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Bell & Howell Information and Learning 300 North Zeeb Road, Ann Artx)r, Ml 48106-1346 USA UMI 800-521-0600 Towards a Molecular Analysis of Associative Learning in the Honey Bee, Apis mellifera, via Massed Conditioning and Genetic Transformation DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Kellie Oline Robinson, 8.S. ***** The Ohio State University 1999 Dissertation Committee: Dr. Brian Smith, Adviser Approved by Dr. David Denlinger Dr. John Oberdick Dr. Amanda Simcox Cdviser / y/ Dr. Harald Vaessin Molecular, Cellular,^d Developmental Biology Graduate Program UMI Number: 9951717 UMI Microform 9951717 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ABSTRACT Understanding conditions under which an organism correlates temporally related events is critical for establishing a link between behavioral mechanisms of associative learning and molecular mechanisms of acquisition and storage of this information. We examined the effect of massed-trial conditioning, which involves short (30 second) intervals between conditioning trials, on learning performance using the Proboscis Extension Conditioning (PEC) paradigm on worker honey bees. Specifically, we investigated the associative nature of the effect by incorporating several control procedures that have not been evaluated in a massed trials conditioning paradigm. A second goal was to define temporal events that occur during acquisition of information and consolidation as memory. Therefore, we tested recall at different post-conditioning intervals in order to investigate the temporal dynamics of memory consolidation. Acquisition of the Proboscis Extension Response (PER) is not dependent on the inter-trial interval (ITI) during forward- paired conditioning. The PER is the behavioral response of the subject (extension of the proboscis) after exposure to sucrose or conditioned stimuli. This response was used to score individual subjects during and after olfactory training utilizing the proboscis extension within various training presentations. Furthermore, the relatively higher response levels in forward-paired conditioning groups are dependent on a specific temporal and predictive relationship between the odor conditioned stimulus and sucrose reinforcement. Our data also indicate that massed conditioning with 30 second ITI produces robust associative recall over 24 hours A full understanding of the molecular basis of learning and memory' will depend upon the functional testing of implicated genes in transgenic honey bees. As a first step towards this goal, strategies to transform the genome of honey bees with foreign DNA plasmids were investigated Linearized plasmids were introduced with semen during instrumental insemination of virgin queen honey bees. Lar\'ae from a few such queens were subsequently identified as positive for the plasmid via PCR amplification of a plasmid fragment and fluorescent expression of green fluorescent protein encoded by the plasmid. These transgenic lines transmitted the transgene for two generations, demonstrating heritability. However, there was no evidence of integration of the construct into the honey bee genome as determined by genomic Southern analysis. For this reason, the use of a transposable element was explored. These experiments tested the utility of a potential universal gene vector for transformation of the honey bee genome. The Tel transposon was originally isolated from C. elegans and is well characterized. The data indicate sperm-mediated introduction of the Tel element via instrumental insemination results in PCR-positive progeny in the FO and the FI generations. Ligation-mediated PCR and plasmid rescue from genomic DNA samples confirmed the persistence of the Tel element through the generations. However, recombination of the Tel element into the bee genome was not detected. Therefore we consider it likely that the Tel element is maintained but persists as an extrachromosomal element. Ill Dedicated to my Mom. Bonnie Robinson, who would never let me give up and My Grandma, Emma Oline Jordan, who always has a good time with life. IV ACKNOWLEDGMENTS I wish to thank my advisor. Dr. Brian Smith, for guidance and infinite patience throughout this research. Brian you have always seen to it that everything that could be done to further my graduate studies was available. Thank you Sue Cobey for detailed instruction in honey bee inseminations. Dr. Holly Furgeson for pioneering the honey bee transformation project, and Dr. Seetha Bhagavan for discussions on honey bee behavior. Thank you Dr. Geraldine Wright for help with the statistical analysis, scientific discussions, and friendship through the most trying of times. Thank you to the Vaessin lab for their collaboration in the genetic transformation of honey bees. Dr. Harald Vaessin, thank you for patiently correcting all 101 graduate student mistakes and for continuous intellectual support. Thank you Dr. Kirsten Bremer, Dr. Julie Pinsonneault, Dr. Terrie Rife, Ling Li and Dr. Amian Ahmed for scientific discussions, encouragement and continuous moral support when it was most desperately needed. Thanks to the Denlinger Lab for my start in scientific research and molecular biology. Specifically thanks goes to Dr. David Denlinger for both serving on my committee and advising me on numerous occasions. Dr. Karl Joplin, Dr. George Yocum, and Dr. Ron Flannagan for advice, friendship and support. Dr. John Oberdick, and Dr. Amanda Simcox, thank you for serving on my graduate committee, advising on my scientific career, reading and committing on my dissertation and manuscripts. VI VITA June 21, 1969 ............................Bom-Crystal City, Missouri 1989............................................A.A., Palm Beach Community College 1992 B.S. Biology, Florida Atlantic University 1992-present.............................. Graduate Teaching and Research .Associate Molecular, Cellular and Developmental Biology, The Ohio State University PUBLICATIONS FIELDS OF STUDY Major Field; Molecular, Cellular, and Developmental Biology emphasis on Apis mellifera VII TABLE OF CONTENTS Paae Abstract............................................................................................................................... ii Dedication..........................................................................................................................iv Acknowledgments .............................................................................................................v Vita..................................................................................................................................... vii List of Tables.....................................................................................................................xi List of Figures.................................................................................................................. xii Chapters: 1. Introduction How is learning related to the formation of memory? .....................1 Wliat are good model animals? Aplysia californien.....................................................................4 Drosophila melanogasier.........................................................7 Why honey bees? ...................................................................................10 Toward a molecular approach to studying learning & memory in the honey bee .................................................................................... 12 Transformation in Apis mellifera.......................................................13 2. Massed Conditioning Produces Associative Learning in the Honey Bee, Apis mellifera Abstract....................................................................................................15 Introduction ............................................................................................. 17 Materials and Methods ......................................................................... 21 viii Honey