REPRODUCTION, SEX RATIO AND BACTERIAL COMMUNITIES OF THE COFFEE BERRY BORER Hypothenemus hampei F. (COLEOPTERA: CURCULIONIDAE) by Yobana Andrea Mariño Cárdenas A thesis submitted to the DEPARTMENT OF BIOLOGY FACULTY OF NATURAL SCIENCES UNIVERSITY OF PUERTO RICO RIO PIEDRAS CAMPUS In partial fulfillment of the requirements for the degree of DOCTOR IN PHILOSOPHY May 2015 San Juan, Puerto Rico © Yobana Andrea Mariño-Cárdenas All rights reserved ii This thesis have been accepted by the faculty of the DEPARTMENT OF BIOLOGY FACULTY OF NATURAL SCIENCES UNIVERSITY OF PUERTO RICO RIO PIEDRAS CAMPUS In partial fulfillment of the requirements for the degree of DOCTOR IN PHILOSOPHY iii TABLE OF CONTENTS LIST OF TABLES ................................................................................................................... vii LIST OF FIGURES ................................................................................................................... ix ABSTRACT .............................................................................................................................. xi DEDICATION ........................................................................................................................ xiv ACKNOWLEDMENTS ........................................................................................................... xv CHAPTER 1. GENERAL INTRODUCTION: Factors that affect the reproduction, sex ratio and bacterial communities in insects in general ......................................................................... 1 Introduction ............................................................................................................................ 2 1. Factors that influence sex ratio and reproduction of insects .............................................. 3 1.1. Biotic factors ............................................................................................................... 3 1.1.1. Infection with endosymbiotic bacteria ..................................................................... 3 1.1.2. Population density and density of conspecifics ........................................................ 5 1.1.3. Food resources (quality and amount) ....................................................................... 5 1.2. Abiotic factors ............................................................................................................. 6 1.2.1. Temperature ............................................................................................................. 6 1.2.2. Relative humidity ..................................................................................................... 8 2. Factors that shape bacterial communities in insects ........................................................... 9 2.1 Biotic factors ................................................................................................................ 9 2.1.1 Food resources or diet ............................................................................................... 9 2.1.2. Stage of development of insect hosts ..................................................................... 10 2.2. Abiotic factors ........................................................................................................... 10 2.2.1 Temperature ............................................................................................................ 10 3. Coffee and Coffee Berry Borers: an ideal system to study the effect of biotic and abiotic factors on reproduction, sex ratio and bacterial communities of insects ............................. 11 3.1. The coffee berry borer (H. hampei) .......................................................................... 11 4. Dissertation thesis structure ............................................................................................. 13 5. References ........................................................................................................................ 16 CHAPTER 2: Sun vs. shade affects infestation, population sizes and sex ratio of the coffee berry borer (Hypothenemus hampei) in Puerto Rico ................................................................ 25 Abstract ................................................................................................................................ 26 1. Introduction ...................................................................................................................... 26 2. Materials and Methods ..................................................................................................... 28 2.1. Study region and site descriptions ............................................................................. 28 2.2. Sampling methods ..................................................................................................... 29 2.3. Statistical analysis ..................................................................................................... 30 3. Results .............................................................................................................................. 31 3.1. Environmental characteristics of plots ...................................................................... 31 3.2. Differences between sun vs. shade on infestation of CBB ....................................... 32 3.3. Differences between sun vs. shade on number of CBB per fruit .............................. 32 3.4. Environmental variables and relationship to number of CBB per bored fruit .......... 33 3.5. Sex ratio ..................................................................................................................... 33 3.6. Differences between sun vs. shade on the penetration positions of colonizing females of H. hampei ....................................................................................................... 34 3.7. Differences between sun vs. shade on trap capture rates .......................................... 34 4. Discussion ........................................................................................................................ 35 4.1. CBB infestation levels compared to other coffee-growing countries ....................... 35 4.2. Coffee fruit development and its association to CBB infestation ............................. 35 4.3. Differences between sun vs. shade coffee on CBB infestation ................................. 36 4.4. Coffee fruit development and number of CBB per fruit ........................................... 36 iv 4.5. Differences between sun vs. shade on number of CBB per fruit .............................. 37 4.6. Differences in CBB infestation and population sizes between years ........................ 39 4.8. Differences between sun vs. shade on sex ratio ........................................................ 41 4.9. Use of traps with ethanol: methanol to monitor CBB populations ........................... 43 5. Conclusions and recommendations .................................................................................. 43 6. Acknowledgments ............................................................................................................ 44 7. References ........................................................................................................................ 44 8. Appendices ....................................................................................................................... 59 CHAPTER 3: Wolbachia affects reproduction, sex ratio and population dynamics of the Coffee Berry Borer Hypothenemus hampei (Coleoptera: Curculionidae) ............................... 63 Abstract ................................................................................................................................ 64 1. Introduction .................................................................................................................. 64 2. Materials and Methods ..................................................................................................... 66 2.1. Insect Rearing. ........................................................................................................... 66 2.1.1. Antibiotic Treatments ............................................................................................. 67 2.2. Detection of Wolbachia ............................................................................................. 67 2.2.1. DNA Extraction, PCR Amplification and DNA sequencing ................................. 67 2.3. Phylogenetic classification in Wolbachia supergroups ............................................. 68 2.4. Effect of antibiotic treatments of CBB Reproduction and Sex Ratio ....................... 68 2.6. Life Tables of H. hampei ........................................................................................... 69 2.7. Statistical analysis ..................................................................................................... 70 3. Results .............................................................................................................................. 71 3.1. Wolbachia detection and classification ..................................................................... 71 3.3. Effect of antibiotic treatment on Wolbachia infection and CBB reproduction ......... 72 3.4. Effect of antibiotic treatment of life table parameters of H. hampei ......................... 73 4. Discussion .......................................................................................................................