AN ODORANT‐BINDING PROTEIN INVOLVED IN PERCEPTION OF HOST PLANT ODORANTS IN LOCUST Locusta migratoria

Locusts, Locusta migratoria (Orthoptera: Acrididae), are extremely destructive agricultural pests, but very little is known of their molecular aspects of perception to host plant odorants including related odorant‐binding proteins (OBPs), though several OBPs have been identified in locust. To elucidate the function of LmigOBP1, the first OBP identified from locust, RNA interference was employed in this study to silence LmigOBP1, which was achieved by injection of dsRNA targeting LmigOBP1 into the hemolymph of male nymphs. Compared with LmigOBP1 normal nymphs, LmigOBP1 knockdown nymphs significantly decreased food (maize leaf, Zea mays) consumption and electro‐antennography responses to five maize leaf volatiles, ((Z)‐3‐hexenol, linalool, nonanal, decanal, and (Z)‐3‐hexenyl acetate). These suggest that LmigOBP1 is involved in perception of host plant odorants.     

Composition and emission dynamics of migratory locust volatiles in response to changes in developmental stages and population density

Chemical communication plays an important role in density‐dependent phase change in locusts. However, the volatile components and emission patterns of the migratory locust, Locusta migratoria, are largely unknown. In this study, we identified the chemical compositions and emission dynamics of locust volatiles from the body and feces and associated them with developmental stages, sexes and phase changes. The migratory locust shares a number of volatile components with the desert locust (Schistocerca gregaria), but the emission dynamics of the two locust species are significantly different. The body odors of the gregarious nymphs in the migratory locust consisted of phenylacetonitrile (PAN), benzaldehyde, guaiacol, phenol, aliphatic acids and 2,3‐butanediol, and PAN was the dominant volatile. Volatiles from the fecal pellets of the nymphs primarily consist of guaiacol and phenol. Principal component analysis (PCA) showed significant differences in the volatile profiles between gregarious and solitary locusts. PAN and 4‐vinylanisole concentrations were significantly higher in gregarious individuals than in solitary locusts. Gregarious mature males released significantly higher amounts of PAN and 4‐vinylanisole during adulthood than mature females and immature adults of both sexes. Furthermore, PAN and 4‐vinylanisole were completely lost in gregarious nymphs during the solitarization process, but were obtained by solitary nymphs during gregarization. The amounts of benzaldehyde, guaiacol and phenol only unidirectionally decreased from solitary to crowded treatment. Aliphatic aldehydes (C7 to C10), which were previously reported as locust volatiles, are now identified as environmental contaminants. Therefore, our results illustrate the precise odor profiles of migratory locusts during developmental stages, sexes and phase change. However, the function and role of PAN and other aromatic compounds during phase transition need further investigation.     

Identification of Components of the Oviposition Aggregation Pheromone of the Gregarious Desert Locust,Schistocerca gregaria (Forskal)

Acetophenone and veratrole have been identified as two major behaviourally active components of the oviposition aggregation pheromone of the desert locust, Schistocerca gregaria. These compounds were identified from the volatiles of egg pod froth using gas chromatography-electroantennographic (GC-EAD) and gas chromatography-mass spectrometric (GC-MS) analysis. Both compounds were shown to elicit aggregation of gravid females in oviposition bioassays; however, they do not act synergistically. Both acetophenone and veratrole individually at optimum doses induced about 70% egg laying, a value similar to that evoked with froth volatiles.     

Fecal volatile components elicit aggregation in the oriental migratory locust,Locusta migratoria manilensis (Orthoptera: Acrididae)

Abstract The aggregation components from fecal volatiles of the oriental migratory locust, Locusta migratoria manilensis were identified with gas chromatographic/electroantennographic detector (GC‐EAD), GC‐MS (mass spectrometry) analyses and behavioral bioassays. Both last instar nymphs and adults of the oriental migratory locust have similar aggregation pheromones in their volatiles. A total of 11 electrophysiologically active compounds, namely, hexanal, cyclohexanol, heptanal, phenol, 2,5‐dimethyl‐pyrazine, benzyl alcohol, benzaldehyde, guaiacol, nonanal, 2,6,6‐trimethyl‐2‐cyclohexene‐1,4‐dione and decyl aldehyde were identified in the fecal volatiles of 2‐day‐old immature adult male locusts. Only hexanal, nonanal, benzaldehyde, cyclohexanol and 2,5‐dimethyl‐pyrazine elicited significant aggregation responses in immature 2‐day‐old adult males. However, adult males had significantly lower behavioral responses to synthetics of five single compounds than the blend of cyclohexanol, 2,5‐dimethyl‐pyrazine, benzaldehyde, nonanal, hexanal in ratios of 100: 100: 2: 60: 30 in the range of 30–60 μg/mL. We propose that it is the blend of these five compounds that plays a key role in eliciting and sustaining aggregation in gregarious oriental migratory locusts. These results also showed that the aggregation pheromones of the oriental migratory locust are significantly different from those found in the desert locust.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

Intriguing similarities between two novel odorant-binding proteins of locusts

Two novel odorant-binding proteins (OBPs) of locust, LmigOBP2 and LmigOBP3 are very different from each other and from the previously reported LmigOBP1 in their amino acid sequences. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, these two proteins exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for LmigOBP2 and LmigOBP3, but clearly different for LmigOBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in LmigOBP2 and LmigOBP3, but not in LmigOBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected LmigOBP3 in any of these tissues. In immunocytochemistry, antibodies against LmigOBP2 strongly stained the outer lymph of sensilla chaetica of the antennae, in contrast with LmigOBP1, previously reported in sensilla basiconica.     

A subset of chemosensory genes differs between two populations of a specialized leaf beetle after host plant shift

Due to its fundamental role in shaping host selection behavior, we have analyzed the chemosensory repertoire of Chrysomela lapponica. This specialized leaf beetle evolved distinct populations which shifted from the ancestral host plant, willow (Salix sp., Salicaceae), to birch (Betula rotundifolia, Betulaceae). We identified 114 chemosensory candidate genes in adult C. lapponica: 41 olfactory receptors (ORs), eight gustatory receptors, 17 ionotropic receptors, four sensory neuron membrane proteins, 32 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSP) by RNA‐seq. Differential expression analyses in the antennae revealed significant upregulation of one minus‐C OBP (ClapOBP27) and one CSP (ClapCSP12) in the willow feeders. In contrast, one OR (ClapOR17), four minus‐C OBPs (ClapOBP02, 07, 13, 20), and one plus‐C OBP (ClapOBP32) were significantly upregulated in birch feeders. The differential expression pattern in the legs was more complex. To narrow down putative ligands acting as cues for host discrimination, the relative abundance and diversity of volatiles of the two host plant species were analyzed. In addition to salicylaldehyde (willow‐specific), both plant species differed mainly in their emission rate of terpenoids such as (E,E)‐α‐farnesene (high in willow) or 4,8‐dimethylnona‐1,3,7‐triene (high in birch). Qualitatively, the volatiles were similar between willow and birch leaves constituting an “olfactory bridge” for the beetles. Subsequent structural modeling of the three most differentially expressed OBPs and docking studies using 22 host volatiles indicated that ligands bind with varying affinity. We suggest that the evolution of particularly minus‐C OBPs and ORs in C. lapponica facilitated its host plant shift via chemosensation of the phytochemicals from birch as novel host plant.     

Volatile compounds of Soumbala, a fermented African locust bean (Parkia biglobosa) food condiment

AIMS: To determine the profile of volatile compounds responsible for the aroma of Soumbala produced spontaneously and with pure and mixed cultures of Bacillus subtilis and Bacillus pumilus. METHODS AND RESULTS: Traditional and controlled fermentation trials of African locust bean with pure and mixed starter cultures of B. subtilis (B7, B9 and B15) and B. pumilus (B10) were performed. Aroma volatiles were analysed using Likens-Nikerson method coupled with gas chromatography and mass spectrophotometry. Sensory analysis of Soumbala as well as rice dishes prepared with each type of Soumbala were carried out by 10 panellists. In total 116 compounds were identified. They included pyrazines, aldehydes, ketones, esters, alcohols, acids, alkanes, alkenes, amines, pyridines, benzenes, phenols, sulphurs, furans and other compounds. Using principal component analysis for comparison, the aroma profiles of the Soumbala samples could be separated into three groups. The sensory evaluation showed variable acceptability. However, it was noticed that Soumbala samples produced with starter cultures were scored higher than traditionally prepared Soumbala. CONCLUSIONS: Aroma volatiles and organoleptic properties of Soumbala vary according to the Bacillus isolates involved in the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus starter cultures for controlled production of Soumbala.     

假眼小绿叶蝉对茶梢挥发物的行为反应

【目的】研究假眼小绿叶蝉Empoasca vitis G?the对健康茶梢和蝉害茶梢挥发物的行为反应,筛选出可引诱假眼小绿叶蝉的茶梢挥发物。【方法】以动态吸附法收集健康茶梢和蝉害茶梢挥发物,用气相色谱–质谱联用仪(GC-MS)进行定性定量分析,借助于Y形嗅觉仪检测多种挥发物引诱假眼小绿叶蝉成虫的活性。【结果】从健康茶梢和蝉害茶梢中共鉴定出30种挥发物组分,其中烯烃类含量较大。健康茶梢和蝉害茶梢挥发物中共有组分有13种,蝉害之后其含量皆上升,其中Z-b-罗勒烯和乙酸叶醇酯的含量分别是健康茶梢中的142.27倍、12.90倍。蝉害茶梢中新出现的组分有12种,其中紫苏烯含量较高。在10-2、10~(-4)、10-6 g/mL浓度下,乙酸叶醇酯表现出极显著的引诱水平(P<0.01);紫苏烯在10~(-2)和10~(-4) g/mL浓度下表现出极显著引诱水平(P<0.01);10~(-4) g/mL浓度下,Z-b-罗勒烯和D-柠檬烯极显著引诱假眼小绿叶蝉(P<0.01);10~(-4) g/mL芳樟醇、10~(-6) g/mL乙酸正丁酯和10~(-6) g/mL D-柠檬烯呈现出显著引诱水平(P<0.05);混合物组分Blend1和Blend2分别表现出极显著和显著引诱水平;而10~(-6) g/mLa-法尼烯显著排斥假眼小绿叶蝉(P<0.05)。【结论】假眼小绿叶蝉成虫对健康茶梢和蝉害茶梢挥发物多种组分具有不同的行为反应,引诱效果较强的单组分或混合组分的选定可为田间引诱效果试验提供参考。     

Molecular and functional characterization of three odorant‐binding proteins from the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana, a periodic but devastating wheat pest, relies on wheat spike volatiles as a cue in selecting hosts for oviposition. Insect odorant‐binding proteins (OBPs) are thought to play essential roles in filtering, binding and transporting hydrophobic odorant molecules to specific receptors. To date, the molecular mechanisms underlying S. mosellana olfaction are poorly understood. Here, three S. mosellana antenna‐specific OBP genes, SmosOBP11, 16 and 21, were cloned and bacterially expressed. Binding properties of the recombinant proteins to 28 volatiles emitted from wheat spikes were investigated using fluorescence competitive binding assays. Sequence analysis suggested that these SmosOBPs belong to the Classic OBP subfamily. Ligand‐binding analysis showed that all three SmosOBPs preferentially bound alcohol, ester and ketone compounds, and SmosOBP11 and 16 also selectively bound terpenoid compounds. In particular, the three SmosOBPs had high binding affinities (K_i < 20 μmol/L) to 3‐hexanol and cis‐3‐hexenylacetate that elicited strong electroantennogram (EAG) response from female antennae. In addition, SmosOBP11 displayed significantly higher binding (K_i < 8 μmol/L) than SmosOBP16 and 21 to 1‐octen‐3‐ol, D‐panthenol, α‐pinene and heptyl acetate which elicited significant EAG response, suggesting that SmosOBP11 plays a major role in recognition and transportation of these volatiles. These findings have provided important insight into the molecular mechanism by which S. mosellana specifically recognizes plant volatiles for host selection, and have facilitated identification of effective volatile attractants that are potentially useful for pest monitoring and trapping.     

Odorants of different chemical classes interact with distinct odorant binding protein subtypes.

The ligand profile for three odorant binding proteins (OBPs) of the rat have been determined using a large number of odorous compounds from different chemical classes. To evaluate the binding spectra of distinct subtypes, all OBPs were produces in Escherichia coli as recombinant His-tagged fusion proteins. The individual binding properties of each OBP subtype were analysed using a large array of organic compounds, representing derivatives of aliphatic and aromatic compounds, as well as terpenes, pyrazines and thiazoles, in a competitive spectroscopic binding assay with various fluorescence chromophores as the specific interacting partner for the OBPs. Most of the compounds were identified to interact only with one OBP subtype. But interestingly, a small change, for example in the 2-methyl or 2-ethoxy side chain in the pyrazine and thiazole derivatives to a 2-isobutyl group, caused overlapping binding affinities to rat-OBP1 and rat-OBP3. However, the data strongly support the notion that each OBP subtype displays a characteristic ligand binding profile and interacts with a different subset of exogenous organic compounds in a micromolar range.     

A Multi-species Bait for Chagas Disease Vectors

Background

Triatomine bugs are the insect vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. These insects are known to aggregate inside shelters during daylight hours and it has been demonstrated that within shelters, the aggregation is induced by volatiles emitted from bug feces. These signals promote inter-species aggregation among most species studied, but the chemical composition is unknown.

Methodology/Principal Findings

In the present work, feces from larvae of the three species were obtained and volatile compounds were identified by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). We identified five compounds, all present in feces of all of the three species: Triatoma infestans, Panstrongylus megistus and Triatoma brasiliensis. These substances were tested for attractivity and ability to recruit insects into shelters. Behaviorally active doses of the five substances were obtained for all three triatomine species. The bugs were significantly attracted to shelters baited with blends of 160 ng or 1.6 µg of each substance.

Conclusions/Significance

Common compounds were found in the feces of vectors of Chagas disease that actively recruited insects into shelters, which suggests that this blend of compounds could be used for the development of baits for early detection of reinfestation with triatomine bugs.     

Induced volatiles in elicitor-treated and rice blast fungus-inoculated rice leaves

The volatiles released from elicitor (copper chloride, jasmonic acid, UV, L-methionine and chitosan oligomer)-treated and rice blast fungus-inoculated rice leaves were collected by the solid-phase microextraction technique and analyzed by GC-MS. (Z)-3-Hexen-1-ol, monoterpenes, methyl salicylate, and sesquiterpenes were identified as elicitor-induced volatiles by a comparison of their GC retention times and mass spectra with those of authentic compounds. The different elicitors resulted in some qualitative and quantitative differences in the production of volatiles. Monoterpenes and sesquiterpenes were identified as the rice blast fungus-induced volatiles.     

Binding specificity of locust odorant binding protein and its key binding site for initial recognition of alcohols

Odorant binding proteins (OBPs) are required for olfaction perception, and thus may be possible targets for controlling the population of pests by interfering with their chemical communication. A single OBP LmigOBP1 has been identified in the antennae of Locusta migratoria, though four isoforms have been detected. Here, we have investigated the ligand-binding specificity of LmigOBP1 using 67 volatile odor compounds. Fluorescence assays indicate that LmigOBP1 does not bind fecal volatiles or green leaf odors, but shows high affinity for some linear aliphatic compounds, with pentadecanol and 2-pentadecanone being the strongest binding ligands. A 3-dimensional (3D) model of LmigOBP1 was built by homology modeling. Docking simulations based on this model suggested that Asn74 of LmigOBP1 is a key binding site, and this was validated by site-directed mutagenesis and fluorescence assays. We suggest that, as a general rule, a hydrophilic amino acid at the entrance of the binding cavity participates in initial recognition of ligands, and contributes to ligand-binding specificity of OBPs.