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

DC Field Value Language
dc.contributor.advisor안용준-
dc.contributor.author정제원-
dc.date.accessioned2017-07-13T08:23:28Z-
dc.date.available2017-07-13T08:23:28Z-
dc.date.issued2015-08-
dc.identifier.other000000066671-
dc.identifier.urihttps://hdl.handle.net/10371/119502-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부(바이오모듈레이션전공), 2015. 8. 안용준.-
dc.description.abstractChemoreception 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
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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.		-
dc.description.tableofcontentsABSTRACT··············································································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
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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
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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
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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
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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
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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···························································207		-
dc.formatapplication/pdf-
dc.format.extent5466807 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectchemoreception-
dc.subject.ddc630-
dc.titleMolecular and Electrophysiological Characterization of Olfactory and Gustatory Perception in Honeybee and Cockroach for Application as Biosensor-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pages211-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2015-08-
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