Electroantennogram Recordings Research Articles

Silencing sensory neuron membrane protein RferSNMPu1 impairs pheromone detection in the invasive Asian Palm Weevil

The red palm weevil (RPW), Rhynchophorus ferrugineus (Olivier), also known as the Asian palm weevil, is an invasive pest that causes widespread damage to palm trees around the globe. As pheromone communication is crucial for their mass attack and survival on palm trees, the olfactory concept of pest control strategies has been widely explored recently. We aim to understand the molecular basis of olfaction in RPW by studying one of the key olfactory proteins in insect pheromone communication, sensory neuron membrane proteins (SNMPs). SNMPs belong to the CD36 (cluster of differentiation 36) family that perform two distinct olfactory roles in insects, either in pheromone (odorant) transfer to the odorant receptors (SNMP1) or in the pheromone clearing process (SNMP2). In this study, we performed antennal transcriptomic screening and identified six SNMPs, mapping them on the R. ferrugineus genome, and confirmed four distinct SNMPs. Both SNMP1 proteins in RPW, viz., RferSNMPu1 and RferSNMPu2, were mapped onto the same scaffold in different loci in the RPW genome. To further understand the function of these proteins, we first classified them using phylogenetic analysis and checked their tissue-specific expression patterns. Further, we measured the relative transcript abundance of SNMPs in laboratory-reared, field-collected adults and pheromone-exposure experiments, ultimately identifying RferSNMPu1 as a potential candidate for functional analysis. We mapped RferSNMPu1 expression in the antennae and found that expression patterns were similar in both sexes. We used RNAi-based gene silencing to knockdown RferSNMPu1 and tested the changes in the RPW responses to aggregation pheromone compounds, 4-methyl-5-nonanol (ferrugineol) and 4-methyl-5-nonanone (ferrugineone), and a kairomone, ethyl acetate using electroantennogram (EAG) recordings. We found a significant reduction in the EAG recordings in the RferSNMPu1 knockdown strain of adult RPWs, confirming its potential role in pheromone detection. The structural modelling revealed the key domains in the RferSNMPu1 structure, which could likely be involved in pheromone detection based on the identified ectodomain tunnels. Our studies on RferSNMPu1 with a putative role in pheromone detection provide valuable insight into understanding the olfaction in R. ferrugineus as well as in other Curculionids, as SNMPs are under-explored in terms of its functional role in insect olfaction. Most importantly, RferSNMPu1 can be used as a potential target for the olfactory communication disruption in the R. ferrugineus control strategies.

Volatiles of different resistant cotton varieties mediate the host preference of Mirid bug Apolygus lucorum.

Cotton, a crucial economic crop, is also the preferred host plant of the mirid bug Apolygus lucorum. In our previous field experiments, we found that cotton cultivars Kelin 08-15 and BR-S-10 (healthy and herbivore-damaged plants) exhibit distinct attraction and repellence to A. lucorum, respectively. However, the key plant volatiles determining attraction or repulsion effects remain unknown. Here, we investigated the volatiles emitted by these two cotton cultivars before and after herbivore infestation. We found that susceptible Kelin 08-15 emitted a greater diversity and quantity of volatiles than those of BR-S-10, with herbivore-damaged cottons releasing more volatile substances. Electroantennogram (EAG) recordings further revealed that 15 representative volatiles identified above could elicited electrophysiological responses in female and male A. lucorum antennae. Among them, behavioral assays showed that two compounds, 1,3-Diethylbenzene and 4-Ethylbenzaldehyde, exhibited attractive properties, whereas six volatiles including Hexyl Acrylate, Cumene, 2,4-Dimethylstyrene, Eucalyptol, Linalool and Butyl Acrylate demonstrated repellent effects on A. lucorum. Taken together, our findings suggest the critical role of volatile compounds in mediating bug-plant interactions and provide a foundation for the development of strategies to prevent and control of A. lucorum in cotton fields.

Circabidian rhythm of sex pheromone reception in a scarab beetle

Animals have endogenous clocks that regulate their behavior and physiology. These clocks rely on environmental cues (time givers) that appear approximately every 24h due to the Earth's rotation; thus, most insects exhibit a circadian rhythm. One notable exception is the scarab beetle, Holotrichia parallela, a severe agricultural pest in China, Japan, South Korea, and India. Females emerge from the soil every other night, reach the canopy of host plants, evert an abdominal gland, and release a pheromone bouquet comprising l-isoleucine methyl ester (LIME) and l-linalool. To determine whether this circa'bi'dian rhythm affects the olfactory system, we aimed to identify H.parallela sex pheromone receptor(s) and study their expression patterns. We cloned 14 odorant receptors (ORs) and attempted de-orphanizing them in the Xenopus oocyte recording system. HparOR14 gave robust responses to LIME and smaller responses to l-linalool. Structural modeling, tissue expression profile, and RNAi treatment followed by physiological and behavioral studies support that HparOR14 is a sex pheromone receptor-the first of its kind discovered in Coleoptera. Examination of the HparOR14 transcript levels throughout the adult's life showed that on sexually active days, gene expression was significantly higher in the scotophase than in the photophase. Additionally, the HparOR14 expression profile showed a circabidian rhythm synchronized with the previously identified pattern of sex pheromone emission. 48h of electroantennogram recordings showed that responses to LIME were abolished on non-calling nights. In contrast, responses to the green leaf volatile (Z)-3-henexyl acetate remained almost constant throughout the recording period.

Attraction of Male Pine Sawflies, Diprion jingyuanensis, to Synthetic Pheromone Candidates: Synergism between Two Stereoisomers

The pine sawfly Diprion jingyuanensis Xiao and Zhang (Hymenoptera: Diprionidae) is a serious pest of Pinus tabulaeformis Carr. in the Shanxi, Gansu, and Inner Mongolia provinces in P. R. China. The sex pheromone of D. jingyuanensis was shown to be the propionate ester of 3,7-dimethyl-2-tridecanol. Virgin females contained an approximate 1:3 blend of the pheromone precursors erythro-(2S,3S,7R/S and 2R,3R,7R/S)-3,7-dimethyl-2-tridecanol and threo-(2S,3R,7R/S and 2R,3S,7R/S)-3,7-dimethyl-2-tridecanol, but the exact stereoisomers were not determined. Males responded the strongest to the propionate ester of the two threo-isomers, (2S,3R,7R) and (2S,3R,7S), in electroantennogram (EAG) recordings, followed by a significant EAG response to the (2S,3R,7R) propionate of diprionol (pheromone component of D. similis), whereas the remaining two isomers (2S,3S,7S and 2S,3S,7R) of the propionate ester of 3,7-dimethyl-2-tridecanol and the acetate of the (2S,3R,7R) isomer (one of the two pheromone components of D. pini) did not elicit any significant increase in antennal response. In the field, the strongly EAG-active (2S,3R,7R)-isomer alone was only weakly (but significantly) attractive to D. jingyuanensis males at 100 µg, while the equally EAG- active (2S,3R,7S)-isomer alone at the same loading was 8–14 times more attractive than was the (2S,3R,7R)-isomer alone. Traps baited with the same amounts of the two threo-isomers ((2S,3R,7R) and (2S,3R,7S), 100 µg + 100 µg) caught significantly more males than did traps baited with other isomers, either of the two isomers alone or other proportions of the two isomers. Thus, the (2S,3R,7S)-isomer is considered as a strong and essential sex-attractant component for D. jingyuanensis males, whereas the (2S,3R,7R)-isomer is a weak but synergistic sex-attractant. This is one of the few examples of a pine sawfly responding significantly stronger to a binary blend of stereoisomers in a synergistic fashion than to a single stereoisomer alone.

Response of Odorant Receptors with Phenylacetaldehyde and the Effects on the Behavior of the Rice Water Weevil (Lissorhoptrus oryzophilus).

The rice water weevil (RWW), Lissorhoptrus oryzophilus Kuschel (Coleoptera: Curculionidae), is a destructive rice pest that threatens the rice industry worldwide. Odorant receptors (ORs) and odorant receptor coreceptors (Orcos) play an important role in the process of insects' whole life activities; however, there are no related functional studies on RWW. On this basis, a heterologous study of LoryOR20/LoryOrco in Xenopus laevis oocytes was performed to detect the effects of certain natural compounds on RWWs and four active compounds were found. Electroantennogram (EAG) recordings and a behavior test showed that RWWs exhibited a significant response to phenylacetaldehyde (PAA) and an EAG measurement of dsRNA-LoryOR20-treated RWWs revealed a significant decrease in response to PAA. Our results revealed an olfactory molecular mechanism for the recognition of PAA by RWWs, thus providing a potential genetic target at the peripheral olfactory sensing level, contributing to the development of novel control strategies for pest management.

Development of a high-efficiency sex pheromone formula to control Euproctis pseudoconspersa

The tea tussock moth (Euproctis pseudoconspersa) is one of the most destructive chewing pests in tea plantations and causes a serious allergic reaction on the skin of tea plantation workers. The sex pheromone components of its Japanese population were first identified as 10,14-dimethylpentadecyl isobutyrate (10Me14Me-15:iBu) and 14-methylpentadecyl isobutyrate (14Me-15:iBu), with a stereogenic center. Only 10Me14Me-15:iBu has been identified in the Chinese E. pseudoconspersa population. However, field tests have shown that 10Me14Me-15:iBu cannot meet the demand of effective pest control in China. To develop a high-efficiency E. pseudoconspersa sex pheromone formula, electroantennogram (EAG) recordings of (S)- and (R)-enantiomers of 10Me14Me-15:iBu and 14Me-15:iBu were obtained in the present study. The results demonstrated that the EAG responses of male antennae to (R)-enantiomers were significantly higher than responses to the (S)-enantiomers, and 14Me-15:iBu also elicited EAG activity. Field tests showed that the catch numbers of male moths by (R)-enantiomers were significantly higher (P<0.05) than those of (S)-enantiomers. Addition of 14Me-15:iBu significantly increased the catch numbers of both the (S)- and (R)-enantiomers. The efficient pheromone formula containing 0.75 mg (R)-10Me14Me-15:iBu and 0.1 mg 14Me-15:iBu showed significantly higher attractiveness than commercial pheromone products. Our study demonstrated that (R)-10Me14Me-15:iBu was the major sex pheromone component of E. pseudoconspersa, and 14Me-15:iBu might be the minor sex pheromone component. Furthermore, a high-efficiency sex pheromone formula for E. pseudoconspersa control was defined in this study.

Sublethal Dose of β-Cypermethrin Impairs the Olfaction of Bactrocera dorsalis by Suppressing the Expression of Chemosensory Genes.

Simple SummaryThe oriental fruit fly, Bactrocera dorsalis, is a pest that causes huge economic losses in the fruit and vegetable industry. β-cypermethrin has been widely used in the orchard to control this major pest. According to a previous study, the oriental fruit fly developed significant resistance against β-cypermethrin in fields, which indicated that the oriental fruit fly has been exposed to sublethal concentrations of β-cypermethrin in the field for a long time. However, the sublethal effect and its mechanism are still unclear. In our present study, after treatment with sublethal concentrations of β-cypermethrin, the olfaction was disrupted significantly and the chemosensory genes were suppressed obviously. Our data demonstrated that the sublethal dose of β-cypermethrin impairs the olfaction of the pest insects by suppressing the expression of chemosensory genes, which provided theoretical guidance for the rational use of pesticides in fields.The oriental fruit fly, Bactrocera dorsalis, is one of the most destructive fruit insect pests. β-cypermethrin has been widely used in the orchard to control this major insect. Based on the resistance monitoring in 2011, B. dorsalis developed significant resistance against β-cypermethrin in fields. This indicated that the B. dorsalis has been exposed to sublethal concentrations of β-cypermethrin in the field for a long time. Thus, it is urgent to understand the sublethal effects of β-cypermethrin on this fly to guide the rational use of an insecticide. According to the olfactory preference assays and electroantennogram (EAG) recording, the B. dorsalis after β-cypermethrin exposure (LD30 = 10 ng/fly) severely decreased the ability to perceive the tested odorants. Moreover, we then performed quantitative real-time PCR and found the chemosensory genes including odorant receptor co-receptor (BdorORco) and ionotropic receptor co-receptors (BdorIRcos) were obviously suppressed. Our results demonstrated that the sublethal dose of β-cypermethrin impairs the olfaction of the pest insects by suppressing the expression of chemosensory genes (BdorORco and BdorIRcos), which expanded our knowledge of the sublethal effects of the pesticide on insects.

Mosquito Electroantennography.

Mosquitoes use multiple cues to locate food sources (animal and plants), mates, and oviposition sites. The sense of smell plays an important role in these behaviors, and olfactory cues are detected primarily by the appendages on the head-in particular, the antennae. Thus, mosquito olfaction can be studied by conducting electroantennogram (EAG) recordings. EAGs have emerged as a reliable technique to screen for bioactive compounds and are useful in the development of attractants and repellents for mosquito population control. Here, we focus on comparing the two main experimental approaches used in the literature (whole-body and whole-head mounting) and highlight key considerations for conducting EAGs in multiple species and for obtaining reliable and reproducible data.

Differences in olfactory sensitivity and odor detection correlate with foraging task specialization in honeybees Apis mellifera

Division of labor is central to the ecological success of social insects. Among honeybees foragers, specialization for collecting nectar or pollen correlates with their sensitivity to gustatory stimuli (e.g. sugars). We hypothesize that pollen and nectar foragers also differ in their sensitivity to odors, and therefore in their likelihood to show odor-mediated responses. To assess foragerś sensitivity to natural odors, we quantified the conditioning of the proboscis extension reflex (PER) to increasing concentrations (0.001; 0.01; 0.1; 1 M) of linalool or nonanal. Furthermore, we compared electroantennogram (EAG) recordings to correlate bees’ conditioned responses with the electrophysiological responses of their antennae. To further explore differences of the antennal response of foragers in relation to task-related odors, we registered EAG signals for two behaviorally ‘‘meaningful’’ odors that mediate pollen collection: fresh pollen odors and the brood pheromone (E)-β-ocimene. Pollen foragers performed better than nectar foragers in PER conditioning trials when linalool and nonanal were presented at low concentrations (0.001, 0.01 M). Consistently, their antennae showed stronger EAG signals (higher amplitudes) to these odors, suggesting that differences in sensitivity can be explained at the periphery of the olfactory system. Pollen and nectar foragers detect pollen odors differently, but not (E)-β-ocimene. Pollen volatiles evoked EAG signals with hyper and depolarization components. In pollen foragers, the contribution of the hyperpolarization component was higher than in nectar foragers. We discuss our findings in terms of adaptive advantages to learn subtle olfactory cues that influence the ability to better identify/discriminate food sources.

Odor-Pollution From Fungicides Disrupts Learning and Recognition of a Common Floral Scent in Bumblebees (Bombus impatiens)

Background and AimsBumblebees provide vital pollination services to both natural and agricultural ecosystems. Consequently their declines in species-diversity and population size over the last five decades is alarming. Direct contributors to these declines include pesticides, habitat loss, and disease. However, given that colony fitness is linked to foraging success, successful conservation requires mitigation of any anthropogenic practices that negatively impact foraging. Previous work has shown that agrochemical odor-pollution, including that of fungicides, can modulate bumblebee foraging behavior. This study investigates how odor pollution from three common fungicides (Safer® Brand Garden Fungicide II, Scotts® Lawn Fungus Control, and Reliant® Systemic Fungicide) affects Bombus impatiens’ floral-odor learning and recognition using an associative learning paradigm.MethodsThe effects of fungicide-odor pollution were tested in three ways: (1) background pollution during floral-odor learning; (2) background pollution during floral-odor recognition; and (3) point (localized) pollution during floral-odor recognition. Electroantennogram (EAG) recordings from B. impatiens confirmed the salience of all odor-stimuli and examined impacts of background fungicide-odor on antennal responses to floral-odor. To better understand how fungicide-odor structure related to behavioral data, scents were sampled (Solid Phase Microextraction) and analyzed using gas chromatography–mass spectrometry. Odors were then characterized using the Compounds Without Borders (CWB) vectorization method.ConclusionAll fungicides tested disrupted floral-odor learning and recognition for at least one concentration tested, and Scotts® was universally disruptive at all tested concentrations. All fungicides induced EAG responses, indicating they provide perceivable odor stimuli. Interestingly, two of three tested fungicides (Scotts® and Reliant®) inhibit antennal responses to Monarda fistulosa odor. Odor characterization supports previous findings that sulfurous scents could be disruptive to odor-driven foraging behaviors. Inability for foraging bumblebees to associate to rewarding floral odors in the presence of fungicidal odor pollution could have negative large-scale implications for colony health and reproductive fitness.

An odorant receptor of the green mirid bug, Apolygus lucorum, tuned to linalool

A highly sensitive olfactory system is required for various insect behaviors, including oviposition site selection, host location, and mate recognition. Odorant receptors (ORs) play a critical role in odorant detection. In this study, we cloned four OR genes referred to as AlucORs (AlucOR4, AlucOR39, AlucOR43, and AlucOR47) from the green mirid bug, Apolygus lucorum, and used Real-time quantitative PCR to show that expression of all four ORs was considerably biased to antennae. Functional analysis, performed using a Xenopus oocyte expression system, revealed that AlucOR47 was robustly and sensitively tuned to the important plant volatile, linalool, and its analogs, linalyl acetate and linalool tetrahydride. Electroantennogram recordings showed that all three ligands elicited obvious responses in male and female mirid bug antennae, with the response to linalool being the strongest. In behavioral assays, male and female mirid bugs displayed significant aversions to linalool. Additionally, the repellent behavior effect of A. lucorum in response to linalool disappeared after knocking down AlucOR47 by RNA interference (RNAi). Taken together, these results indicate that AlucOR47 is necessary for linalool perception in A. lucorum. Our results suggest that AlucOR47 may play a role in plant-insect interactions and provide insight into potential means of biological control against mirid bugs.

Acute ozone exposure impairs detection of floral odor, learning, and memory of honey bees, through olfactory generalization

Air pollution stemming from human activities affects the environment in which plant and animal species live and interact. Similar to primary air pollutants which are emitted, secondary air pollutants, such as tropospheric ozone (O3) formed from nitrogen oxides, are also harmful to human health and plant physiology. Yet, few reports studied the effects of O3 on pollinators' physiology, despite that this pollutant, with its high oxidative potential, likely affects pollinators behaviors, especially the perception of signals they rely on to navigate their environment. Volatile Organic Compounds (VOCs) released by plants are used as signals by different animals. For pollination services, VOCs attract different insects to the flowers and strengthen these interactions. Here, we used the honey bee Apis mellifera as a model to characterize the effects of acute exposure to different realistic mixing ratios of O3 (80-, 120-, and 200-ppb) on two crucial aspects: first, how exposed honey bees detect VOCs; and second, how O3 affects these pollinators' learning and memory processes. With electroantennogram (EAG) recordings, we showed that increasing O3 mixing ratios had a biphasic effect: an initial 25% decrease of the antennal activity when bees were tested directly after exposure (O3 direct effect), followed by a 25% increase in activity and response when bees were allowed a two-hour rest after exposure (O3 delayed effect). In parallel, during olfactory conditioning, increasing O3 mixing ratios in both exposure protocols scarcely affected olfactory learning, followed by a decrease in recall of learned odors and an increase of response to new odors, leading to a higher generalization rate (i.e., discrimination impairment). These results suggest a link between O3-related oxidative stress and olfactory coding disturbance in the honey bee brain. If ozone affects the pollinators' olfaction, foraging behaviors may be modified, in addition with a possible long-term harmful effect on pollination services.

Biosynthesis of artemisinic acid in engineered Saccharomyces cerevisiae and its attractiveness to the mirid bug Apolygus lucorum

Artemisia annua is an important preferred host of the mirid bug Apolygus lucorum in autumn. Volatiles emitted from A. annua attract A. lucorum. Volatile artemisinic acid of A. annua is a precursor of artemisinin that has been widely investigated in the Chinese herbal medicine field. However, little is known at this point about the biological roles of artemisinic acid in regulating the behavioral trends of A. lucorum. In this study, we collected volatiles from A. annua at the seedling stage by using headspace solid phase microextraction (HS-SPME). Gas chromatography-mass spectrometry (GC-MS) analysis showed that approximately 11.03±6.00 and 238.25±121.67 ng h−1 artemisinic acid were detected in volatile samples and milled samples, respectively. Subsequently, a key gene for artemisinic acid synthesis, the cytochrome P450 gene cyp71av1, was expressed in engineered Saccharomyces cerevisiae to catalyze the production of artemisinic acid. After the addition of exogenous artemisinic alcohol or artemisinic aldehyde, artemisinic acid was identified as the product of the expressed gene. In electroantennogram (EAG) recordings, 3-day-old adult A. lucorum showed significant electrophysiological responses to artemisinic alcohol, artemisinic aldehyde and artemisinic acid. Furthermore, 3-day-old female bugs were significantly attracted by artemisinic acid and artemisinic alcohol at a concentration of 10 mmol L−1, whereas 3-day-old male bugs were attracted significantly by 10 mmol L−1 artemisinic acid and artemisinic aldehyde. We propose that artemisinic acid and its precursors could be used as potential attractant components for the design of novel integrated pest management strategies to control A. lucorum.

Detection of Volatile Organic Compounds by Antennal Lamellae of a Scarab Beetle

Chemoreceptive sensilla are abundantly distributed on antennal lamellae of scarab beetles. Olfactory reception by these sensory lamellae plays a major role in feeding behaviors and sexual communication of these beetles. A new electroantennogram (EAG) recording technique is here described for evaluation of electrophysiological responses of antennal lamellae of Pseudosymmachia flavescens to sex pheromones and host plant-related compounds. EAG responses were recorded simultaneously from each lamella and the closed antennal club. All test stimuli elicited similar EAG depolarization profiles in all the three lamellae and the closed club although EAG amplitudes from the same lamella or the club varied widely among different chemical stimuli. The mid lamella tended to produce significantly greater EAG responses. EAG responses evoked by a sex pheromone component, anisole, showed a significant correlation with the density of sensilla placodea subtype 1 (SP1). However, no general patterns were obtained for correlations between the density of any test sensilla type and EAG amplitudes evoked by all the six plant volatiles. Single sensillum recordings are needed to elucidate the specific roles of these sensilla in intraspecific sexual communication and perception of host plant volatiles.

GOBP1 from the Variegated Cutworm Peridroma saucia (Hübner) (Lepidoptera: Noctuidae) Displays High Binding Affinities to the Behavioral Attractant (Z)-3-Hexenyl acetate.

Simple SummaryThe variegated cutworm Peridroma saucia (Hübner) is a polyphagous pest that originated in Europe and North America and has gradually become an important agricultural pest worldwide since the 1970s. In 2017, this pest experienced a serious outbreak in the suburbs of Luoyang, Henan Province, China. Odorant-binding proteins (OBPs) are small soluble proteins expressed in insect olfactory tissues and involved in the first step of odorant reception. Characterization of the function of P. saucia OBPs may contribute to identifying new attractants/repellents and assist in the development of more efficient and environmentally acceptable controlling strategies of this pest. In this study, we cloned an antennae-specific gene PsauGOBP1 which encodes for a general odorant-binding protein (GOBP) of P. saucia. We expressed PsauGOBP1 in bacteria and found that the purified recombinant PsauGOBP1 binds robustly to (Z)-3-hexenyl acetate. Electroantennogram tests demonstrate that (Z)-3-hexenyl acetate elicits strong responses from the antennae of both males and females of P. saucia. Y-tube olfactometer bioassays show that (Z)-3-hexenyl acetate is attractive to adult P. saucia of both sexes. This study increases our understanding of the olfactory mechanism of P. saucia and provides a theoretical basis for the design of attractants for the control of P. saucia.The variegated cutworm Peridroma saucia (Hübner) is a worldwide pest that causes serious damage to many crops. To recognize sex pheromones and host plant volatiles, insects depend on olfactory chemoreception involving general odorant-binding proteins (GOBPs). In this study, PsauGOBP1 was cloned from the adult antennae of P. saucia. RT-qPCR and Western-blot analysis showed that PsauGOBP1 was specifically and equally expressed in the adult antennae of both females and males. Fluorescence competitive-binding assays with sex pheromones and host plant volatiles demonstrated that PsauGOBP1 bound to six host plant volatiles: (Z)-3-hexenyl acetate (KD = 4.0 ± 0.1 μM), citral (KD = 5.6 ± 0.4 μM), farnesol (KD = 6.4 ± 0.6 μM), nonanal (KD = 6.8 ± 0.3 μM), (Z)-3-hexen-1-ol (KD = 8.5 ± 0.6 μM), and benzaldehyde (KD = 9.4 ± 0.5 μM). Electroantennogram recordings with the six host plant volatiles indicated that (Z)-3-hexenyl acetate elicited the strongest responses from both male and female antennae. Further bioassays using Y-tube olfactometers showed that (Z)-3-hexenyl acetate was attractive to adult P. saucia of both sexes. These results suggest that PsauGOBP1 might be involved in detecting host plant volatiles and that (Z)-3-hexenyl acetate might serve as a potential attractant for the biological control of P. saucia.

Behavioral and Antennal Responses of Tribolium confusum to Varronia globosa Essential Oil and Its Main Constituents: Perspective for Their Use as Repellent.

Essential oils of aromatic plants represent an alternative to classical pest control with synthetic chemicals. They are especially promising for the alternative control of stored product pest insects. Here, we tested behavioral and electrophysiological responses of the stored product pest Tribolium confusum, to the essential oil of a Brazilian indigenous plant, Varronia globosa, collected in the Caatinga ecosystem. We analyzed the essential oil by GC-MS, tested the effects of the entire oil and its major components on the behavior of individual beetles in a four-way olfactometer, and investigated responses to these stimuli in electroantennogram recordings (EAG). We could identify 25 constituents in the essential oil of V. globosa, with anethole, caryophyllene and spathulenole as main components. The oil and its main component anethole had repellent effects already at low doses, whereas caryophyllene had only a repellent effect at a high dose. In addition, the essential oil abolished the attractive effect of the T. confusum aggregation pheromone. EAG recordings revealed dose-dependent responses to the individual components and increasing responses to the blend and even more to the entire oil. Our study reveals the potential of anethole and the essential oil of V. globosa in the management of stored product pests.

Functional Characterization of Sex Pheromone Neurons and Receptors in the Armyworm, Mythimna separata (Walker).

Pheromone receptors (PRs) of moths are expressed on the dendritic membrane of odorant receptor neurons (ORNs) housed in the long trichoid sensilla (TS) of antennae and are essential to sex pheromone reception. The function of peripheral neurons of Mythimna separata in recognizing sex pheromones is still unclear. In this study, electroantennogram recordings were performed from male and female antennae of M. separata, and showed that the major component of sex pheromones, (Z)-11-hexadecenal (Z11–16:Ald), evoked the strongest response of male antennae with significant differences between sexes. Single sensillum recording was used to record responses of neurons housed in TS of male M. separata. The results revealed four types of TS with three neurons housed in each type, based on profiles of responses to sex pheromone components and pheromone analogs. ORN-B of type-I TS was specifically tuned to the major sex pheromone component Z11–16:Ald; ORN-Bs in type-III and type-IV TSs were, respectively, activated by minor components (Z)-11-hexadecen-1-yl acetate (Z11–16:OAc) and hexadecenal (16:Ald); and ORNs in type-II TS were mainly activated by the sex pheromone analogs. We further cloned full-length sequences of six putative PR genes and an Orco gene. Functional characterization of PRs in the Xenopus oocyte system demonstrated that male antennae-biased MsepPR1 responded strongly to (Z)-9-tetradecenal (Z9-14:Ald), suggesting that MsepPR1 may be expressed in type-II TS. MsepPR6 was exclusively tuned to (Z)-9-tetradecen-1-yl acetate (Z9–14:OAc). MsepPR2 and MsepPR4 showed no responses to any tested components. Female antennae-biased MespPR5 was broadly tuned to Z9–14:Ald, Z9–14:OAc, Z11–16:Ald, and (Z)-11-hexadecen-1-ol (Z11–16:OH). Our results further enriched the sex pheromone recognition mechanism in the peripheral nervous system of moth M. separata.

Pyrethrins elicit olfactory response and spatial repellency in Aedes albopictus.

Pyrethrum from dry flowers of Chrysanthemum is a well-known botanical insecticide and repellent. Its insecticidal activity attributes to its six insecticidal esters, collectively known as pyrethrins. Pyrethrins and its synthetic analogs pyrethroids exert their toxic action by modifying the function of voltage-gated sodium channels. Aside from insecticidal activity, pyrethrum has also been used to repel mosquitoes for centuries. Today, pyrethrum continues to be used as an active ingredient in mosquito coils and other mosquito-repellent devices globally. However, the mechanism of pyrethrum repellency remains largely unknown. Here we report that pyrethrum vapor induced spatial (non-contact) repellency in Aedes albopictus, a major vector of dengue and West Nile viruses. Using electroantennogram (EAG) recordings from adult antennae, we found that pyrethrum elicited EAG response in a dose-dependent manner. We then isolated the six insecticidal esters, pyrethrins I and II, cinerins I and II, jasmolins I and II from pyrethrum extract and discovered that five of the six esters, except jasmolin I, all elicited EAG responses. Furthermore, pyrethrins I and II, cinerin II and jasmolin II induced repellency, whereas cinerin I and jasmolin I did not. Of the six pyrethrins, four of them, pyrethrins I and II, cinerin II and jasmolin II, activate olfactory-receptor neurons and elicit spatial repellency in Ae. albopictus. Our study provided a foundation for future structure-function studies of pyrethrins, their cognate olfactory receptors and efficacies of repellency and for the development of new and more effective mosquito repellents for controlling vector-borne human diseases. © 2021 Society of Chemical Industry.

Comparative dissection of the peripheral olfactory system of the Chagas disease vectors Rhodnius prolixus and Rhodnius brethesi.

American trypanosomiasis, or Chagas disease, is transmitted by both domestic and sylvatic species of Triatominae which use sensory cues to locate their vertebrate hosts. Among them, odorants have been shown to play a key role. Previous work revealed morphological differences in the sensory apparatus of different species of Triatomines, but to date a comparative functional study of the olfactory system is lacking. After examining the antennal sensilla with scanning electronic microscopy (SEM), we compared olfactory responses of Rhodnius prolixus and the sylvatic Rhodnius brethesi using an electrophysiological approach. In electroantennogram (EAG) recordings, we first showed that the antenna of R. prolixus is highly responsive to carboxylic acids, compounds found in their habitat and the headspace of their vertebrate hosts. We then compared responses from olfactory sensory neurons (OSNs) housed in the grooved peg sensilla of both species, as these are tuned to these compounds using single-sensillum recordings (SSRs). In R. prolixus, the SSR responses revealed a narrower tuning breath than its sylvatic sibling, with the latter showing responses to a broader range of chemical classes. Additionally, we observed significant differences between these two species in their response to particular volatiles, such as amyl acetate and butyryl chloride. In summary, the closely related, but ecologically differentiated R. prolixus and R. brethesi display distinct differences in their olfactory functions. Considering the ongoing rapid destruction of the natural habitat of sylvatic species and the likely shift towards environments shaped by humans, we expect that our results will contribute to the design of efficient vector control strategies in the future.

An odorant-binding protein of Asian citrus psyllid, Diaphorina citri, participates in the response of host plant volatiles.

Odorant-binding proteins (OBPs) in insects contribute to the sensitivity of the olfactory system and connect external odorants to olfactory receptor neurons. Determination of the chemosensory functions in Diaphorina citri, a vector of the citrus Huanglongbing pathogen, may help in developing a potential target for pest management. Diaphorina citri showed dose-dependent electroantennogram recording (EAG) responses to 12 host plant volatiles. A two-choice behavioral trap experiment showed that four compounds (methyl salicylate, linalool, citral and R-(+)-limonene) that elicited high EAG responses also had significant attraction to adults. The expression profiles induced by these compounds were detected in nine OBP genes, DcitOBP1-9. DcitOBP3, DcitOBP6 and DcitOBP7 commonly showed significant upregulation or downregulation compared with the control. Microscale thermophoresis (MST) showed that the recombinant protein DcitOBP7 had high in vitro binding affinities (Kd < 10μm) to methyl salicylate, linalool and R-(+)-limonene, and moderate binding affinity to citral with a Kd value of 15.95 μm. Furthermore, RNA interference (RNAi)-suppressed messenger RNA (mRNA) expression of DcitOBP7 resulted in a significant reduction in EAG activity and in adult D. citri behavioral responses to tested volatiles and the preferred host, Murraya paniculata. The hydrophilic residue Arg107 of DcitOBP7 may have a key role in binding odorants via formation of hydrogen bonds. These results show that DcitOBP7 plays an important role in the olfactory response. This finding may provide new insight into the functions of OBP families in D. citri and aid in the development of safe strategies for managing D. citri populations. © 2021 Society of Chemical Industry.