The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects

Odor experiences during preimaginal stages cause behavioral and neural plasticity in adult honeybees

In eusocial insects, experiences acquired during the development have long-term consequences on mature behavior. In the honeybee that suffers profound changes associated with metamorphosis, the effect of odor experiences at larval instars on the subsequent physiological and behavioral response is still unclear. To address the impact of preimaginal experiences on the adult honeybee, colonies containing larvae were fed scented food. The effect of the preimaginal experiences with the food odor was assessed in learning performance, memory retention and generalization in 3–5- and 17–19 day-old bees, in the regulation of their expression of synaptic-related genes and in the perception and morphology of their antennae. Three-five day old bees that experienced 1-hexanol (1-HEX) as food scent responded more to the presentation of the odor during the 1-HEX conditioning than control bees (i.e., bees reared in colonies fed unscented food). Higher levels of proboscis extension response (PER) to 1-HEX in this group also extended to HEXA, the most perceptually similar odor to the experienced one that we tested. These results were not observed for the group tested at older ages. In the brain of young adults, larval experiences triggered similar levels of neurexins (NRXs) and neuroligins (Nlgs) expression, two proteins that have been involved in synaptic formation after associative learning. At the sensory periphery, the experience did not alter the number of the olfactory sensilla placoidea, but did reduce the electrical response of the antennae to the experienced and novel odor. Our study provides a new insight into the effects of preimaginal experiences in the honeybee and the mechanisms underlying olfactory plasticity at larval stage of holometabolous insects.Fil: Ramirez, Gabriela Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Fagundez, Carol Betiana. Instituto Universitario del Hospital Italiano; ArgentinaFil: Grosso, Juan Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Argibay, Pablo Francisco. Hospital Italiano. Instituto de Ciencias Básicas y Medicina Experimental; ArgentinaFil: Arenas, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Farina, Walter Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Elemental and configural olfactory coding by antennal lobe neurons of the honeybee (Apis mellifera)

When smelling an odorant mixture, olfactory systems can be analytical (i.e. extract information about the mixture elements) or synthetic (i.e. creating a configural percept of the mixture). Here, we studied elemental and configural mixture coding in olfactory neurons of the honeybee antennal lobe, local neurons in particular. We conducted intracellular recordings and stimulated with monomolecular odorants and their coherent or incoherent binary mixtures to reproduce a temporally dynamic environment. We found that about half of the neurons responded as ‘elemental neurons’, i.e. responses evoked by mixtures reflected the underlying feature information from one of the components. The other half responded as ‘configural neurons’, i.e. responses to mixtures were clearly different from responses to their single components. Elemental neurons divided in late responders (above 60 ms) and early responder neurons (below 60 ms), whereas responses of configural coding neurons concentrated in-between these divisions. Latencies of neurons with configural responses express a tendency to be faster for coherent stimuli which implies employment in different processing circuits

Hyperbolic odorant mixtures as a basis for more efficient signaling between flowering plants and bees

Animals use odors in many natural contexts, for example, for finding mates or food, or signaling danger. Most analyses of natural odors search for either the most meaningful components of a natural odor mixture, or they use linear metrics to analyze the mixture compositions. However, we have recently shown that the physical space for complex mixtures is ‘hyperbolic’, meaning that there are certain combinations of variables that have a disproportionately large impact on perception and that these variables have specific interpretations in terms of metabolic processes taking place inside the flower and fruit that produce the odors. Here we show that the statistics of odorants and odorant mixtures produced by inflorescences (Brassica rapa) are also better described with a hyperbolic rather than a linear metric, and that combinations of odorants in the hyperbolic space are better predictors of the nectar and pollen resources sought by bee pollinators than the standard Euclidian combinations. We also show that honey bee and bumble bee antennae can detect most components of the B. rapa odor space that we tested, and the strength of responses correlates with positions of odorants in the hyperbolic space. In sum, a hyperbolic representation can be used to guide investigation of how information is represented at different levels of processing in the CNS

Molecular and Electrophysiological Characterization of Olfactory and Gustatory Perception in Honeybee and Cockroach for Application as Biosensor

학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부(바이오모듈레이션전공), 2015. 8. 안용준.Chemoreception is an essential sensory modality for the survival and reproduction of many animals, especially insects. Food sources, mate and oviposition sites are located and evaluated through the use of the chemosensory apparatus. Despite growing knowledge of insect olfactory and gustatory detection and processing very little is known about the modulation and receptor function of the chemosensory system. This objective of this thesis was to explore olfactory modulation of the peripheral olfactory system in the American cockroach, Periplaneta americana and to compare olfactory sensitivity between Asian honeybee, Apis cerana and European honeybee, Apis mellifera. In addition, this research concentrated on 2 determining the natural ligands such as sugar and amino acids of gustatory receptors of honeybee, Apis mellifera, in order to understand the gustatory world of insect. Finally, we developed a bioelectronics tongue using honeybee taste receptor for the detection of umami taste compounds with human like performance. Through the use of morphological, behavioral, electrophysiological, pharmacological, neuroanatomical, and biophysical techniques we have begun to elucidate the modulatory effects of internal factors, neuromodulators, on insect chemoreception. Also, we demonstrated functional role and characterization of gustatory receptors related to sweet and amino acids compounds in the honeybee, Apis mellifera. These studies provide a foundation for understanding the molecular and cellular basis of olfactory and gustatory coding and behavior.ABSTRACT··············································································1 CONTENTS··············································································3 LIST OF TABLES·····································································9 LIST OF FIGURES··································································10 INTRODUCTION····································································13 CHAPTER I Neuromodulation of olfactory sensitivity in the peripheral olfactory organs of the American cockroach, Periplaneta americana Abstract·················································································24 Introduction···········································································26 Materials and Methods···························································29 1. Insect and tissue preparation·····················································29 2. Gene Cloning of octopamine receptor, tachykinin, and tachykinin 4 receptor··············································································29 3. Quantitative real-time PCR·······················································30 4. In situ Hybridization, immunostaining, and imaging·························31 5. Injection of tachykinins, dsRNAs, and octopamine agonist and antagonist···········································································32 6. Electroantennogram·······························································32 7. Single sensillum recording························································33 8. Data analysis········································································33 Results 1. Cloning of octopamine receptor, tachykinin, and tachykinin receptor of P. Americana···········································································35 2. Localization of PaOA1, PaTK, and PaTKR genes in antennae···············35 3. Effects of tachykinin (TK) on olfactory sensitivity in antennae ·············36 4. Alternation of olfactory sensitivity by dsRNA·································37 Discussion··············································································39 CHAPTER II Structural and functional differences in the antennal olfactory system of worker honey bees of Apis mellifera and Apis cerana Abstract···················································································59 Introduction·············································································60 Materials and Methods·····························································63 5 1. Insect preparation··································································63 2. Observation of antennal sensilla using SEM···································63 3. Surface areas and densities·······················································64 4. Electroantennogram·······························································65 5. Western blot·········································································66 6. Data analysis········································································67 Results 1. Differences of Sensilla Number and Density in Antennae ···················69 2. Number of Olfactory Sensilla in Antenna Segment····························69 3. Olfactory Responses Measured by Electroantennogram (EAG) ············71 4. Protein Expression of Orco·······················································71 Discussion ·············································································73 Chapter III Identification and characterization of sugar receptors and amino acids receptor in the western honey bee, Apis mellifera Abstract···················································································88 Introduction·············································································89 Materials and Methods 1. Insect and tissue preparation·····················································93 2. Scanning Electron Microscopy (SEM) ·········································93 6 3. RNA Isolation and cDNA synthesis·············································94 4. Quantitative real-time PCR (qRT-PCR) ········································95 5. Gene cloning of gustatory receptors 1, 2, and 10···························95 6. In situ hybridization, immunostaining, and imaging ······················96 7. Receptor expression in Xenopus oocytes and two-electrode voltage-clamp electrophysiological recordings ············································98 8. Tip recordings of antennal sensilla··········································99 9. Phylogenetic analysis························································100 10. Immunofluorescence analysis··················································101 11. Intracellular Calcium assay·····················································101 12. Calcium imaging·································································102 13. Scanning electron microscopy··················································102 14. Data analysis··································································103 Result 1. Differences of number of sensillum chaetica per antennae segments··········································································104 2. Responses of contact chemoreceptors to sugars······························104 3. AmGr1 and AmGr2 are highly expressed in the distal segment of antennae···········································································105 4. AmGr1 is a specific receptor for sugar compounds·························106 5. Fructose is a specific ligand for an AmGr3··································107 6. Localization of sugar receptors AmGr1 and AmGr2 in 7 antennae···········································································107 7. AmGr10 is ubiquitous expressed in external and internal organs of honeybee···········································································108 8. L-amino acids are ligands for an A. mellifera gustatory receptor, AmGr10············································································108 9. IMP can strongly potentiate the umami taste intensity in AmGr10············································································110 Discussion·············································································111 Chapter IV Discrimination of Umami Substances using Floating Electrode Sensor mimicking Insect Taste Systems Abstract·················································································133 Introduction···········································································134 Materials and Methods 1. Materials and Insect preparation················································138 2. HEK-293 cell culture····························································138 3. RNA extraction, cDNA synthesis, RT-PCR.··································138 4. Heterologous expression of AmGr10 into HEK-293 cells··················139 5. Construction of nanovesicles from HEK-293 cell expressing honeybee umami receptor ···································································140 6. Western blot·······································································140 8 7. Intracellular Calcium assay·····················································141 8. Calcium imaging·································································142 9. Fabrication of a CNT-FET with Floating Electrodes························142 10. Immobilization Nanovesicles on the Floating Electrodes of the CNT-FET·················································································143 11. Electrical Measurements ························································144 12. Preparation of Tastants···························································144 RESULTS AND DISCUSSION················································145 GENERAL CONCLUSIONS···················································157 LITERATURAL CITED·························································160 KOREAN ABSTRACT···························································207Docto

Olfactory mechanisms of host selection in phytophagous insects

The most challenging tasks for phytophagous insects are the location and selection of mates, food sources, and oviposition sites, all crucial for survival and reproduction. To perform these tasks insects rely largely on their sense of smell (olfaction). I address how the insect olfactory system discriminates between components of complex odor mixtures, modulating behavior and fitness. I have studied modulation of attraction in the moth Spodoptera littoralis and the bark beetle Ips typographus by separation of pheromone (Ph) and anti-attractants, and of Ph components alone. An antagonist reduced male moth attraction towards the female sex Ph, and a blend of non-host volatiles (NHV) reduced attraction of both sexes of I. typographus towards their Ph, insect catches decreased with decreasing odor-source distance. Conversely, increasing distance between Ph components decreased attraction in both insect species. However, moths were more sensitive to small-scale spacing. Reproductive behaviors as well as fecundity and longevity of S. littoralis moths were negatively affected in the presence of volatiles from leaves of non-host plants, Picea abies or Adhatoda vasica. The presence of non-host plants strongly modulated male moths’ behavior, reducing their attraction towards the Ph source in flight assays. Gas chromatography-electroantenno-graphic detection (GC-EAD) by female S. littoralis antennae with headspace volatile collections from P. abies and A. vasica revealed eight active compounds, with seven new actives. Single sensillum recordings (SSR) created a functional-morphological map of 49 olfactory sensory neuron (OSN) functional types in six morphological sensillum types in female S. littoralis. Proximally located OSNs showed a higher sensitivity, shorter latency, and displayed more phasic responses than distally located OSNs of the same class. GC-SSRs with volatiles from a larval host, cotton plants, and the adult nectar source, lilac flowers, revealed 38 active compounds for female OSNs, including 12 new actives. The odor response specificities of four olfactory receptor (OR) genes of S. littoralis were deorphanized by expression in the Empty Neuron System (ENS) of Drosophila melanogaster using SSR and GC-SSR (GC-SSR-ENS). Two of the ORs responded specifically to single odorants, while the other two responded similarly to the same 9 compounds, but dose-response experiments with new compounds, identified by GC-SSR, revealed specific odor-response profiles

Transcriptome And Expression Profiling Analysis Link Patterns Of Gene Expression To Antennal Responses In Spodoptera Litura

Background: The study of olfaction is key to understanding the interaction of insects with their environment and provides opportunities to develop novel tactics for control of pest species. Recent developments in transcriptomic approaches enable the molecular basis of olfaction to be studied even in species with limited genomic information. Here we use transcriptome and expression profiling analysis to characterize the antennal transcriptome of the noctuid moth and polyphagous pest Spodoptera litura. Results: We identify 74 candidate genes involved in odor detection and recognition, encoding 26 ORs, 21 OBPs, 18 CSPs and 9 IRs. We examine their expression levels in both sexes and seek evidence for their function by relating their expression with levels of EAG response in male and female antennae to 58 host and non-host plant volatiles and sex pheromone components. The majority of olfactory genes showed sex-biased expression, usually male-biased in ORs. A link between OR gene expression and antennal responses to odors was evident, a third of the compounds tested evoking a sex-biased response, in every case also male-biased. Two candidate pheromone receptors, OR14 and OR23 were especially strongly expressed and male-biased and we suggest that these may respond to the two female sex pheromone components of S. litura, Z9E11-14:OAc and Z9E12-14:OAc, which evoked strongly male-biased EAG responses. Conclusions: Our results provide the molecular basis for elucidating the olfactory profile of moths and the sexual divergence of their behavior and could enable the targeting of particular genes, and behaviors for pest management

Host location in a specialist parasitoid wasp via olfactory cues – a physiological, behavioural and morphological study

For successful host location, parasitoids are thought to have evolved different strategies to filter relevant olfactory cues which indicate the presence of the host. Because of their versatility in their ecology and behaviour, as well as their fine tuned olfactory system to volatile compounds of the host and host plant, they have gained increasing recognition as model organisms to study learning and behaviour in an adaptive ecological context. However, neural and cellular mechanisms of olfactory detection and processing in parasitoids are mainly unknown.In this thesis physiological, behavioural and morphological experiments were used to determine neural and behavioural mechanisms of host location via olfactory cues in the specialist parasitoid Cotesia vestalis. C. vestalis showed significant antennal responses to a range of odour compounds. Behavioural experiments, however, have demonstrated that only the herbivore-induced plant volatile linalool attracts C. vestalis males and females, but 1-nonanol has a repulsive effect on females. A morphological study of the antennal lobe, the first brain area where olfactory information is processed, revealed 40 ordinary glomeruli in both males and females. In addition, a complex of 2-3 enlarged glomeruli (MGC) was found in males. The courtship behaviour observed in males and the MGC suggest that males could use sex pheromones to locate females. Finally, calcium imaging studies showed glomerular activity to olfactory stimulation in bees but not in parasitoids. In conclusion, the degree of host specialisation in C. vestalis appears to influence olfactory learning in males and females, which favours learning of volatiles related to its host and host plant, as well as the morphological organisation of the antennal lobe. Larger, fewer and possibly specialised glomeruli could enhance processing of odour cues which are important for this parasitoid

Studies on olfactory systems of two parasitoid wasps: Diadegma semiclausum (Hellén) (Hymenoptera: Ichneumonidae) and Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) of the Diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae)

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most destructive insect pests of brassicaceous crops, which has shown problematic resistance to almost every common insecticide. In certain parts of the world, the economic production of crucifers has become nearly impossible due to the failure in controlling DBM. Consequently, increased efforts worldwide have been undertaken to implement biological control programs that are principally employing its natural enemies such as Diadegma semiclausum and Trichogramma pretiosum. D. semiclausum is a specialist DBM larvae parasitoid and one of the most common parasitoids in Australia. T. pretiosum is a generalist egg parasitoid, and it inserts its eggs into the host eggs, including DBM. However, limited studies have been conducted on these two parasitoid wasps regarding how they detect DBM, how their olfactory systems guide them to localize the DBM and what olfactory genes are involved in these DBM-seeking and oviposition behaviours. Without this knowledge, it is difficult to utilize them at maximum efficiency and effectiveness in DBM management programs. In this study, analytical chemistry, electrophysiology, scanning electron microscopy, genomics, transcriptomics, bioinformatics and molecular biology approaches were applied to investigate the olfactory systems of D. semiclausum and T. pretiosum. Eight candidate attractants were identified from DBM-infested canola Brassica napus, while some of them were found to be able to initiate significant antennal responses from the parasitoid wasp D. semiclausum. Male and female D. semiclausum exhibited different antennal responses to various tested volatile compounds. Candidate olfactory genes, including 17 odorant-binding proteins (OBPs) and 67 odorant receptors (ORs), were identified and characterized from D. semiclausum. Similarly, a total of 22 OBPs and 121 ORs were identified and characterized from T. pretiosum. These genes may play pivotal roles in the host-seeking and oviposition behaviours. This study improves our understanding of the olfactory systems of these two wasps and their host-seeking behaviours, which will assist in developing more efficient and environmentally friendly biological control strategies to manage DBM

Distinct Expression Profiles and Different Functions of Odorant Binding Proteins in Nilaparvata lugens Stål

Background: Odorant binding proteins (OBPs) play important roles in insect olfaction. The brown planthopper (BPH), Nilaparvata lugens Sta˚l (Delphacidae, Auchenorrhyncha, Hemiptera) is one of the most important rice pests. Its monophagy (only feeding on rice), wing form (long and short wing) variation, and annual long distance migration (seeking for rice plants of high nutrition) imply that the olfaction would play a central role in BPH behavior. However, the olfaction related proteins have not been characterized in this insect. Methodology/Principal Findings: Full length cDNA of three OBPs were obtained and distinct expression profiles were revealed regarding to tissue, developmental stage, wing form and gender for the first time for the species. The results provide important clues in functional differentiation of these genes. Binding assays with 41 compounds demonstrated that NlugOBP3 had markedly higher binding ability and wider binding spectrum than the other two OBPs. Terpenes and Ketones displayed higher binding while Alkanes showed no binding to the three OBPs. Focused on NlugOBP3, RNA interference experiments showed that NlugOBP3 not only involved in nymph olfaction on rice seedlings, but also had non-olfactory functions, as it was closely related to nymph survival. Conclusions: NlugOBP3 plays important roles in both olfaction and survival of BPH. It may serve as a potential target fo