. We cannot exclude, however, that the distinctness of sting-pheromone representation with respect to that of its components constitutes a form of specialized neural processing strategy for this kind of substance.     

蛾类昆虫性信息素受体及其作用机理

蛾类昆虫性信息素受体首先从烟芽夜蛾Heliothis virescens和家蚕Bombyx mori中鉴定出来, 到目前为止已经克隆得到了19种蛾类昆虫的几十种性信息素受体基因, 并且这些基因在系统发育树中聚成一个亚群。性信息素受体从蛾类蛹期开始表达, 主要表达在雄性触角的毛形感器中, 少部分受体在雌性触角、 雄性触角其他感器以及身体其他部位中也有表达。大部分蛾类性信息素受体的配体并不是单一的, 而是能够对多种性信息素组分有反应, 部分性信息素受体还能够识别性信息素以外的其他物质, 还有一部分性信息素受体的识别配体目前尚不清楚。另外发现在雌性蛾类触角中也存在一些嗅觉受体能够识别雄性分泌的性信息素。在蛾类性信息素受体与性信息素识别的过程中, 性信息素结合蛋白不仅能够特异性地运送配体到嗅觉神经元树状突上, 还能够提高性信息素与性信息素受体之间的结合效率。另外, OrCo类受体与性信息素受体共表达在嗅觉神经元中, 在蛾类性信息素受体与配体的识别过程中扮演了重要角色。但是蛾类信息素对神经元刺激的终止并非由性信息素受体控制, 而是由细胞中的气味降解酶等其他因子调控。蛾类性信息素受体研究中还有很多疑问需要解答, 其过程可能比我们想象的更为复杂。     

Pheromonal communication in amphibians

Pheromonal communication is widespread in salamanders and newts and may also be important in some frogs and toads. Several amphibian pheromones have been behaviorally, biochemically and molecularly identified. These pheromones are typically peptides or proteins. Study of pheromone evolution in plethodontid salamanders has revealed that courtship pheromones have been subject to continual evolutionary change, perhaps as a result of co-evolution between the pheromonal ligand and its receptor. Pheromones are detected by the vomeronasal organ and main olfactory epithelium. Chemosensory neurons express vomeronasal receptors or olfactory receptors. Frogs have relatively large numbers of vomeronasal receptors that are transcribed in both the vomeronasal organ and the main olfactory epithelium. Salamander vomeronasal receptors apparently are restricted to the vomeronasal organ. To date, no chemosensory ligands have been matched to vomeronasal receptors or olfactory receptors so it is unknown whether particular receptor types are (1) specialized for detection of pheromones versus other chemosignals, or (2) specialized for detection of volatile, nonvolatile, or water-borne chemosignals. Despite progress in understanding amphibian pheromonal communication, only a small fraction of amphibian species have been examined. Study of additional species of amphibians will indicate which traits related to pheromonal communication are evolutionarily conserved and which traits have diverged over time.     

Novel odorant-binding proteins expressed in the taste tissue of the fly

A taste tissue cDNA library of the fleshfly Boettcherisca peregrina was screened with a subtracted cDNA probe enriched with taste-receptor-tissue-specific cDNA. Seven genes were identified with sequence similarity to insect odorant-binding protein (OBP) genes. The predicted amino acid sequences of the genes contain the putative signal peptide sequence at the N-terminal and most of them conserve the six cysteines common to known insect OBPs. These genes show a high degree of sequence divergence with approximately 20% amino acid identity. The most striking feature was that all seven of these genes are expressed mainly in the taste tissues, such as the labellum and tarsus, unlike the known insect OBP genes expressed in olfactory tissue. The predicted amino acid sequences had the highest degree of sequence similarity to the Drosophila melanogaster OBPs named pheromone binding protein-related proteins (PBPRPs). These gene products are here referred to as gustatory PBP-related proteins (GPBPRPs) 1-7. Homologous GPBPRP genes were found also in D. melanogaster by database search and are shown to be expressed in Drosophila taste tissues.     

Proteomic analysis of castor bean tick Ixodes ricinus: a focus on chemosensory organs

In arthropods, the large majority of studies on olfaction have been focused on insects, where most of the proteins involved have been identified. In particular, chemosensing in insects relies on two families of membrane receptors, olfactory/gustatory receptors (ORs/GRs) and ionotropic receptors (IRs), and two classes of soluble proteins, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). In other arthropods, such as ticks and mites, only IRs have been identified, while genes encoding for OBPs and CSPs are absent. A third class of soluble proteins, called Niemann-Pick C2 (NPC2) has been suggested as potential carrier for semiochemicals both in insects and other arthropods.Here we report the results of a proteomic analysis on olfactory organs (Haller's organ and palps) and control tissues of the tick Ixodes ricinus, and of immunostaining experiments targeting NPC2s. Adopting different extraction and proteomic approaches, we identified a large number of proteins, and highlighted those differentially expressed. None of the 13 NPC2s known for this species was found. On the other hand, using immunocytochemistry, we detected reaction against one NPC2 in the Haller's organ and palp sensilla. We hypothesized that the low concentration of such proteins in the tick's tissues could possibly explain the discrepant results. In ligand-binding assays the corresponding recombinant NPC2 showed good affinity to the fluorescent probe N-phenylnaphthylamine and to few organic compounds, supporting a putative role of NPC2s as odorant carriers.     

A putative pheromone receptor gene is expressed in two distinct olfactory organs in goats

Mammals possess two anatomically and functionally distinct olfactory systems. The olfactory epithelium (OE) detects volatile odorants, while the vomeronasal organ (VNO) detects pheromones that elicit innate reproductive and social behavior within a species. In rodent VNO, three multigene families that encode the putative pheromone receptors, V1Rs, V2Rs and V3Rs, have been expressed. We have identified the V1R homologue genes from goat genomic DNA (gV1R genes). Deduced amino acid sequences of gV1R genes show 40-50% and 20-25% identity to those of rat and mouse V1R and V3R genes, respectively, suggesting that the newly isolated goat receptor genes are members of the V1R gene family. One gene (gV1R1 gene) has an open reading frame that encodes a polypeptide of 309 amino acids. It is expressed not only in VNO but also in OE. In situ hybridization analysis revealed that gV1R1 -expressing cells were localized in neuropithelial layers of VNO and OE. These results suggest that the goat may detect pheromone molecules through two distinct olfactory organs.     

Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

Cell signaling systems transmit information by post-translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-protein coupled receptor (GPCR). We used published mass spectrometry-based proteomics data to identify putative sites of phosphorylation on pheromone pathway components, and we used evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of putative phosphorylation events that contribute to adjust the input-output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results suggest that relatively small quantitative influences from individual phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.     

A Drosophila protein specific to pheromone-sensing gustatory hairs delays males' copulation attempts

In insects, increasing evidence suggests that small secreted pheromone binding proteins (PBPs) and odorant binding proteins (OBPs) are important for normal olfactory detection of airborne pheromones and odorants far from their source. In contrast, it is unknown whether extracellular ligand binding proteins participate in perception of less volatile chemicals, including many pheromones, that are detected by direct contact with chemosensory organs. CheB42a, a small Drosophila melanogaster protein unrelated to known PBPs or OBPs, is expressed and likely secreted in only a small subset of gustatory sensilla on males' front legs, the site of gustatory perception of contact pheromones. Here we show that CheB42a is expressed specifically in the sheath cells surrounding the taste neurons expressing Gr68a, a putative gustatory pheromone receptor for female cuticular hydrocarbons that stimulate male courtship. Surprisingly, however, CheB42a mutant males attempt to copulate with females earlier and more frequently than control males. Furthermore, CheB42a mutant males also attempt to copulate more frequently with other males that secrete female-specific cuticular hydrocarbon pheromones, but not with females lacking cuticular hydrocarbons. Together, these data indicate that CheB42a is required for a normal gustatory response to female cuticular hydrocarbon pheromones that modulate male courtship.     

A flux capacitor for moth pheromones

In this issue of Chemical Senses, Baker et al. propose a provocative and intriguing explanation for a commonly observed phenomenon in moth chemocommunication. Sex pheromones in moths typically consist of mixtures of long-chain unsaturated compounds in specific ratios. These ratios are correspondingly detected by male moths using separate olfactory sensory neurons for each pheromone component housed singly or multiply in long trichoid sensilla on the antennal surface. These neurons are often present in different proportions, typically with the neuron responding to the highest ratio component present in greatest abundance or with the largest dendritic diameter. In their article, Baker et al. postulate that these physical differences in neuron magnitudes arise to compensate for the higher molecular flux present with the most abundant pheromone components. Such a suggestion raises several questions concerning the physiological and behavioral nature of pheromone communication. Specifically, is the flux in a natural pheromone plume high enough to warrant increased flux detection for the most abundant components? Second, how can changes in neuronal number or size lead to increased flux detection? And finally, how would this increased flux detection be accomplished at molecular, cellular, and ultimately network scales? We address each of these questions and propose future experiments that could offer insight into the stimulating proposition raised by Baker et al.     

Identification and Characterization of Pheromone Receptors and Interplay between Receptors and Pheromone Binding Proteins in the Diamondback Moth,Plutella xyllostella

Moths depend on olfactory cues such as sex pheromones to find and recognize mating partners. Pheromone receptors (PRs) and Pheromone binding proteins (PBPs) are thought to be associated with olfactory signal transduction of pheromonal compounds in peripheral olfactory reception. Here six candidate pheromone receptor genes in the diamondback moth, Plutella xyllostella were identified and cloned. All of the six candidate PR genes display male-biased expression, which is a typical characteristic of pheromone receptors. In the Xenopus-based functional study and in situ hybridization, PxylOR4 is defined as another pheromone receptor in addition to the previously characterized PxylOR1. In the study of interaction between PRs and PBPs, PxylPBPs could increase the sensitivity of the complex expressing oocyte cells to the ligand pheromone component while decreasing the sensitivity to pheromone analogs. We deduce that activating pheromone receptors in olfactory receptor neurons requires some role of PBPs to pheromone/PBP complex. If the chemical signal is not the pheromone component, but instead, a pheromone analog with a similar structure, the complex would have a decreased ability to activate downstream pheromone receptors.     

Sex pheromone blend of Manduca sexta: Responses of central olfactory interneurons to antennal stimulation in male moths

Recently, chemical analysis of solvent rinses of the external surfaces of pheromone glands from female Manduca sexta revealed a blend of 12 aldehydes, including the previously identified sex pheromone component, (E,Z)-10,12-hexadecadienal (bombykal). Previous electrophysiological studies showed that olfactory (deutocerebral) interneurons in the antennal lobes of males exhibited a wide range of responsiveness to pheromonal stimulation of the ipsilateral antenna. These experiments were performed with crude extracts of pheromone glands as well as two synthetic compounds: the major pheromone component, bombykal, and (E,Z)-11,13-pentadecadienal, a mimic of a second component of the female's pheromone blend. Using intracellular methods, we have now reexamined similar olfactory interneurons, using each of the 12 chemically identified components as well as synthetic blends of various combinations of them. Eight of the 12 components isolated from female glands elicited some form of response in olfactory interneurons in males. In accordance with biochemical and behavioral data, the most potent are bombykal and two trienals, (E,E,E)- and (E,E,Z)-10,12,14-hexadecatrienal. We also conclude that the C₁₅ dienal is selective for one of the trienal receptors on the antenna, but is much less potent than the natural trienal stimulant.     

Mixtures of Two Bile Alcohol Sulfates Function as a Proximity Pheromone in Sea Lamprey

Unique mixtures of pheromone components are commonly identified in insects, and have been shown to increase attractiveness towards conspecifics when reconstructed at the natural ratio released by the signaler. In previous field studies of pheromones that attract female sea lamprey (Petromyzon marinus, L.), putative components of the male-released mating pheromone included the newly described bile alcohol 3,12-diketo-4,6-petromyzonene-24-sulfate (DkPES) and the well characterized 3-keto petromyzonol sulfate (3kPZS). Here, we show chemical evidence that unequivocally confirms the elucidated structure of DkPES, electrophysiological evidence that each component is independently detected by the olfactory epithelium, and behavioral evidence that mature female sea lamprey prefer artificial nests activated with a mixture that reconstructs the male-released component ratio of 30:1 (3kPZS:DkPES, molar:molar). In addition, we characterize search behavior (sinuosity of swim paths) of females approaching ratio treatment sources. These results suggest unique pheromone ratios may underlie reproductive isolating mechanisms in vertebrates, as well as provide utility in pheromone-integrated control of invasive sea lamprey in the Great Lakes.     

G protein Gs alpha (GNAS 1), the probable candidate gene for Albright hereditary osteodystrophy, is assigned to human chromosome 20q12-q13.2

Guanine nucleotide-binding proteins, also known as G proteins, mediate intracellular responses to a wide variety of extracellular stimuli. A variety of genes that specify the synthesis of the components of guanine nucleotide proteins have been identified. One of these proteins, termed Gs alpha (GNAS1), is the G protein component of the olfactory signal transduction cascade. Mutations in the GNAS1 gene leading to Gs alpha protein deficiency are known to be associated with pseudohypoparathyroidism Ia (Albright hereditary osteodystrophy) and certain pituitary tumors with acromegaly. Studies on the human--mouse somatic cell hybrids provisionally assigned this gene to chromosome 20. We have now confirmed this localization on chromosome 20 and regionally assigned the GNAS1 gene to 20q12-q13.2 by in situ hybridization.     

Staining for protease activity on polyacrylamide gels

A high-performance liquid chromatographic procedure has been developed for the detailed analysis of amino acids and related compounds in 10-μl samples of perilymph from the guinea pig cochlea (inner ear). The procedure employs an Aminco amino acid analyzer and combines the use of a single chromatographic column, lithium citrate buffers for elution, a change of column temperature, and fluorometric detection of o-phthaldialdehyde/2-mercaptoethanol adducts of primary amines. Sensitivity is about 0.2 pmol referenced to leucine. Fifty-four primary amine components are detectable in perilymph collected in relative silence. Twentynine compounds have been identified, and six are putative amino acid neurotransmitters. The present method provides new information about the chemical composition of perilymph and is suitable for the analysis of physiological fluids available only in volumes of several microliters.     

Characterization of comQ and comX, Two Genes Required for Production of ComX Pheromone in Bacillus subtilis

Many microbes use secreted peptide-signaling molecules to stimulate changes in gene expression in response to high population density, a process called quorum sensing. ComX pheromone is a modified 10-amino-acid peptide used by Bacillus subtilis to modulate changes in gene expression in response to crowding. comQ and comX are required for production of ComX pheromone. We found that accumulation of ComX pheromone in culture supernatant paralleled cell growth, indicating that there was no autoinduction of production of ComX pheromone. We overexpressed comQ and comX separately and together and found that overexpression of comX alone was sufficient to cause an increase in production of ComX pheromone and early induction of a quorum-responsive promoter. These results indicate that the extracellular concentration of ComX pheromone plays a major role in determining the timing of the quorum response and that expression of comX is limiting for production of ComX pheromone. We made alanine substitutions in the residues that comprise the peptide backbone of ComX pheromone. Analysis of these mutants highlighted the importance of the modification for ComX pheromone function and identified three residues (T50, G54, and D55) that are unlikely to interact with proteins involved in production of or response to ComX pheromone. We have also identified and mutated a putative isoprenoid binding domain of ComQ. Mutations in this domain eliminated production of ComX pheromone, consistent with the hypothesis that ComQ is involved in modifying ComX pheromone and that the modification is likely to be an isoprenoid.