MOLECULAR DISCRIMINATION OF PHYTOSEIIDS ASSOCIATED WITH THE RED PALM MITE RAOIELLA INDICA (ACARI: TENUIPALPIDAE) FROM MAURITIUS AND SOUTH FLORIDA By HEIDI MARIE BOWMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010 1 © 2010 Heidi Marie Bowman 2 To my family and friends who have supported me throughout my academic endeavors 3 ACKNOWLEDGMENTS I thank my advisor and chair of my graduate committee, Dr. Marjorie Hoy, for providing scientific guidance and financial support. I thank my committee members Dr. Jorge Peña and Dr. Amy Roda for their professional and academic advice, their contributions to my research proposal, and for reviewing this thesis. I would like to acknowledge Dr. A. Jeyaprakash for his guidance in the laboratory and phylogenetic analysis. I would like to extend appreciation to Dr. Denmark and Dr. Welbourn for their taxonomic assistance. I would like to acknowledge Daniel Carrillo and Dr. Jorge Peña for their assistance in collecting phytoseiid specimens in South Florida. I thank Michael Dornburg, Karol Krey, Ryan Tanay, and Reggie Wilcox for their assistance in rearing and maintaining the phytoseiid and prey colonies. I thank my family for their constant support and encouragement of my academic endeavors. I also wish to give thanks to Robert Cating for being an excellent friend, motivating labmate, and inspiration. Finally, I wish to acknowledge my partner, Jorge Pérez Gallego, for his steadfast patience, love, and support. Funding for this research has been provided by the USDA-APHIS and the Davies, Fisher, and Eckes Endowment for Biological Control to Dr. M. A. Hoy. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................. 4 LIST OF TABLES............................................................................................................ 7 LIST OF FIGURES.......................................................................................................... 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 11 Introduction ............................................................................................................. 11 The Red Palm Mite Raoiella indica (Acari: Tenuipalpidae)..................................... 13 RPM Dispersal and Range............................................................................... 13 RPM Hosts and Symptomology........................................................................ 14 RPM Description and Development ................................................................. 15 U.S.A. RPM Regulatory Response................................................................... 17 RPM Biological Control .................................................................................... 18 RPM Natural Enemies...................................................................................... 19 Classical Biological Control and the Phytoseiidae .................................................. 20 The Phytoseiidae .................................................................................................... 22 Phytoseiid Biology ............................................................................................ 22 Amblyseius....................................................................................................... 24 Arthropod Identification........................................................................................... 26 Species Concepts................................................................................................... 26 Taxonomic........................................................................................................ 28 Biological.......................................................................................................... 29 Phylogenetic..................................................................................................... 31 Molecular Markers .................................................................................................. 33 The Mitochondrial Genome .............................................................................. 34 The Nuclear Genome ....................................................................................... 35 Rate of Evolution .............................................................................................. 36 Single-Copy Genes .......................................................................................... 37 Availability of Related Sequence Data and Primer Design............................... 38 Molecular Markers of Interest for This Study .......................................................... 38 12S rRNA ......................................................................................................... 38 Cytochrome Oxidase I (COI) ............................................................................ 39 Elongation Factor-I Alpha (EF-I)..................................................................... 40 Random Amplified Polymorphic DNA (RAPD) PCR ......................................... 42 Research Objectives............................................................................................... 45 Research Aim................................................................................................... 45 Main Objectives................................................................................................ 45 5 2 MOLECULAR DISCRIMINATION OF PHYTOSEIIDS ASSOCIATED WITH THE RED PALM MITE FROM MAURITIUS AND SOUTH FLORIDA ............................. 59 Introduction ............................................................................................................. 59 Methods.................................................................................................................. 62 Phytoseiid Collection and Colony Maintenance................................................ 62 DNA Extractions ............................................................................................... 63 Amplification and Sequencing of Partial Mitochondrial 12S rRNA and Nuclear EF-I Genes .................................................................................... 64 Sequence Editing and Alignment ..................................................................... 65 Phylogenetic Analysis ...................................................................................... 66 Pairwise Distance Analysis............................................................................... 67 High-fidelity-RAPD-PCR................................................................................... 67 Mitochondrial 12S rRNA Population-Specific Primers ...................................... 69 Results.................................................................................................................... 70 ‘Amblyseius largoensis’ Bayesian Analysis and Sequence Divergence ........... 70 12S rRNA Sequences................................................................................ 70 Elongation Factor-I Alpha Sequences........................................................ 73 High-fidelity-RAPD-PCR Analysis of ‘A. largoensis’ Populations...................... 76 Mitochondrial 12S rRNA Population-Specific Primers ...................................... 77 Discussion .............................................................................................................. 77 APPENDIX: PERSPECTIVES................................................................................... 112 LIST OF REFERENCES ............................................................................................. 114 BIOGRAPHICAL SKETCH.......................................................................................... 136 6 LIST OF TABLES Table page 1-1 A list of Raoiella indica hosts reported in Florida................................................ 47 1-2 A list of viruses transmitted by Brevipalpus mites to their host plants................. 48 1-3 List of South Florida Amblyseius 'largoensis' collection sites.............................. 49 1-4 The partial 12S rRNA sequences from Phytoseiulus spp. submitted by Tixier et al. (2009) to GenBank .................................................................................... 50 1-5 Corrected pairwise distances between the partial 12S rRNA sequences from Phytoseiulus spp. submitted by Tixier et al. (2009) to GenBank......................... 51 2-1 List of molecular markers used and expected PCR product size........................ 82 2-2 List of GenBank accession numbers for clones and taxa included in the 12S rRNA Bayesian analysis. .................................................................................... 83 2-3 List of GenBank accession numbers for clones and taxa included in the EF- I Bayesian analysis........................................................................................... 86 2-4 Corrected pairwise distances between unique mitochondrial 12S rRNA sequences obtained from the Mauritius and S. Florida ‘A. largoensis’ populations* with additional phytoseiid GenBank accessions using PAUP 4.0b8 with Kimura 2-parameter and among-site rate variation