Evidence for male-produced aggregation pheromone in American palm weevil,Rhynchophorus palmarum (L.) (Coleoptera: Curculionidae):

Field trapping of the American palm weevil (APW),Rhynchophorus palmarum, showed that the combination of caged male APWs and palm stem was much more attractive to APWs of both sexes than palm stem alone. Caged female APWs did not enhance the attractiveness of the palm. Caged APWs without palm stem were not attractive. Virgin laboratory-bred males were highly attractive to APWs of both sexes in a two-choice pitfall olfactometer, whereas virgin laboratory-bred females were not. Adsorbenttrapped volatiles from virgin laboratory-bred males reproduced the effect of living males, giving evidence for a male-produced aggregation pheromone in this species. Wild-mated APWs of both sexes were as responsive to the aggregation pheromone as virgin laboratory-bred APWs. This is the first record of chemical communication in this species. These results have prompted investigations into the chemical identification of the aggregation pheromone.     

Identification of Pheromone Synergists in American Palm Weevil, Rhynchophorus Palmarum, and Attraction of Related Dynamis Borassi

Thirteen host-plant kairomone blends, including 28 compounds, were tested and showed moderate to high synergy with rhynchophorol. The blends plus rhynchophorol also attracted the related Dynamis borassi. Ethanol–ethyl acetate blends in various ratios showed moderate synergy. Two blends, including "characteristic coconut" odor molecules, were as efficient as sugarcane in synergizing rhynchophorol and field luring American Palm weevils (APWs). Preliminary olfactometer tests of natural host-plant volatiles demonstrated the role of fermentation in primary APW attraction. The synergists were chosen from a comparative study of the odors emitted by four plant materials attractive to the APW: sugarcane, coconut, Jacaratia digitata tree and Elaeis guineensis (Oil palm). The volatiles were isolated during 6 days of sequential trappings onto Supelpak-2 adsorbent. The highly volatile fraction of sugarcane volatiles was sampled by solid-phase microextraction (SPME). Odors were analyzed and identified by gas chromatography and mass spectrometry. Electroantennogram responses to the plant odors were recorded to help in screening for bioactivity. The odor compositions between plants prior to and during fermentation were compared using a principal component analysis (PCA) to determine common odor features of the plants and to design simplified blends for field activity screening. About 100 components were identified in the >4-carbon fraction of the odors, among which 65% were fermentation volatiles. Fermentation generated a strong increase in the amount and variety of the volatiles emitted. The palm materials emitted two- to threefold greater odor amounts than the other plants. The odors from each plant were distinct according to PCA, with few common abundant components: isopentanol, 2-methylbutanol, their acetates, acetoin, isobutyl acetate, 2,3-butanediol, and 2-phenylethanol. Ethanol and ethyl acetate accounted for 80–90% in the highly volatile fraction of sugarcane odors. Coconut odor was mainly characterized by phenol, guaiacol, 1,2-dimethoxybenzene, ethyl esters of tiglic and 3,3-dimethylacrylic acids, 2-hexanone, 2-nonanone; and, to a lesser extent, by 2-heptanone, menthone, -phellandrene, ethyl octanoate and decanoate, which were also present in other plants.     

Chemical ecology of the palm weevilRhynchophorus palmarum (L.) (Coleoptera: Curculionidae): Attraction to host plants and to a male-produced aggregation pheromone

Attraction to host plants by adultRhynchophorus palmarum (L.) palm weevils was studied in the field and in the laboratory. Chemical analysis revealed the presence of ethanol and ethyl-acetate in stems of coco palms and in pineapple fruits and of pentane, hexanal, and isopentanol in coco stems. In the olfactometer, the first two compounds and isoamyl-acetate were attractive to the insects and the last three compounds, although not attractive by themselves, increased attractiveness when mixed with the first two compounds. Mixtures of these compounds, in proportions similar to the one occurring in attractive plant tissue, were as attractive as natural coconut tissue. In the field, the chemical compounds, either presented alone or as a mixture, did not attract the weevil. Males produce an aggregation pheromone when smelling ethyl-acetate. Rhynchophorol, 2(E)-6-methyl-2-hepten-4-ol, the known active component of the aggregation pheromone, attracts weevils in the olfactometer and in the field only if plant tissue, ethyl-acetate, or the above-mentioned odor mix are present. We propose that a complex mix of ethanol, ethyl-acetate, pentane, hexanal, isolamyl-acetate, and/or isopentanol serve as a short-range orientation cue to fresh wounds on the plant and that additional host odors, attracting weevils from a distance, have still to be discovered. Rhynchophorol can be considered to be a Synergist, having an anemotactic action at a distance. We recommend the use of retention traps baited with rhynchophorol, ethyl-acetate, and sugar cane as an alternative control method for the pest.     

Identification and Molecular Cloning of Putative Odorant-Binding Proteins and Chemosensory Protein from the Bethylid Wasp, <Emphasis Type="Italic">Scleroderma guani</Emphasis> Xiao et Wu

Two putative odorant-binding proteins (OBPs) and one putative chemosensory protein (CSP) from females of the ant-like bethylid wasp, Scleroderma guani Xiao et Wu (Hymenoptera: Bethylidae), were identified and cloned. The putative OBPs and CSP were identified by nondenaturing polyacrylamide gel electrophoresis (native-PAGE). 3′ rapid amplification of cDNA ends (3′RACE) was performed to obtain the sequences of the mature proteins by using degenerate primers designed from N-terminal sequences. Gene-specific primers for 5′ rapid amplification of cDNA ends (5′RACE) were designed according to 3′RACE results and used in polymerase chain reaction (PCR) to obtain full-length sequences. The proteins (Sgua-OBP1, Sgua-OBP2, and Sgua-CSP1) encode 133, 142, and 129 amino acid-deduced sequences, respectively. Prediction of signal peptide sequences matches the N-terminal amino acid sequence of the isolated proteins. Database searches suggest that the Sgua-OBP1 and Sgua-OBP2 are homologs of OBPs from other insects, and Sgua-CSP1 shares a high level of identity with previously described CSPs. Daguang Lu and Xiangrui Li contributed equally to this work     

Identification and Cloning of a Pheromone-Binding Protein from the Oriental Beetle, Exomala orientalis

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 Popillia japonica (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.     

Host Attractants for Red Weevil, Rhynchophorus ferrugineus: Identification, Electrophysiological Activity, and Laboratory Bioassay

A steam distillate from the freshly cut young bark of coconut palm Cocos nucifera was analyzed by gas chromatography, combined gas chromatography–electroantennographic detection (GC-EAD) and GC-MS to detect host attractants for the curculionid weevil Rhynchophorus ferrugineus, one of the major coconut pests in Sri Lanka. A twin FID peak consisting of a minor and a major component was shown to possess electrophysiological (EAG) activity. The minor peak was identified as -nonanoic lactone 1, while the major peak was identified as 4-hydroxy-3-methoxystyrene 2. In an EAG assay the synthetic racemic nonanoic lactone 1 did not elicit a considerable response in the antenna of R. ferrugineus, whereas the laboratory synthesized 2 showed activity. In a laboratory bioassay using a Y-type olfactometer, synthetic 1 and 2 elicited moderate attractant properties to R. ferrugineus, whereas a 1:1 mixture of the compounds showed increased attraction over that of the individual compounds.     

Male-produced aggregation pheromone of the american palm weevil,Rhynchophorus palmarum (L.) (Coleoptera,Curculionidae): Collection,identification, electrophysiogical activity,and laboratory bioassay

Male American palm weevils (APWs),Rhynchophorus palmarum (L.) produced two sex-specific compounds, which were disclosed by volatile collections on Supelpak-2 and gas chromatography. One was a minor compound, not always detected. The major male-produced volatile was identified as (2E)-6-methyl-2-hepten-4-ol through coupled gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared spectrometry, proton nuclear magnetic resonance spectrometry, and rational synthesis. We propose the trivial name rhynchophorol for this new molecule, which proved to be the essential component of the APW aggregation pheromone by electroantennography, coupled gas chromatography-electroantennography and behavioral bioassays.Research supported in part by the IRHO-CIRAD, Avenue du Val de Montferrand, 34032 Montpellier CEDEX, France.     

Aggregation pheromone of palmetto weevil,Rhynchophorus cruentatus (F.) (Coleoptera: Curculionidae)

5-Methyl-4-octanol is the major aggregation pheromone of the palmetto weevil,Rhynchophorus cruentatus (F.). The pheromone (cruentol) was identified by coupled gas chromatographic-electroantennographic (GC-EAD) analysis of male-produced volatiles, coupled GC-mass spectrometry (MS) in electron impact and chemical ionization mode, and coupled GC-high resolution MS. In laboratory and field assays, a diastereomeric mixture of synthetic cruentol greatly enhanced attraction of weevils to cabbage palmetto,Sabal palmetto (Walter), stem tissue, indicating that cruentol and host volatiles are synergistically attractive. An attractive lure in combination with efficient traps should facilitate development of semiochemical-based management forR. cruentatus.     

Ethyl propionate: Synergistic kairomone for african palm weevil,Rhynchophorus phoenicis L. (Coleoptera: Curculionidae)

Small trunk pieces of a freshly felled 10-year-old oil palm,Elaeis quineensis (Jacq.), were placed in a modified Nalgene desiccator, and volatiles captured for six days on Porapak Q. Gas chromatographic (GC) analysis of Porapak-Q-trapped volatiles with both flame ionization (FID) and electroantennographic detection (EAD) using male or femaleR. phoenicis antennae revealed several EAD-active compounds. They were identified as: ethyl acetate, ethyl propionate, isobutyl propionate, ethyl butyrate, and ethyl isobutyrate. In field experiments in the La Me Research Station, Côte d'Ivoire, ethyl propionate (50 mg/24 hr) but not all esters combined (50 mg/24 hr each) significantly increased capture ofR. phoenicis in pheromone-baited (3 mg/24 hr) traps. One kilogram of 1- to 3-day-old palm tissue was significantly more effective than ethyl propionate in enhancing pheromone attraction. Superior attraction of palm tissue may be attributed to additional as yet unknown semiochemicals. Alternatively, release rates and/or ratios of synthetic volatiles differed from those of palm tissue at peak attraction.     

Pheromone chirality of african palm weevil,Rhynchophorus phoenicis (F.) and palmetto weevil,Rhynchophorus cruentatus (F.) (Coleoptera: Curculionidae)

There are four stereoisomers of both 3-methyl-octan-4-ol, the aggregation pheromone of the African palm weevil,Rhynchophorus phoenicis (F.) and 5-methyl-octan-4-ol, the aggregation pheromone of the palmetto weevil,Rhynchophorus cruentatus (F.). Synthetic stereoisomers of 3-methyl-octan-4-ol and 5-methyl-octan-4-ol were baseline-separated on a Cyclodex-B fused silica column. Use of this column in gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometric (GC-MS) analyses revealed that only one stereoisomer, (3S,4S)-3-methyl-octan-4-ol and (4S,5S)-5-methyl-octan-4-ol, is produced by maleR. phoenicis and maleR. cruentatus, respectively, and elicits good antennal responses by conspecific male and female weevils. In field trapping experiments, withR. phoenicis in Côte d'Ivoire andR. cruentatus in Florida, (3S,4S)-3-methyl-octan-4-ol and (4S,5S)-5-methyl-octan-4-ol strongly enhanced attraction of fresh palm tissue, whereas other stereoisomers were behaviorally benign. Stereoisomeric 3-methyl-octan-4-ol and 5-methyl-octan-4-ol may be utilized to monitor and/or manage populations of these two palm weevils.     

Identification of Proteins Modulated in the Date Palm Stem Infested with Red Palm Weevil (Rhynchophorus ferrugineus Oliv.) Using Two Dimensional Differential Gel Electrophoresis and Mass Spectrometry

A state of the art proteomic methodology using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI TOF) has been employed to characterize peptides modulated in the date palm stem subsequent to infestation with red palm weevil (RPW). Our analyses revealed 32 differentially expressed peptides associated with RPW infestation in date palm stem. To identify RPW infestation associated peptides (I), artificially wounded plants (W) were used as additional control beside uninfested plants, a conventional control (C). A constant unique pattern of differential expression in infested (I), wounded (W) stem samples compared to control (C) was observed. The upregulated proteins showed relative fold intensity in order of I > W and downregulated spots trend as W > I, a quite interesting pattern. This study also reveals that artificially wounding of date palm stem affects almost the same proteins as infestation; however, relative intensity is quite lower than in infested samples both in up and downregulated spots. All 32 differentially expressed spots were subjected to MALDI-TOF analysis for their identification and we were able to match 21 proteins in the already existing databases. Relatively significant modulated expression pattern of a number of peptides in infested plants predicts the possibility of developing a quick and reliable molecular methodology for detecting plants infested with date palm.     

Proteins from Crambe abyssinica oilseed. II. Biochemical and functional properties

Little information is available concerning the properties of proteins that constitute a major component of the seed of Crambe abyssinica. Therefore, a method was developed to isolate these proteins. This procedure resulted in two fractions, an isoelectric precipitate (Fraction 1) and a retentate after ultrafiltration (Fraction 2). Biochemical and functional properties of both fractions were studied. Gel permeation chromatography revealed that high-molecular-weight proteins (>669,000 Da) are present only in Fraction 1, whereas Fraction 2 consists of proteins with lower molecular weights (<200,000 Da). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in the presence of mercaptoethanol, showed that both fractions consist of proteins that range mainly from 40,000 to less than 14,400 Da. Fraction 1 was highly soluble only at acid and alkaline pH values, while the solubility of Fraction 2 remained high (>80%) over the whole pH range tested. Addition of NaCl did not have any profound effect on the solubility of Fraction 2, but it increased significantly that of Fraction 1 in the isoelectric range. Foaming properties of Fraction 1 were better than those of chicken egg white only at pH 9, whereas those of Fraction 2 were superior at almost every pH value studied. Addition of NaCl improved significantly the foaming properties of Fraction 1 at all pH values tested but did not have a profound effect on the foaming properties of Fraction 2. Both fractions had good emulsifying properties only at alkaline pH values.     

Identification and field evaluation of the compounds comprising the sex pheromone of the female boll weevil

The terpenoid compounds (+)-cis-2-isopropenyl-1-methylcyclo-butaneethanol (I), (Z)-3,3-dimethyl-A-cyclohexaneethanol (II), and -caryophyllene were isolated from frass of the female boll weevil (Anthonomus grandis Boheman). In laboratory bioassays, a mixture of these components attracted primarily males, whereas the male pheromone, grandlure, attracted primarily females. The addition to the cotton bud hydrocarbons, -pinene, myrcene, andl-lim-nene, improved the response by males so that the potency of the mixture was comparable to that of grandlure for females. In field tests, I + II + hydrocarbons attracted both sexes, but grandlure alone and grandlure + hydrocarbons were more effective.Coleoptera: Curculionidae.In cooperation with the Mississippi Agricultural and Forestry Experiment Station, Mississippi State, Mississippi 39762.Mention of a commercial or proprietary product in this paper does not constitute endorsement of this product by USDA.     

Identification and Expression Profile Analysis of Odorant Binding Proteins in the Oriental Fruit Fly Bactrocera dorsalis

Olfaction is crucial in many insects for critical behaviors, including those regulating survival and reproduction. Insect odorant-binding proteins (OBPs) function in the first step of the olfactory system and play an essential role in the perception of odorants, such as pheromones and host chemicals. The oriental fruit fly, Bactrocera dorsalis, is a destructive fruit-eating pest, due to its wide host range of up to 250 different types of fruits and vegetables, and this fly causes severe economic damage to the fruit and vegetable industry. However, OBP genes have not been largely identified in B. dorsalis. Based on our previously constructed B. dorsalis cDNA library, ten OBP genes were identified in B. dorsalis for the first time. A phylogenetic tree was generated to show the relationships among the 10 OBPs of B. dorsalis to OBP sequences of two other Dipteran species, including Drosophila melanogaster and the mosquito Anopheles gambiae. The expression profiles of the ten OBPs in different tissues (heads, thoraxes, abdomens, legs, wings, male antennae and female antenna) of the mated adults were analyzed by real-time PCR. The results showed that nine of them are highly expressed in the antenna of both sexes, except BdorOBP7. Four OBPs (BdorOBP1, BdorOBP4, BdorOBP8, and BdorOBP10) are also enriched in the abdomen, and BdorOBP7 is specifically expressed in leg, indicating that it may function in other biological processes. This work will provide insight into the roles of OBPs in chemoreception and help develop new pest-control strategies.     

Contact Sex Pheromone Components of the Cowpea Weevil, Callosobruchus maculatus

The cowpea weevil, Callosobruchus maculatus, is a major pest of stored pulses. Females of this species produce a contact sex pheromone that elicits copulation behavior in males. Pheromone was extracted from filter-paper shelters taken from cages that housed females. Crude ether extract stimulated copulation in male C. maculatus. Initial fractionation showed behavioral activity in acidic and neutral fractions. Furthermore, bioassay-guided fractionation and gas chromatography-mass spectroscopy (GC-MS) analysis of active fractions revealed that the active components of the acidic fraction were 2,6-dimethyloctane-1,8-dioic acid and nonanedioic acid. These components along with the hydrocarbon fraction, a mixture of C₂₇–C₃₅ straight chain and methyl branched hydrocarbons, had a synergistic effect on the behavior of males. Glass dummies treated with an authentic pheromone blend induced copulation behavior in males. The potential roles of the contact sex pheromone of C. maculatus are discussed.