ABSTRACT
Chemosensory proteins (CSPs) have been considered to play a key role in chemoreception in insects. As stated in our earlier study, three CSP genes from rice leaf folder Cnaphalocrocis medinalis have been identified and showed potential physiological functions in olfaction. Here, we conducted western blot, immunolocalization, competitive binding assay and knockdown assay by RNA interference both in vitro and in vivo to reveal the functions of these three CSPs in C. medinalis. Results showed that both CmedCSP1 and CmedCSP2 are housed in sensilla basiconica and showed high binding affinities to a wide range of host-related semiochemicals. On the other hand, CmedCSP3 is highly expressed in sensilla trichodea of males and sensilla basiconica of females. It showed binding affinities to plant volatiles, especially terpenoids, as well as two of the C. medinalis sex pheromone components, Z11-16:Ac and Z11-16:Al. The transcript expression level of the three CSP genes significantly decreased after injecting target double-stranded RNAs and resulted in remarkably down-regulation on electroantennogram responses evoked by host-related semiochemicals and one sex pheromone compound, which have high binding affinities with CmedCSPs. In conclusion, the three CmedCSPs tested are involved in C. medinalis reception of semiochemicals, including host attractants and sex pheromones.
Subject(s)
Insect Proteins/metabolism , Moths/metabolism , Sensilla/metabolism , Sex Attractants/metabolism , Smell , Animals , Female , Insect Proteins/isolation & purification , Male , RNA InterferenceABSTRACT
Re-emergence of vector-borne diseases such as dengue and yellow fever, which are both transmitted by the Aedes aegypti mosquito, has been correlated with insecticide resistance. P-glycoproteins (P-gps) are ATP-dependent efflux pumps that are involved in the transport of substrates across membranes. Some of these proteins have been implicated in multidrug resistance (MDR). In this study, we identified a putative P-glycoprotein in the Ae. aegypti database based on its significantly high identity with Anopheles gambiae, Culex quinquefasciatus, Drosophila melanogaster and human P-gps. The basal ATPase activity of ATP-binding cassette transporters in larvae was significantly increased in the presence of MDR modulators (verapamil and quinidine). An eightfold increase in Ae. aegypti P-gp (AaegP-gp) gene expression was detected in temephos-treated larvae as determined by quantitative PCR. To analyse the potential role of AaegP-gp in insecticide efflux, a temephos larvicide assay was performed in the presence of verapamil. The results showed an increase of 24% in temephos toxicity, which is in agreement with the efflux reversing effect. RNA interference (RNAi)-mediated silencing of the AaegP-gp gene caused a significant increase in temephos toxicity (57%). In conclusion, we have demonstrated for the first time in insects that insecticide-induced P-gp expression can be involved in the modulation of insecticide efflux.
Subject(s)
Aedes/drug effects , Larva/drug effects , Temefos , ATP Binding Cassette Transporter, Subfamily B , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Animals , Gene Expression/drug effects , Insecticide Resistance/genetics , Molecular Sequence Data , Mortality , Quinidine/pharmacology , Verapamil/pharmacologyABSTRACT
Using molecular- and sensory physiology-based approaches, three novel natural products, a simple ester, and a behavioral antagonist have been identified from the pheromone gland of the navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae). In addition to the previously identified (Z,Z)-11,13-hexadecadienal, the pheromone blend is composed of (Z,Z,Z,Z,Z)-3,6,9,12,15-tricosapentaene, (Z,Z,Z,Z,Z)-3,6,9,12,15-pentacosapentaene, ethyl palmitate, ethyl-(Z,Z)-11,13-hexadecadienoate, and (Z,Z)-11,13-hexadecadien-1-yl acetate. The C(23) and C(25) pentaenes are not only novel sex pheromones, but also new natural products. In field tests, catches of A. transitella males in traps baited with the full mixture of pheromones were as high as those in traps with virgin females, whereas control and traps baited only with the previously known constituent did not capture any moths at all. The navel orangeworm sex pheromone is also an attractant for the meal moth, Pyralis farinalis L. (Pyralidae), but (Z,Z)-11,13-hexadecadien-1-yl acetate is a behavioral antagonist. The new pheromone blend may be highly effective in mating disruption and monitoring programs.
Subject(s)
Lepidoptera/physiology , Sex Attractants/chemistry , Animals , Behavior, Animal , Chromatography, Gas , Larva , Lepidoptera/growth & development , Pupa , Sex Attractants/physiology , Sexual Behavior, AnimalABSTRACT
The sex pheromone of the citrus fruit borer Ecdytolopha aurantiana has been identified by gas chromatography coupled to an electroantennographic detector (GC-EAD). The electron impact mass spectral (EI-MS) fragmentation of the major EAD-active peak gave identifying features for a monounsaturated acetate. Further analyses by chemical ionization mass spectrometry (CI-MS), vapor-phase infrared spectroscopy (GC-IR), along with chemical derivatization (DMDS reaction), led to full characterization of the major component as (E)-8-dodecenyl acetate (E8-12 : Ac). The second constituent was identified as the related alcohol, (E)-8-dodecenol (E8-12 : OH). The two compounds were indistinguishable from the authentic synthetic standards in chemical and EAD analyses. Samples of the two compounds were obtained by a facile synthesis utilizing lithium chemistry. Field tests showed that captures in traps baited with a mixture of E8-12 : Ac and E8-12 : OH at 100 : 1 and 10 : 1 ratios were not significantly different from the catches in traps having two virgin females. Dosage tests showed better performance of traps baited with 1 mg than those with 0.1 mg of the pheromone blend, either in 100 : 1 or 10 : 1 ratio.
Subject(s)
Acetates/pharmacology , Fatty Acids, Monounsaturated/chemistry , Fatty Acids, Monounsaturated/pharmacology , Lepidoptera , Sex Attractants/chemistry , Sex Attractants/pharmacology , Acetates/isolation & purification , Alcohols/chemistry , Alcohols/isolation & purification , Alcohols/pharmacology , Animals , Chromatography, Gas , Fatty Acids, Monounsaturated/isolation & purification , Gas Chromatography-Mass Spectrometry , Larva , Movement , Sex Attractants/isolation & purification , SmellABSTRACT
Males and virgin females of the citrus fruit borer Ecdytolopha aurantiana Lima, displayed two flight peaks during a 24-hr period, one at dawn and the other at dusk in an orange grove near Gavião Peixoto, São Paulo, Brazil. During the day, when temperatures were highest and relative humidity lowest, most individuals rested on leaves in the lower and middle crown. Moths rapidly moved higher in the crown after sunset, and many were observed flying above the tree canopy. This behavior was mainly associated with mating. Males and virgin females marked with fluorescent powder of different colors were observed in the dark with the aid of a black light. Mating was observed only in the upper crown of citrus trees from 6:00 to 9:00 PM, with a peak (64%) between 7:00 and 8:00 PM. Males of E. aurantiana were captured in traps baited either with virgin females or female extracts, suggesting the use of a long-range sex pheromone. At close distance (1-2 cm), males and females displayed a short-range communication behavior, with males exposing hairpencils and vibrating their wings. Females were frequently stimulated to contact the body of a male before copulation. The mean duration of copulation was 1 hr 40 min.
Subject(s)
Animal Communication , Lepidoptera , Sex Attractants , Sexual Behavior, Animal , Animals , Circadian Rhythm , Female , Flight, Animal , Humidity , Male , Movement , Temperature , Vibration , Wings, AnimalABSTRACT
Odorants are transmitted by small hydrophobic molecules that cross the aqueous sensillar lymph surrounding the dendrites of the olfactory neurons to stimulate the olfactory receptors. In insects, the transport of pheromones, which are a special class of odorants, is mediated by pheromone-binding proteins (PBPs), which occur at high concentrations in the sensillar lymph. The PBP from the silk moth Bombyx mori (BmPBP) undergoes a pH-dependent conformational transition between the forms BmPBP(A) present at pH 4.5 and BmPBP(B) present at pH 6.5. Here, we describe the NMR structure of BmPBP(A), which consists of a tightly packed arrangement of seven alpha-helices linked by well defined peptide segments and knitted together by three disulfide bridges. A scaffold of four alpha-helices that forms the ligand binding site in the crystal structure of a BmPBP-pheromone complex is preserved in BmPBP(A). The C-terminal dodecapeptide segment, which is in an extended conformation and located on the protein surface in the pheromone complex, forms a regular helix, alpha(7), which is located in the pheromone-binding site in the core of the unliganded BmPBP(A). Because investigations by others indicate that the pH value near the membrane surface is reduced with respect to the bulk sensillar lymph, the pH-dependent conformational transition of BmPBP suggests a novel physiological mechanism of intramolecular regulation of protein function, with the formation of alpha(7) triggering the release of the pheromone from BmPBP to the membrane-standing receptor.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Insect Proteins/chemistry , Insect Proteins/metabolism , Sex Attractants/metabolism , Amino Acid Sequence , Animals , Binding Sites , Bombyx/genetics , Bombyx/metabolism , Carrier Proteins/genetics , Female , Hydrogen-Ion Concentration , Insect Proteins/genetics , Intercellular Signaling Peptides and Proteins , Magnetic Resonance Spectroscopy , Male , Models, Molecular , Molecular Sequence Data , Olfactory Receptor Neurons/metabolism , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
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.
ABSTRACT
We have identified and cloned a pheromone-binding protein (EoriPBP) from the Japanese and American populations of the Oriental beetle, Exomala orientalis (Coleoptera: Scarabaeidae). The protein showed more than 90% amino acid identity to the previously identified pheromone-binding proteins from Popilliajaponica (PjapPBP) and Anomala osakana (AosaPBP), as well as to one of the odorant-binding proteins from Phyllopertha diversa (PdivOBP1). EoriPBP has 116 amino acids, with a calculated molecular mass of 12,981 Da. pI of 4.3, and six highly conserved cysteine residues. 5'-RACE amplifications led to the characterization of a signal peptide with 19 amino acids. The signal peptide showed high amino acid identity to the signal peptide for AosaPBP. Comparison of the amino acid sequences of the PBPs involved in the detection of similar ligands, i.e., monounsaturated lactones and ketone, suggests that the most variable residues among the PBPs from E. orientalis, P. japonica, and A. osakana are probably the most discriminating residues. As with the pheromone-binding protein from Bombyx mori, the residues at positions 61, 64, 71, and 82 in EoriPBP, PajpPBP, and AosaPBP, which are either valine, leucine, isoleucine, or methionine, are likely to be specificity determinants.
Subject(s)
Carrier Proteins/chemistry , Coleoptera/chemistry , Insect Proteins , Pheromones/metabolism , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/genetics , Cloning, Molecular , DNA, Complementary/genetics , Molecular Sequence Data , Polymerase Chain Reaction/veterinary , Protein Sorting SignalsABSTRACT
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.
Subject(s)
Coleoptera/physiology , Olfactory Receptor Neurons/drug effects , Olfactory Receptor Neurons/physiology , Plant Extracts/pharmacology , Acetates/pharmacology , Affinity Labels , Animals , Female , Ligands , Male , Plant Extracts/chemistry , Sex Attractants/pharmacology , Sex Attractants/physiologyABSTRACT
Three related nucleotide sequences, encoding mature proteins of 108-113 amino acids, have been obtained from antennal cDNA of the Phasmid Eurycantha calcarata. Among these, one is also expressed in the tarsi as demonstrated by N-terminal sequence and mass spectrometric analyses of protein samples isolated from both organs. PCR experiments performed with specific primers, showed that this species is also expressed in the mouth organs and in the cuticle, while the other two are antennal specific. All three isoforms are similar to Drosophila OS-D and other proteins reported in several insect orders, but one of them is significantly different from the other two. The best conserved elements are the N-terminal region and the four cysteine residues. Accurate ESMS measurements indicated that all cysteines are involved in two disulphide bonds and ruled out the occurrence of additional post-translational modifications. Polyclonal antibodies, raised against the purified protein, did not react with proteins of the same class expressed in another Phasmid species, Carausius morosus, and in the orthopteran Schistocerca gregaria, nor did antibodies against these proteins recognise those of E. calcarata.
Subject(s)
Chemoreceptor Cells/chemistry , Insect Proteins/genetics , Insecta/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA Primers , DNA, Complementary/genetics , Insect Proteins/chemistry , Insecta/chemistry , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single "acid form" (A), and a homogeneous "basic form" (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, "closed" conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.
Subject(s)
Bombyx/chemistry , Carrier Proteins/chemistry , Insect Proteins/chemistry , Animals , Chromatography, High Pressure Liquid , Circular Dichroism , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Protein Conformation , Protein Isoforms , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , SoftwareABSTRACT
(-)-Periplanones C and D were obtained in reproducible yields by modifying reported procedures. Our synthetic sample of (-)-periplanone D showed somewhat different physical and spectroscopic properties from those reported in the literature.
Subject(s)
Ketones/analysis , Sex Attractants/analysis , Animals , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular/methods , Periplaneta/chemistry , Spectroscopy, Fourier Transform Infrared/methodsABSTRACT
BACKGROUND: Insects use volatile organic molecules to communicate messages with remarkable sensitivity and specificity. In one of the most studied systems, female silkworm moths (Bombyx mori) attract male mates with the pheromone bombykol, a volatile 16-carbon alcohol. In the male moth's antennae, a pheromone-binding protein conveys bombykol to a membrane-bound receptor on a nerve cell. The structure of the pheromone-binding protein, its binding and recognition of bombykol, and its full role in signal transduction are not known. RESULTS: The three-dimensional structure of the B. mori pheromone-binding protein with bound bombykol has been determined by X-ray diffraction at 1.8 A resolution. CONCLUSIONS: The pheromone binding protein of B. mori has six helices, and bombykol binds in a completely enclosed hydrophobic cavity formed by four antiparallel helices. Bombykol is bound in this cavity through numerous hydrophobic interactions, and sequence alignments suggest critical residues for specific pheromone binding.
Subject(s)
Bombyx/chemistry , Fatty Alcohols/chemistry , Sex Attractants/metabolism , Amino Acid Sequence , Animals , Binding Sites , Bombyx/physiology , Crystallization , Fatty Alcohols/metabolism , Female , Hydrogen-Ion Concentration , Male , Models, Molecular , Molecular Sequence Data , Pheromones/metabolism , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
The analysis of a recombinant pheromone-binding protein from the silkworm moth, Bombyx mori, by native gel electrophoresis with Coomassie staining showed one single band with a molecular mass consistent with a monomer. A slow migrating band, detected in the recombinant and native samples by a polyclonal antibody, was indistinguishable from the monomer in the mass spectrum fragmentation pattern and chromatographic behavior. Flow injection analyses of the protein by mass spectrometry in the negative mode showed fragments of a dimer. The dimeric form was also supported by estimation of the molecular mass by gel filtration at basic pH. A cross-linked dimer coeluted with the noncovalent dimer on a gel filtration column. The molecular mass of the protein changed in a pH-dependent way with a dramatic transition from dimer to monomer between pH 6 and 4.5. A low pH induced not only dissociation of the dimer, but also a conformational change in the protein. In marked contrast to denaturation with guanidinium chloride, the emission maxima of tryptophan was not significantly changed at low pH. BmPBP is thus a dimer at slightly acid, neutral, and basic pH, which dissociates and then undergoes conformational change at low pH.
Subject(s)
Bombyx/chemistry , Carrier Proteins/chemistry , Insect Proteins/chemistry , Animals , Blotting, Western , Carrier Proteins/metabolism , Cross-Linking Reagents/chemistry , Dimerization , Insect Proteins/metabolism , Intercellular Signaling Peptides and Proteins , Mass Spectrometry , Molecular Weight , Spectrometry, FluorescenceABSTRACT
All of the four possible stereoisomers of 5,9-dimethylpentadecane, the major sex pheromone component of the coffee leaf miner moth (Perileucoptera coffeella), were synthesized by using the methyl esters of (S)- and (R)-3-hydroxy-2-methylpropanoic acid as chiral sources for the purpose of determining the stereochemistry of the pheromone.
Subject(s)
Alkanes/chemical synthesis , Moths , Sex Attractants/chemical synthesis , Alkanes/chemistry , Animals , Biochemistry/methods , Sex Attractants/chemistry , StereoisomerismABSTRACT
The pheromone-detecting sensilla placodea are significantly more numerous than other sensory structures in the antennae of the Japanese beetle, Popillia japonica (Coleoptera: Scarabaeidae). Their abundance in males is nearly twice of that in females, showing a clear sexual dimorphism. Externally, they have a tortoise shell-like round cuticular plate containing a few polygonal plates separated by narrow ridges. Internally, they house two long dendrites that branch and terminate near fine cuticular pores. They have a system of two bipolar neurons accompanied by three enveloping cells, resembling sensilla trichodea in moths. The conspicuous difference with the latter is that the sensillum-lymph cavity near the outer cuticle is funnel-shaped, into which the tormogen cell projects numerous microvilli whose tips approach the terminal branches of the dendrites.
ABSTRACT
Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, followed by digestion with endoproteinase Lys-C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and protein sequencing supported the assignment of disulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.
Subject(s)
Bombyx/chemistry , Carrier Proteins/chemistry , Disulfides/chemistry , Insect Hormones/chemistry , Insect Proteins/chemistry , Amino Acid Sequence , Animals , Chymotrypsin/pharmacology , Cyanogen Bromide/pharmacology , Indicators and Reagents/pharmacology , Intercellular Signaling Peptides and Proteins , Mass Spectrometry , Metalloendopeptidases/pharmacology , Molecular Sequence Data , Phosphines/pharmacology , Recombinant Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
The pale-brown chafer, Phyllopertha diversa, utilizes an unusual alkaloid, 1,3-dimethyl-2,4-(1H,3H)-quinazolinedione, as its sex pheromone. This compound is rapidly degraded in vitro by the antennal protein extracts from this scarab beetle. Demethylation at the N-1 position and hydroxylation of the aromatic ring have been identified as the major catabolic pathways. The enzyme responsible for the pheromone degradation is membrane-bound, requires NAD(P)H for activity and is sensitive to cytochrome P450 inhibitors, such as proadifen and metyrapone. The ability to metabolize this unusual pheromone was not detected in 12 species tested, indicating that the P450 system, specific to male P. diversa antennae, has evolved as a mechanism for olfactory signal inactivation.
Subject(s)
Coleoptera/metabolism , Cytochrome P-450 Enzyme System/metabolism , Insect Proteins/metabolism , Quinazolines/metabolism , Sex Attractants/metabolism , Alkaloids/metabolism , Animals , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Mass Spectrometry , Metyrapone/pharmacology , Microsomes/metabolism , Molecular Structure , Proadifen/pharmacology , Quinazolines/chemistryABSTRACT
The pheromone-binding protein (PBP) from Bombyx mori was expressed in Escherichia coli periplasm. It specifically bound radiolabeled bombykol, the natural pheromone for this species. It appeared as a single band both in native and SDS-polyacrylamide gel electrophoresis and was also homogeneous in most chromatographic systems. However, in ion-exchange chromatography, multiple forms sometimes appeared. Attempts to separate them revealed that they could be converted into one another. Analysis of the protein by circular dichroism and fluorescence spectroscopy demonstrated that its tertiary structure was sensitive to pH changes and that a dramatic conformational transition occurred between pH 6.0 and 5.0. This high sensitivity to pH contrasted markedly with its thermal stability and resistance to denaturation by urea. There was also no significant change in CD spectra in the presence of the pheromone. The native protein isolated from male antennae displayed the same changes in its spectroscopic properties as the recombinant material, demonstrating that this phenomenon is not an artifact arising from the expression system. This conformational transition was reproduced by interaction of the protein with anionic (but not neutral) phospholipid vesicles. Unfolding of the PBP structure triggered by membranes suggests a plausible mechanism for ligand release upon interaction of the PBP-pheromone complex with the surface of olfactory neurons. This pH-linked structural flexibility also explains the heterogeneity reported previously for B. mori PBP and other members of this class of proteins.