Pheromone Biosynthesis by Selected Species of Grain and Bark Beetles by DdsirBe Vanderwel B. Sc. (Honors), University of Victoria, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR IN PHILOSOPHY in the Department of Chemistry @ DdsirBe Vanderwel, 1991 SIMON FRASER UNIVERSITY MARCH 1991 All rights reserved. This work may not be reproduced in whole or in part, by photocopy or other means, without permission of the author. APPROVAL Name: DWi6e Vanderwel Degree: DOCTOR OF PHILOSOPHY Title of Thesis: Pheromone Biosynthesis in Selected Species of Grain and Bark Beetles Examining Committee: Chairman: Dr. F.W.B. Einstein Dr. A. C. Oehlschlage( Senior Supervisor Dr. J?. H. Borden, Supervisory Committee Dr. K. k Slessor, &emisory Committee Dr. B. L. Funt, Supervisory Committee Dr. T. J. ~or&'&d, kternal Examiner Dr. W. L. Roelofs, drnalExaminer Department of Entomology, Cornell University Date Approved: (1 PARTIAL COPYRIGHT LICENSE I hereby grant to Simon Fraser Unlverslty the rlght to lend my thesis, project or extended essay (the title of which is shown below) to users of the Simon Fraser University L Ibrary, and to make part ial or single copies only for such users or In response to a request from the library of any other university, or other educational Institution, on its own behalf or for one of its users. I further agree that perm i ss ion for multiple copying of this work for scholarly purposes may be granted by me or the Dean of Graduate Studies. It is understood that copying or publication of this work for financial gain shall not be allowed without my written permission. Title of Thesis/Project/Extended Essay Pheromone Biosynthesis by Selected Species of Grain and Bark Beetles Author: I-signature) Desiree Vanderwel ( name April 26, 1991 (date) ABSTRACT The overall goal of this thesis was to investigate pheromone biosynthesis in grain and bark beetles. Insects were exposed to either radio- or stable isotope-labelled precursors. Incorporation of label into pheromones was determined by scintillation counting or gas chromatography/selected ion monitoring-mass spectroscopy, respectively. Most known aggregation pheromones of cucujid grain beetles are macrolides, called "cucujolides". Objectives of this project were to determine the biosynthetic origin of these cucujolides and to study the mechanism of cyclization. Studies were performed on Oryzaephilus mercator (Fauvel) and/or Cryptolestes ferrugineus (Stephens). In C. ferrugineus, cucujolide I (numbered according to J. Chem. Ecol., 1988, 14:2069-2096) was of terpenoid origin. Cucujolides 11, 111, IV and V were found to be of fatty acid origin. The de novo biosynthesis of all cucujolides (I through V) was detected. The cyclizations of the hydroxy acid precursors to form I in C. ferrugineus, and I1 in C. ferrugineus and 0. mercator, proceeded with retention of the hydroxyl oxygens. The cyclic ketals exo- and ends-brevisomin are pher~m0ne~of many economically important scolytids. The objective of this project was to study brevicomin biosynthesis in the mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins and the western balsam bark beetle, Dryocoetes confusus Swaine. Insects exposed to (2)- or (E)-D2-nonen-2-one produced exo- or endo-brevicomin, respectively, that were clearly enriched in two deuterium atoms. The D2-exo-brevicomin produced by male MPB was found to be of the natural (+)-chirality by complexation chromatography. The biosynthetic transformation apparently proceeds through a keto-epoxide intermediate. Both oxygens of brevicomin are derived from molecular oxygen. Ipsdienol is a common bark beetle pheromone. The chirality of ipsdienol varies widely between different species, populations and even individuals. The objective of this project was to test the hypothesis iii that increased activity of the oxidation/reduction equilibrium between ipsdienol and ipsdienone effects a larger proportion of S-(+)-ipsdienol. The study insects were Dendroctonus ponderosae Hopkins, two populations of Ips pini (Say), and Ips paraconfusus Lanier. The hypothesis was disproved. ACKNOWLEDGEMENTS First and foremost I would like to thank my senior supervisor, Professor Cam Oehlschlager, for his enthusiasm, exciting ideas, and for providing an excellent work environment. I would also like to thank Professor John Borden for his guidance and encouragement, and Professor Mario Pinto for the constructive criticism of this thesis and for the many helpful discussions held during the course of this work. Additionally I owe a debt of gratitude to my family for their support. Many individuals have persistantly and generously shared both their time and technical expertise with me. I would like to thank Dr. Harold Pierce, Jr. for many hours of patient instruction and stimulating discussions. I also enjoyed fruitful discussions with Regina Gries, Dr. Gerhard Gries and Dr. Dan Miller, and would additionally like to thank them for supplying many of the insects used in this study. I greatly appreciate helpful discussions with Dr. Blair Johnston and Dr. Mohan Singh, and would like to thank them for supplying some of the chemicals used in this study. I would also like to thank Dr. Rob Currie for the constructive editing of this thesis and for assisting with the statistical analyses. Helpful input by Dr. Sowrnianarayanan Ramaswamy, Dr. Brian Morgan and Jean-Pierre Lafontaine was also much appreciated. I would also like to thank the many people who helped to make my experience as a graduate student invigorating and fun as well as educational. These especially include the individuals in the laboratories of Professors Oehlschlager, Borden and Winston. I would like to thank Art Stock, Leslie Chong and Steve Seybold for generously supplying the many of the insects used in this study; Marcy Tracy for assisting with the NMR analyses; Greg Owen for assisting with the GC/MS analyses; Denyse Zenner and Penny Snell for administrative assistance; and Professor Robert Cushley and Pherotech Inc. for the gifts of some of the chemicals used in this study. I would also like to thank the Natural Sciences and Engineering Research Council of Canada, Simon Fraser University, and the R. H. Wright Foundation for the financial support of this research. TABLE OF CONTENTS Approval ............................. ii ABSTRACT ............................. iii ACKNOWLEDGEMENTS ......................... v List of Tables ........................... vi ListofFigures ........................ xvii List of Synthetic Schemes ..................... xxii GENERAL INTRODUCTION ........................ 1 CHAPTER I: LITERATURE REVIEW A) Pheromone bioaynthesis in the boll weevil .......... B) Pheromone biosynthesis in bark beetles ............ 1 . Biosynthesis of terpenoid alcohol and ketone pheromones a) Pheromones derived from myrcene ......... b) Pheromones derived from a-pinene ......... c) Pheromones synthesized de novo .......... 2 . Biosynthesis of 2-phenylethanol ............. 3 . Biosynthesis of cyclic ketal and acetal pheromones ... C) Role of microorganisms in "pheromone" biosynthesis ...... CHAPTER 11: MACROLIDE (CUCUJOLIDE) BIOSYNTHESIS IN CUCUJID GRAIN BEETLES A)INTRODUCTION ........................... 26 B) EXPERIMENTAL .......................... 30 1 . Insects: .......................... 30 2 . Preparation of experimental cultures: ........... 30 3 . Capture and isolation of culture volatiles: ........ 31 4 . Analyses of cucujolides: .................. 31 5 . Isolation. purification and analyses of cucujolides: .... 31 vii a) Analyses of incorporation of radiolabelled precursors: .................... b) Analyses of incorporation of stable isotope-labelled precursors: .................... c) Analyses of chirality of cucujolide I1 ........ d) Isolation. purification. and analyses of unknowns: ... 6 . Lipid analyses of oats: .................. a) Method 1: Bligh and Dyer extraction of "defatted" oats ....................... b) Method 2: Saponification of extracted lipids .... 7 . Purchased/donated chemicals: ................ 8 . Synthesis of stable isotope-labelled chemicals: ...... a)Analyses: ...................... b) Syntheses of chemicals: ............... C) RESULTS AND DISCUSSION ...................... 1.Methodology ........................ a) Radiolabels versus stable isotope labels: pros and cons b) Increased incorporation efficiency .......... c) Trial of stable isotope-labelling method ....... 2 . Investigation of cucujolide biosynthesis .......... a) Determination of biosynthetic origin of cucujolides . b) Biosynthesis of cucujolide 11: further studies ... c) Biosynthesis of cucujolide I: further studies .... D)CONCLUSIONS .......................... CHAPTER 111: BREVICOMIN BIOSYNTRESIS IN THE MOUNTAIN PINE BEETLE. DBNDROCTONUS PONDEROSABI AND TEE BALSAM BARK BEETLE. DRPOCOETES CONFUSUS (COLEOPTERA: SCOLYTIDAE) A)INTRODUCTION .......................... I B)EXPERIMENTAL .......................... viii 1 . Insects ......................... 2 . Treatment of Insects ................... a) Exposure to I8o2 .................. b) Exposure to "0~~................. 3 . Analyses of exo- and endo-brevicomin in extracts: .... 4 . Determination of the chirality of the exo-brevicomin produced ....................... 5 . Statistical analyses ................... 6 . Purchased/donated chemicals: .............. 7 . Synthesis of stable isotope labelled chemicals ...... a)Analyses: ..................... b) Syntheses of chemicals: .............. C) RESULTS AND DISCUSSION ..................... 1 . Biosynthesis