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Molecular Discrimination of Phytoseiids Associated with the Red Palm Mite Raoiella Indica (acari

Permanent Link: http://ufdc.ufl.edu/UFE0041714/00001

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Title: Molecular Discrimination of Phytoseiids Associated with the Red Palm Mite Raoiella Indica (acari Tenuipalpidae) from Mauritius and South Florida
Physical Description: 1 online resource (136 p.)
Language: english
Creator: Bowman, Heidi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 12s, amblyseius, bayesian, biological, classical, control, elongation, factor, indica, largoensis, mite, palm, paralogous, phylogenetic, phytoseiidae, raoiella, rapd, red, rrna, tenuipalpidae
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Since the red palm mite invasion into South Florida and the Caribbean, endemic phytoseiids have been unable to suppress populations or spread of this invasive pest. In response, phytoseiids were imported from Mauritius for evalutation for a classical biological control program. The Mauritius and Florida phytoseiid populations were both morphologically identified as Amblyseius largoensis Muma. Bayesian analysis and sequence divergence calculations of the mitochondrial 12S rRNA and nuclear EF-I alpha genes and Neighbor-Joining analysis of High-fidelity-RAPD-PCR markers were used to discriminate between the two populations. Variability within the 12S gene also was used to develop population-specific primers for identifying the Mauritius phytoseiid in the event it is released in South Florida. Bayesian and sequence divergence analyses of the 12S rRNA sequences suggest that the Mauritius and S. Florida populations represent two different species. These results were supported by the High-fidelity-RAPD-PCR markers that indicate the two populations' genomes are genetically distinct. However, the EF-I alpha gene Bayesian analysis places the two populations within the same clade. The degenerate EF-I alpha primers used to survey the phytoseiids amplified two different elongation factor sequences with distinct amino acid translations identified as the putative EF-I alpha and an unknown elongation factor. As a result of the incongruence between the 12S, EF-I alpha, and RAPD analyses, a conclusion cannot be made as to whether the Mauritius and S. Florida populations are cryptic species or biotypes of 'A. largoensis' without weighing the results of one analysis as more significant than the other.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Heidi Bowman.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Hoy, Marjorie A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041714:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041714/00001

Material Information

Title: Molecular Discrimination of Phytoseiids Associated with the Red Palm Mite Raoiella Indica (acari Tenuipalpidae) from Mauritius and South Florida
Physical Description: 1 online resource (136 p.)
Language: english
Creator: Bowman, Heidi
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: 12s, amblyseius, bayesian, biological, classical, control, elongation, factor, indica, largoensis, mite, palm, paralogous, phylogenetic, phytoseiidae, raoiella, rapd, red, rrna, tenuipalpidae
Entomology and Nematology -- Dissertations, Academic -- UF
Genre: Entomology and Nematology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Since the red palm mite invasion into South Florida and the Caribbean, endemic phytoseiids have been unable to suppress populations or spread of this invasive pest. In response, phytoseiids were imported from Mauritius for evalutation for a classical biological control program. The Mauritius and Florida phytoseiid populations were both morphologically identified as Amblyseius largoensis Muma. Bayesian analysis and sequence divergence calculations of the mitochondrial 12S rRNA and nuclear EF-I alpha genes and Neighbor-Joining analysis of High-fidelity-RAPD-PCR markers were used to discriminate between the two populations. Variability within the 12S gene also was used to develop population-specific primers for identifying the Mauritius phytoseiid in the event it is released in South Florida. Bayesian and sequence divergence analyses of the 12S rRNA sequences suggest that the Mauritius and S. Florida populations represent two different species. These results were supported by the High-fidelity-RAPD-PCR markers that indicate the two populations' genomes are genetically distinct. However, the EF-I alpha gene Bayesian analysis places the two populations within the same clade. The degenerate EF-I alpha primers used to survey the phytoseiids amplified two different elongation factor sequences with distinct amino acid translations identified as the putative EF-I alpha and an unknown elongation factor. As a result of the incongruence between the 12S, EF-I alpha, and RAPD analyses, a conclusion cannot be made as to whether the Mauritius and S. Florida populations are cryptic species or biotypes of 'A. largoensis' without weighing the results of one analysis as more significant than the other.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Heidi Bowman.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Hoy, Marjorie A.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041714:00001


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1 MOLECULAR DISCRIMINATION OF PHYT OSEIIDS 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 FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Heidi Marie Bowman

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3 To my family and friends who have supported me throughout my academic endeavors

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4 ACKNOWLEDGMENTS I thank my advisor and chair of my gr aduate committee, Dr. Ma rjorie Hoy, for providing scientific guidance and financial support. I thank my committee members Dr. Jorge Pea and Dr. A my Roda for their professional and academic advice, their contributions to my research proposal, and fo r 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 ack nowledge Daniel Carrillo and Dr. Jorge Pea 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 coloni es. 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 excell ent friend, motivating labmat e, and inspiration. Finally, I wish to acknowledge my partner, Jorge Pre z 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 Biol ogical Control to Dr. M. A. Hoy.

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5 TABLE OF CONTENTS page ACKNOWLEDG MENTS .................................................................................................. 4LIST OF TABLES............................................................................................................ 7LIST OF FIGURES.......................................................................................................... 8ABSTRACT..................................................................................................................... 9 CHA PTER 1 LITERATURE REVIEW .......................................................................................... 11Introduc tion............................................................................................................. 11The Red Palm Mite Raoiella indica (Acari: Tenui palpidae)..................................... 13RPM Dispersal and Ra nge............................................................................... 13RPM Hosts and Sy mptomo logy........................................................................ 14RPM Description and Development................................................................. 15U.S.A. RPM Regul atory Re sponse................................................................... 17RPM Biological Control.................................................................................... 18RPM Natural Enemies...................................................................................... 19Classical Biological Cont rol and the Ph ytoseiidae.................................................. 20The Phytos eiidae.................................................................................................... 22Phytoseiid Biology............................................................................................ 22Amblyseius ....................................................................................................... 24Arthropod Ident ification........................................................................................... 26Species Co ncepts................................................................................................... 26Taxonom ic........................................................................................................ 28Biological.......................................................................................................... 29Phylog enetic ..................................................................................................... 31Molecular Markers.................................................................................................. 33The Mitochondr ial Ge nome.............................................................................. 34The Nuclear Genome....................................................................................... 35Rate of Ev olution.............................................................................................. 36Single-Cop y Genes.......................................................................................... 37Availability of Related Seque nce Data and Primer De sign............................... 38Molecular Markers of In terest for This Study.......................................................... 3812S rRNA......................................................................................................... 38Cytochrome Oxidase I ( COI )............................................................................ 39Elongation Factor-I Alpha (EF-I)..................................................................... 40Random Amplified Polymo rphic DNA (R APD) PCR......................................... 42Research Ob jectives............................................................................................... 45Research Aim................................................................................................... 45Main Obje ctives................................................................................................ 45

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6 2 MOLECULAR DISCRIMINATION OF PH YTOSEIIDS ASSOCIATED WITH THE RED PALM MITE FROM MAURI TIUS AND SOUT H FLORIDA ............................. 59Introduc tion............................................................................................................. 59Methods.................................................................................................................. 62Phytoseiid Collection and Colony Ma intenance................................................ 62DNA Extractions............................................................................................... 63Amplification and Sequencing of Partial Mitochondrial 12S rRNA and Nuclear EF-I Genes.................................................................................... 64Sequence Editing and Ali gnment..................................................................... 65Phylogenetic Analys is...................................................................................... 66Pairwise Dist ance Anal ysis............................................................................... 67High-fidelityRAPD-P CR................................................................................... 67Mitochondrial 12S rRNA Population-S pecific Pr imers...................................... 69Results.................................................................................................................... 70 Amblyseius largoensis Bayesian Analysis and Sequence Divergence........... 7012S rRNA S equences................................................................................ 70Elongation Factor-I Alpha Sequences........................................................ 73High-fidelity-RAPD-PCR Analysis of A. largoensis Populations...................... 76Mitochondrial 12S rRNA Population-S pecific Pr imers...................................... 77Discuss ion.............................................................................................................. 77APPENDIX: PERSPECTI VES ................................................................................... 112LIST OF RE FERENCES............................................................................................. 114BIOGRAPHICAL SKETCH.......................................................................................... 136

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7 LIST OF TABLES Table page 1-1 A list of Raoiella indic a hosts reported in Florida................................................ 471-2 A list of viruses transmitted by Brevipalpus mites to thei r host plants................. 481-3 List of South Florida Amblyseius 'largoensis' colle ction sites.............................. 491-4 The partial 12S rRNA sequences from Phytoseiulus spp. submitted by Tixier et al. (2009) to G enBank.................................................................................... 501-5 Corrected pairwise distances between t he partial 12S rRNA sequences from Phytoseiulus spp. submitted by Tixier et al. (2009) to GenBank......................... 512-1 List of molecular markers us ed and expected PCR product size........................ 822-2 List of GenBank accession numbers for clones and taxa included in the 12S rRNA Bayesi an analy sis..................................................................................... 832-3 List of GenBank accession numbers for clones and taxa included in the EFI Bayesian analysi s........................................................................................... 862-4 Corrected pairwise distances between unique mitochondrial 12S rRNA sequences obtained from the Ma uritius and S. Florida A. largoensis populations* with additional phytoseiid GenBank accessions using PAUP 4.0b8 with Kimura 2-parameter and am ong-site rate variation distance settings ............................................................................................................... 882-5 BLAST searchs performed for the EF-I consensus tree clade-1 and clade-2 nucleotide sequences using the disc ontiguous megablast and the megablast algorithms in the GenBank database.................................................................. 892-6 List of GenBank accession numbers for clones and taxa included in the putative EF-I Bayesian analysi s....................................................................... 902-7 List of GenBank accession numbers for clones and taxa included in the unknown elongation factor sequence group..................................................... 912-8 Corrected pairwise distances bet ween the Mauritius and S. Florida A. largoensis clones* putative EF-I amino acid and nucleotide sequences with additional phytoseiid GenBank accessions usin g PAUP 4.0b8................... 92

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8 LIST OF FIGURES Figure page 1-1 Map of current Raoiella indi ca range in Flor ida (Saeger 2010). ......................... 531-2 Map of Mauritius Amblyseius 'largoensis' colle ction sites................................... 541-3 CLUSTAL X DNA al ignment for partial 12S rRNA sequences from Phytoseiulus spp. submitted by Tixier et al. (2009) to GenBank......................... 552-1 CLUSTAL X DNA alignment for partial mitochondrial 12S rRNA gene sequences from the S. Florida and Mauritius A. largoensis populations with additional phytoseiid GenBank accessi ons........................................................ 932-2 CLUSTAL X amino acid alignmen t translated from partial nuclear EF-I gene sequences from the S. Florida and Mauritius A. largoensis populations with additional phytoseiid GenBank accessions for the EF-I Bayesian analysis...... 982-3 CLUSTAL X DNA alignm ent for partial nuclear EF-I gene sequences from the S. Florida and Mauritius A. largoensis populations with additional phytoseiid GenBank accessions for the EF-I Bayesian analysis.................... 1002-4 The 12S rRNA concensus tree was inferred using the MCMC method in MrBayes........................................................................................................... 1072-5 The EF-I concensus tree was inferred using the MCMC method in MrBayes with the assumption that all s equences represent a singl e gene...................... 1082-6 The putative EF-I concensus tree was inferred using the MCMC method in MrBayes assuming two diffe rent genes ar e pres ent......................................... 1092-7 The evolutionary histor y obtained from the high-fidelity-RAPD-PCR markers 196 and 199 for S. Florida and Mauritius Amblyseius largoensis populations was inferred using the Neighbo r-Joining method in PAUP 4.0b10................... 1102-8 High-fidelity PCR products obtained from the Mauritiu s and S. Florida colonies using 12S rRNA population-s pecific primers...................................... 111

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9 Abstract of Thesis Pres ented to the Graduate School of the University of Florida in Partial Fulf illment of the Requirements for t he Degree of Master of Science MOLECULAR DISCRIMINATION OF PHYT OSEIIDS ASSOCIATED WITH THE RED PALM MITE RAOIELLA INDICA (ACARI: TENUIPALPIDAE) FROM MAURITIUS AND SOUTH FLORIDA By Heidi Marie Bowman May 2010 Chair: Marjorie A. Hoy Major: Entomology Since the red palm mite invasion into South Florida and the Caribbean, endemic phytoseiids have been unable to suppress populat ions or spread of this damaging pest. In response, phytoseiids were imported from Mauritius for evaluation for a classical biological control program. The Mauritius and Florida phytose iid populations were both morphologically identified as Amblyseius largoensis Muma. Bayesian analysis and sequence divergence calculati ons of the mitochondrial 12S rRNA and nuclear EF-I genes and Neighbor-Joining analysis of High-fi delity-RAPD-PCR markers were used to discriminate between the two popul ations. Variability within the 12S gene also was used to develop population-specific primers for identifying the Mauritiu s phytoseiid in the event it is released in South Florida. Bayesian and sequence divergence analyses of the 12S rRNA sequences suggest that the M auritius and S. Florida populations represent two different species. These re sults were supported by the High-fidelityRAPD-PCR markers that indica te the two populations genomes are genetically distinct. However, the EF-I gene Bayesian analysis places the two populations within the same clade. The degenerate EF-I primers used to survey the phytoseiids amplified two

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10 different elongation factor sequences with distinct amino acid translations identified as the putative EF-I and an unknown elongation factor As a result of the incongruence between the 12S, EF-I and RAPD analyses, a conclusion cannot be made as to whether the Mauritius and S. Fl orida populations are cryptic species or biotypes of A. largoensis without weighing the resu lts of one analysis as more significant than the other.

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11 CHAPTER 1 LITERATURE REVIEW Introduction The globalization of trade and travel has resulted in an increased movement of exotic pestiferous arthropods on infested plant materials into Amer icas major ports of entry (US Bureau of the Census 1998; Bry an 1999; Pimentel et al. 2000). In 2004, the red palm mite (RPM) Raoiella indica Hirst (Acari: Tenu ipalpi dae) was detected for the first time in the Western He misphere on the Caribbean Island of Saint Lucia (Kane et al. 2005). It subsequently spread rapidly through out the Caribbean and was detected in South Florida in 2007. The RPM is a destr uctive pest known to attack numerous species of commercial and ornamental palms, plantains, bananas, and gingers (Welbourn 2009). Feeding damage results in reduced commodity yield and value (Borchert and Morgosian 2007) and, as a quarantinable pest, its spread within the Caribbean and Florida has resulted in the loss of domestic and foreign markets for nursery stock and propagative materials (Meissner et al. 2009). In response to the threat of RPM to t he USA and Puerto Rico, a Red Palm Mite Technical Working Group was developed. The group consists of experts in biological control, acarology and regulat ion, with the mission to prov ide scientific and technical information for surveying, detection, identif ication, and management of the RPM (DeFeo 2006). An integrated approach in cluding biological control was determined to be the best option for widescale mitigation (Roda et al. 2008). Pea et al. (2009) conducted surveys of natural enemies associated with the pest in the Caribbean and South Florida and identified the ent omopathenogenic fungus Hursutella several phytoseiids, and generalist arthropod natural enemies. A classica l biological control approach also was

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12 adopted and Dr. Marjorie A. Hoy collected ph ytoseiid populations associated with the RPM on coconut palms on the island of Maurit ius where it is not known as a pest. The Mauritius phytoseiids and the S outh Florida phytoseiid populat ions associated with the RPM on coconut palms were identified using morphological characters as Amblyseius largoensis Muma by taxonomists Dr. Cal Welbourn (Florida Department of Agriculture and Consumer Services (FDACS), Division of Plant Industry (DPI), Gainesville, Florida) and Dr. H. A. Denmark. Correct identification of candidate organi sms in classical biological control programs is critical. Misident ifications within the Phytos eiidae can occur due to the presence of relatively few taxonomic characters and cryptic species (McMurtry et al. 1976; Hoying and Croft 1977; Da Silva Noronha and de Moraes 2004; Nijhout 2003; Tixier et al. 2006a, 2008b). Therefore, it was proposed that t he imported Mauritius phytoseiid populations morphological ident ification should be followed by other methods of identification, such as reproducti ve compatibility and molecular markers. If the Mauritius phytoseiid populations are able to feed, survive and reproduce on a diet consisting only of RPM, and found to be more efficacious RPM predators than the phytoseiids currently in South Florida, then methods for discrim inating between two morphologically similar populations must be developed to evaluate their establishment, dispersal, and efficacy in t he field (de Len et al. 2006). The objective of this research is to discriminate between phy toseiids associated with the red palm mite on coconut palms from Mauritius and South Florida using partial sequences from the mitochondrial 12S gene and a nuclear gene, elongation factor-1 ( EF-I), to evaluate Random Amp lified Polymorphic DNA-PCR markers as tools for

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13 discriminating between the two populations, and to develop a molecular marker for identifying the Mauritius phytoseiid populations in the event they are released in South Florida. The Red Palm Mite R aoiella indica (Acari: Tenuipalpidae) RPM Dispersal and Range RPM females are more mobile than other life stages and are considered to be mainly responsible for dispersal ( Kane et al. 2005). It is believed that the RPM dispersed throughout the Caribbean through movement of nursery material, seed coconuts, and handicrafts constructed from infested coconut leaves for the tourist industry on Caribbean islands (Mendonca et al. 2005; Pea et al. 2006; Welbourn 2009). The RPM also is believed to disperse on the wind so that a strong tropical storm or hurricane could distribute the RPM over a wide area (Hoy et al. 2006; Pea et al. 2006). The RPM occurs mostly in tropical and subt ropical climates (Dowling et al. 2008). The RPM was first described from India in 1924 from coconut palms and is found throughout the Asiatic region (I ndia, Egypt, Israel, the Philippines, Mauritius, Reunion, Malaysia, Sudan, Oman, Pakist an, and the United Arab Emirat es) (Hirst 1924; Kane et al. 2005; Hoy et al. 2006; Dowling et al. 2008; Roda et al. 2008). Dow ling et al. (2008) hypothesize that the genus Raoiella originated in the Austra lasian region, spread to occupy the Middle East, and that the United Ar ab Emirates and Iran may be the origin of the species R. indica The RPM was first detected in the Wester n Hemisphere during survey work on the Caribbean islands of Martinique and St. Lucia in 2004 and by 2005 it was detected on the neighboring island of Dominica (Kane et al. 2005; Pea et al. 2009). In 2006, RPM

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14 was reported in the Dominican Republic, Puerto Rico (Rodrigues et al. 2007), Guadeloupe, St. Martin, and Trinidad-T obago (Kane et al. 2005; Etienne and Fletchmann 2006). Uncontrolled, this pest c ontinued to spread and, by 2007, it was detected in Grenada, Venezuela, the US Virgin Islands, Haiti, and Floridas Palm Beach counties (Pea et al. 2006). Since 2007, it has been detected in Cuba and Bahamas, Brazil, and Venezuela (Roda et al. 2008; Va squez et al. 2008; Jorge Pea, pers. comm). As of September 2009, the RPM has been found in 439 sites in six Florida counties: Broward, Lee, Martin, Monroe, Miami-Dade, and Palm Beach (Figure 1-1) (Caps surveys 2010; Welbourn 2009). It is predi cted that the reported host range for the RPM limits their potential spread to areas wit h a USDA Plant Hardiness Zone of 9 or greater. Within the USA, Lousiana, Arizona, Texas, Alabama, Mississippi, South Carolina, Georgia, Nevada, and California may be at risk (Borchert and Morgosian 2007). Mexico, Southern and Central America also are suitable for a RPM invasion (Borchert and Morgosian 2007). RPM Hosts and Symptomology The RPM is primarily found on the abaxial surface of host leaves along the midrib (Jepson et al. 1975; Etienne and Fletchmann 2006; Rodrigues et al. 2007). It attacks 27 known palm hosts (Palmaceae) including the areca palm ( Dypsis lutescens H. Wetland), coconut palm ( Cocos n ucifera L.), canary island date palm ( Phoenix canariensis Chabaud), date palm ( Phoenix dactylifera L.), as well as bananas, plantains (Musaceae: Musa spp.), and gingers (Zingiberaceae: Zingiber spp.) in the Caribbean and South Florida (Welbour n 2009) (Table 1-1). On coconut, there is a higher density of the RPM on older (lower) leaves, with infestations sometimes reaching 4,000 individual s of all life stages per leaflet (Kane et

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15 al. 2005; Pea et al. 2009). Feeding by RPM on healthy coconut fronds results in bright green leaflets turning to pale green; yello w spots soon appear, next chlorotic spots coalesce, and eventually the leaflets develop large copper-brown necrotic spots (Rodrigues et al. 2007). Depending on the seve rity of infestation, the bronzing symptoms can be exhibited wit hin two to three months of RPM feeding on coconut (Roda et al. 2008). Kane et al. (2005) suggest that plants infested with RPM exhibit symptoms similar to plants with nutrient deficiencies or lethal yellowing. It is unknown whether the RPM can vector plant pathogens; however, several species belonging to the genus Brevipalpus (Tenuipalpidae), are associated with plant viruses in the family Rhabdoviridae (Table 1-2) (Child ers et al. 2003; Rodrigues et al. 2004; Kitajima et al. 2003). This suggests that the potential of the RPM as a virus vector needs to be investigated. RPM Description and Development All life stages of the R PM, including eggs, are varying shades of red, although the adult females exhibit dark patches on their dorsum after feeding. The RPM has a flat body with long, spatulate, and slightly serra te dorsal setae on both sexes. The body of the adult RPM does not have striae and is sm ooth except for the presence of punctae (Sayed 1942). The first pair of dorsocentra l hysterosomal setae is longer than the others; the fourth pair of dorsosublateral setae is shorte r than the first pair (Sayed 1942; Pea et al. 2006; Hoy et al. 2006) A droplet of fluid produced at the tip of dorsal setae can be observed on immature and adult RP M (Welbourn 2009). The function of these droplets is unknown but, anecdotally, is belie ved to be repellent to predators. The female is approximately 0.32 mm long (245 microns), 0.18 mm wide (182 microns) and oval in shape (Nageshachandra and Channabasav anna 1984). The male is distinctly

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16 smaller than the female and triangular in shape. Larvae are 0.18 to 0.20 mm long and the nymphs are 0.18 to 0.25 mm long. The larval stage has 3 pairs of legs in contrast to the protonymph and adults, which have 4 (Wel bourn 2009). Eggs of the RPM are ovoid with one end slightly broadened and smooth, averaging 0.1 mm in length (100 microns) and 0.08 mm (80 microns) in width (NageshaChandra and ChannaBasavanna 1984; Hoy et al. 2006). They are deposited in patc hes of 110-330 eggs on the abaxial leaf surface (Moutia 1958; Kane et al. 2005; Pea et al. 2006). The egg is attached directly to the leaf surface and a slender white stipe, as long as or longer (170-210 m) than the egg and with a coiled tip, is located on t he free end of the egg (Nageshachandra and Channabasavanna 1984). Like the setae of the adults, a droplet of fluid may be present on the tip of the egg stipe. It is not known if the egg produces the droplet or if the adult female provides it during oviposition. The incubation period averages 8 days for fertilized and 7.3 days for unfertilized eggs (Hoy et al. 2006; Welbourn 2009). Studies of RPM development and ecology on the coconut palm were published by Moutia (1958), Nageshachandr a and Channabasavanna (1984), and Zaher et al. (1969). Nageshachandra and Channabasavanna (1984 ) propose that the RPM genetic system is arrhenotoky, which they described as mated females producing only female progeny and unmated females producing only males. In Egypt, the RPM developed all year with a generation time of 3 to 4 weeks at 23 to 28 C (Gerson et al. 1983). The development time for females from egg to adult is 23 to 28 days and the adult life span is approximately 30 days at 23.9-25.7 C. Mated females have a 5 to 6 day preoviposition period, can la y on average 28 to 38 eggs (Zaher et al. 1969), and oviposit over 47 days under laboratory conditions. Unma ted females have a 2-day preoviposition

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17 period, can deposit on average 18 eggs, and oviposit for 40 days. The development time for males from egg to adult is 20 to 22 days, with a lif e span of approximately 26.5 days at 23.9-25.7 C (Nageshachandra and Channabasavanna 1984; Hoy et al. 2006; Welbourn 2009). U.S.A. RPM Regulatory Response In response to the threat of RPM to t he USA and Puerto Rico, a Red Palm Mite Technical Working Group, consis ting of expert s in biological control, acarology and regulation, was created in 2006 (DeFeo 2006). It was given the task of developing a protocol for surveying RPM distribution and a plan for mitigating its spread and damage. The group determined that eradication wa s not a viable option, but, instead, an integrated management plan including biological control and limited use of pesticides would be the most appropriate and feasible management strategy. The United States Department of Agricult ures Animal and Plant Health Inspection Service (USDA-APHIS) Center for Plant Heal th Science and Technology (Plant Pest Quarantine) and Cooperative Ag ricultural Pest Survey (CAPS) conducted field surveys in nurseries, residential areas, marinas, and maritime ports in Florida (only here). Surveys involved visual inspection of hos ts for damage and presence of RPM colonies, as well as sample collection for identification by Dr. Cal Welbourn (FDACS-DPI). Permanent survey sites (Sentinel Sites) were developed in high-risk areas and totaled 579 sites in 11 Florida counties (CAPS surveys 2008). Once RPM is detected in an area, delimiting surveys of the environment and host nur series are conducted. As of 2009, once RPM is detected in a Flor ida nursery or a plant broker site, a Compliance Agreement (required for handling regulated material) must be signed and

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18 followed (Bronson 2009). A low-level RPM tolerance approach has been adopted because complete control using acaricides is no longer considered feasible due to pressures from RPM-infested palms in the ar eas surrounding south Florida nurseries. A combination of approved acaric ide treatment(s) and biological control is suggested to reduce RPM populations (Bronson 2009). Chem ical control tactics and a clean certification are required fo r the movement of host pl ants to a non-infested state (Bronson 2009; Pea et al. 2009). For instance, the miticides approved by Texas for RPM treatment are bifenazate, spiromesifen, acequinocyl, efoxazole, and milbemectin (Bronson 2009). RPM Biological Control The RPM Technical Working Group dete rmined biological control was the best option for widesc ale mitigation of the pes t (Roda et al. 2008). Biological control of arthropod pests is the reduction of populati ons by their natural enemies, thereby reducing the environmental, human health, and application costs associated with pesticide application(s) (Van Drieshe and Bellows 2001). The three approaches commonly used in biological control program s are conservation of natural enemies, classical biological cont rol, and augmentation (McMurt ry 1982; Van Drieshe and Bellows 2001; Naranjo 2001). Multiple studies of natur al enemies have been conducted throughout the range of the RP M within the Eastern and Western Hemispheres (Daniel 1981; Somchoudhury and Sarkar 1987; Gassouma 200 6; Pea et al. 2009). In addition, a classical biological control approach utiliz ing phytoseiid populations collected in Mauritius (by M. A. Hoy) is being eval uated. Augmentation has been considered, but multiple releases of commercially av ailable phytoseiid predators may not be economically feasible for widescale mitigatio n efforts, especially in the landscape.

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19 RPM Natural Enemies In the Eastern Hemisphere, surveys of local natural enemies associated with the pest have been conducted (Daniel 1981; Somchoudhur y and Sarkar 1987; Gassouma 2006). In the United Arab Emirates, two arth ropods are considered to do an excellent job in suppressing the RPM, a phytoseiid mite Cydnoseius negevi Swirski-Amitai that is active all year and a neuropteran ( Chrysopa sp.) that is active during the winters (Gassouma 2006). In 1958, Moutia observed that Amblyseius caudatus Berlese was an important predator of RPM eggs on coconut palms in Mauritius. Somchoudhury and Sarkar (1987) found Oligota sp. (Coleoptera: Staphylinidae), Phytoseiulus sp. (Acari: Phytoseiidae), and Amblyseius sp. (Acari: Phytoseiidae) to be the prevalent RPM natural enemies on coconut palms in India. Daniel (1981) considered a phytoseiid, Amblyseius channabasavanni Gupta, and a co ccinellid beetle, Stethorus keralicus Kapur, to be the most important predators of the RPM on palms in India. Once the RPM was detected in the We stern Hemisphere, its range rapidly increased throughout the Caribbean. During survey work, indigenous natural enemies in Puerto Rico and Trinidad and Tobago were obs erved feeding on the RPM on coconut palms (Pea et al. 2009). These include Amblyseius spp. (Phytoseiidae), the thrips Aleurodothrips fasciapennis Franklin (Thysanoptera: Ph laeothripidae), cecidomyiid (Diptera) larvae, the coccinellid Telsimia ephippiger Chapin (Coleoptera), lacewings (Neuroptera), and entomopathogenic fungi (Hirsutella) (Pea et al. 2009). In Trinidad and Tobago, Amblyseius largoensis (Muma) populations were found to increase in response to the RPM population increases as well as follow the mo vement of the pest within coconut palm canopies (Roda et al. 2008; Pea et al. 2009). The predatory mite A. largoensis also has been found in association with the RPM in Puerto Rico, but it has

PAGE 20

20 not been shown to reduce RPM infestations to an acceptable level (Pea et al. 2009) and the RPM continues to increase it s population density and range throughout the Caribbean (Roda et al. 2009). Therefore, it may be surmised that the natural enemies found associated with the RPM in the Caribbean are not doing an adequate job in suppressing the RPM at this time. In anticipation of the RPM entering Florida, Pea et al. (2009) surveyed predacious arthropods inhabiting palms and bananas in the Homestead area between December 2006 and January 2008. He found Amblyseius largoensis Bdella distincta Baker and Balock (Acari: Bdellidae), Stethorus utilis Horn (Coleoptera: Coccinellidae), an unidentified cecidomyiid, and an unident ified predaceous thrips (Phaeothripidae) associated with the RPM. Pea et al. (2009) question the capability of A. largoensis to maintain the RPM at low densities in S. Florida. Subsequently Dr. Pea and Daniel Carrillo have been studying the efficacy of Florida A. largoensis populations as control agents of the RPM. The observed inability of local natural enemies in the Caribbean to control the RPM populations to date raises concerns as to whether Floridas natural enemies will be sufficient. In the situation where the endemic natural enemy species are unable to control the RPM, a classical biological contro l program could provide additional control. Classical Biological Contro l and the Ph ytoseiidae Classical biological control involves im portation of natural enemies believed to have coevolved with the pest and, t herefore, are believed to be effective at controlling the pest populations (Van Driesche and Bellows 2001). To determine where to collect a candidate for biological control, one must l ook to where the pest is not considered a problem. This is because, in theory, the natural enemies that have evolved with them

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21 keep the pest populations in check. Phytos eiid predators are considered an appropriate choice for biological control due to their small size and ease of rearing. Of the 1000 phytoseiid species known, approximately 30 have been successfully utilized to control tetranychid mites in agronomic systems such as greenhouses, vineyards, citrus, cassava, apple, almonds, cotton, corn and strawberries (McMurtry and Croft 1997; Logan 1981; Hoy 1982, 1985). To determine w hether a phytoseiid or phytoseiid complex will provide adequate co ntrol of a pest, several characteristics must be considered: the intrinsic rate of increase s hould be higher than that of the pest; habitat and prey preferences should be appropriate; and ability to disperse and persist in the new environment is essential (Kiritani and De mpster 1973; Luck et al. 1988; Herren and Neuenschwander 1991; Bellows et al. 1992; Yaninek et al. 1992; Yaninek 2007). An historic example of a successful classi cal biological control program utilizing a phytoseiid predator is that of the cassava green mite Mononychellus tanajoa (Bondar) (Acari: Tetranychidae) (Yaninek and He rren 1988). The cassava green mite was a devastating introduced pest of cassava ( Manihoti esculenta Crantz (Euphorbiaceae)) in Africa. The pest was responsible for direct losses of 60% within a short period of time after its introduction and created a famine (G utierrez et al. 1988; Yaninek and Herren 1988; Lhr et al. 1990; Herren and Neuenschwander 1991). In several regions of South America the cassava green mite was not c onsidered a significant pest of cassava. Those areas were surveyed for candidate natural enemies, resulting in the discovery of over 50 phytoseiids associated with the pes t. Of those 50, three species became established in Africa, two of which spread beyond the original release sites (Yaninek et al. 1992). One, Typhlodromalus aripo DeLeon, was found to be an effective predator of

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22 the cassava green mite and became establis hed in over 20 countries (Yaninek 2007). Onzo et al. (2003) determined T. aripo was the best predator because of its ability to establish, disperse, and persist due partially to its ability to use cassava leaf tips as refugia. The Phytoseiidae The family Phytoseiidae Berlese (Acari: Mes ostigmata or Gamasida) has a worldwide distribution and consists of approximately 1,984 valid species within 89 genera and three subfamilies ( de Moraes et al. 2004b; Keit er and Tixier 2006; Chant and McMurtry 2007; Tixier and Kreiter 2009) The Phytoseiidae have been studied at great length with over 4000 publications between the y ears 1960 to 1994 (Kostiainen and Hoy 1996). Most of these publications focu sed on their role as biological control agents of phytophagous mites (McMurtry 1982; Helle and Sabelis 1985; Pickett and Gilstrap 1986; James et al. 1995; McMurtry and Croft 1997; Gerson et al. 2003; de Moraes et al. 2004a). Phytoseiid Biology The chromosome number of phytoseiids may vary (2n=6 or 8) and most are parahaploid (Hoy 1979; Nelson-Rees et al. 1980; Hoy 1985). The life stages include: egg, larva, protonymph, deutonymph, and adult female or male. The sex ratio varies between species and is affected by population density and food availability, but tends to be female biased. Male phytoseiids sele ct female mates through contact with sex pheromones (Hoy and Smilanick 1979) emitted from macropores located on the dorsal shield of protonymphs, deutonymphs, adult virgins, and gravid females (Rock et al. 1976). Males can be observed hovering ov er females selected for reproduction and

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23 mating occurs with males venter to venter with females (Rock et al. 1976; Hoy and Smilanick 1979). Females will oviposit in clutches (Nage lkerke et al. 1996). Egg production is affected by temperature and substrate, species, biotype, number and species of prey consumed, prey stage consumed, species of pollen and/or combination of plant-animal diet and their relative seasonal availabi lity and abundance (Ragusa and Swirski 1977; Bruce-Oliver and Hoy 1990; Gnanavossou et al. 2003; Emmert et al. 2008). The developmental period for most studied phytose iid species averages 6.08 days at 27C and, depending on the abundance of food, a si ngle female may produce 2-4 eggs / female / day (Ragusa and Swirski 1977; Tani goshi 1981; Bruce-Oliver and Hoy 1990; Gnanavossou et al. 2003; Emmert et al. 2008). If optimal condition s are present, this translates to ca. 50 generations in one year. Prey preference and biology will differ between species of the Phytoseiidae (McMurtry and Croft 1997) and also may oc cur between feral and colonized strains (Knipling 1984; Mwansat 2001). S earching for prey is elicited by physical and chemical cues (kairomones, excreta, exuviae) ( Hislop et al. 1978; Hoy and Smilanick 1981). Phytoseiid populations may be locally adapted to certain hosts, host plants, and climatic regimes (Hoy 1975a, 1975b, 1984). The Phytoseiidae may be composed of lo cal populations, perhaps because they have a relatively low mobility and rely on wi nd currents and movement of plant materials by natural or human means. These characterist ics make phytoseiids susceptible to local selection pressures from the natural envir onment and agricultural practices (Hoy and Knop 1979; Hoy et al. 1979). Local populations may exhibit varying degrees of pesticide

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24 resistance and differences in mating behaviors (H oy and Cave 1985). With this in mind, it may be more useful to approach the utility of phytoseiids as biological control agents by population characteristics instead of the traditional species approach. Amblyseius Am blyseius species are used as biological c ontrol agents in organic and IPM systems to control broad mites ( Polyphagotarsonemus latus (Banks), Acari: Tarsonemidae) (Jovicich 2007), two-spotted spider mite ( Tetranychus urticae Koch), chili thrips ( Scriptothrips dorsalis Hood) (Arthurs et al. 2009), flower thrips ( Frankliniella occidentalis Pergande) (Ship and Wang 2003), greenhouse whitefly ( Trialeurodes vaporariorum (Westw.)), and tobacco whitefly ( Bemisia tabaci Gennadius) (Nomikou et al. 2001). The placement, status, and content of the genus Amblyseius have been in debate for the past 35 years (Denmark and Muma 1989). The genus Amblyseius (Amblyseius ) Berlese (Phytoseiidae: Amblyseiinae) contains 295 species organized into 16 species groups (and one unassigned species group) and is the largest genus within the subfamily Amblyseiinae Muma (Chant and Mc Murtry 2006; Tixier et al. 2008a). The Neotropical region (South and C entral America, Caribbean islands, and Florida) has the highest percentage of total endemic Amblyseiinae species and therefore, is hypothesized as the center of origin for t he subfamily (Chant and McMurtry 2006; Tixier et al. 2008a). Amblyseius largoensis. Native populations of phytoseiid mites associated with the RPM in Florida, Trinidad, Tobago, and Puerto Rico have been morphologically identified as A. largoensis. It is considered to be a type-III generalist predator, consuming prey, pollen, and nectar (Yue and Tsai 1996; McMurtry and Croft 1997). In

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25 addition to association with the RPM in Fl orida and the Caribbean, it has been observed preying on two pests of coconut palm, Aceria guerreronis Keifer (Acari: Eriophyidae) in Brazil (Yue and Tsai 1996) and Rarosiella cocosae Rimando, a synonym of R. indica (Acari: Tenuipalpidae) in the Philippines (Pea et al. 2009). The Amblyseius largoensis species group is geographically widespread and includes 11 closely related species: A. largoensis, A. sakalava Blommers, A. herbicoloides McMurtry and de Moraes, A. herbicolus (Chant), A. nambourensis Schicha, A. phillipsi McMurtry and Schicha, A. fletcheri Schicha, A. vazimba Blommers, A. adhatodae Muma, and A. ankaratrae Blommers (Denmark and Muma 1989). This species group is distinguished morphologically by slight differences in setal lengths, shapes of spermathecal cervices, and the posterior margin of the sternal shield (McMurtry and de Moraes 1984; Denmark and Muma 1989). In 19 52, M. H. Muma collected the A. largoensis female holotype from key lime leaves in Key Largo, Florida, but its original geographic r ange is unknown. The species A. sakalava (female holotype, Madagascar) is the most morphol ogically similar species to A. largoensis among the species currently within the largoensis group but differs in having the spermatheca approximately 1/3 longer and the L2 setae twice as long (Denmark and Muma 1989). Amblyseius largoensis and A. herbicolu s (female holotype, Portugal) are the only two species within the largoensis group that are considered cosmopolitan in distribution (McMurtry and de Moraes 1984; de Moraes et al. 2004b), which could be due to movement of plants by humans. Some distribution overlap is known for A. largoensis and A. herbicolus because both have confirmed records in Florida (USA) (Denmark and Muma 1989). However, the range of A. herbicolus is considered to extend to higher

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26 latitudes (confirmed in CA, USA) than A. largoensis and, although both are reported in Florida, they have not been reported together at the same site (McMurtry and de Moraes 1984). Arthropod Identification In biological control, i dentification is an essential step to selecting the most suitable nat ural enemy, evaluating estab lishment, and improving mass production (Mahr and McMurtry 1979; Gordh and B eardsley 1999; de Len et al. 2006). In 2007, M. A. Hoy collected phytoseiid populations associ ated with the RPM on coconut palms on the island of Mauritius (Figure 1-2). T hese populations were identified as Amblyseius largoensis Muma by Dr. Cal Welbourn (FDACS-DPI, Ga inesville, Florida) and Dr. H. A. Denmark using morphological characters. Populations of phytoseiid predators associated with the RPM also were collected from several si tes in South Florida (Table 1-2) and identified as A. largoensis by Dr. Cal Welbourn and Dr. H. A. Denmark. Interestingly, the RPM is not considered a pe st in Mauritius but in South Florida, its population and range has increased since its detection in 2007. This may suggest that the Mauritius populations are more efficient predators of the RPM than the S. Florida populations. If so, the Maurit ius and Florida populations coul d be biotypes or cryptic species but cannot be discriminated using morphological characters alone. An approach utilizing reproductive compatibility as well as morphological and molecular techniques may be best, especially in discerning bioty pes and cryptic species (de Len et al. 2006), but first, we must answer the question, how do we define a species? Species Concepts A definition of species is considered ess ential for testing biological theories. A species is the basic unit of taxonomy and is made up of evolut ionarily distinct

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27 populations. However, defining a species may not be as easy as identifying a definitive morphological character, reproductive isol ation, or distingui shable genotypes or genomic homology (Mallet 2006). This is becaus e evolution is constant and events such as introgression, hybridization, and cryptic species (phenotypically similar species which do not interbreed) occur (King and Stansfiel d 1985; Prowell et al 2004; Rubinoff and Holland 2005; Buckley et al. 2006). Taxonomic, biological, and phylogenetic operat ional species definitions are just a few of the many used today (King and Stans field 1985). All three species concept approaches require the taxonomist to judge how similar or different populations must be to be considered a species. In the end, the def inition of a species can be different from scientist to scientist, resulting in some being considered "sp litters" and some "lumpers". Each definition has its strengt hs and limitations, however. There are three types of systematic e rrors inherent in every species concept: overestimation and underestimation of the number of species, and misrepresenting their phylogenetic relationships (Adams 1998). The fi rst two errors are, simply, incorrect estimations of the number of species. T he third occurs when an interpretation of phylogenetic relationships among species is incongruent with recovered evolutionary history (Adams 1998). If two populations ar e determined separate species, it is predicted that they are on independent evolutionary trajectories that will continue to be independent in the future (i.e. hybridizat ion will not occur) (A dams 1998). To avoid making such predictions, some scientists appr oach the definition of a species as only a snapshot of constant evolutionary change and that haplogroups may represent

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28 evolutionary lineages (William s et al. 2006; Coleman 20 09). In summation, all operational species definitions have advant ages, caveats, and appropriate applications. Taxonomic The taxonomic species concept is based on morphological or phenetic characters and consist s of phenotypically distinguis hable groups of coexisting organisms. Distinguishable morphological characters develop when divergent natural selection leads to gaps in the distribution of at l east one or more morphological traits (Darwin 1859). Characters such as dorsal seta lengt h, leg chaetotaxy, spermatheca shape, and cheliceral dentition typically are used to identify the Phytoseiidae to species (Chant and McMurtry 1994, 2006). However, there ar e disagreements between taxonomists on what morphological characters should be used as the major criteria for species distinction and genus status (Tixier et al. 2008b, 2008c). In additi on, taxonomists may have opposing views on how similar organisms must be to be defined a species. The Phytoseiidae have relatively few morphologica l characteristics to differentiate between closely related or cryptic species (Edwards and Hoy 1993; Hoy et al. 2000; Da Silva Noronha and de Moraes 2004; Tixier et al. 2006a, 2006c). Identification can be tedious because the time required for mo rphological identification of taxa scales inversely with body size (Lawton 1998). The lack of discrim inating morphological characters within a group can lead to an underestimation of spec ies diversity (Fukami et al. 2004). Furthermore, polyphenisms (intraspecies or population variation) within a species can lead to misidentifications (McMurtry et al. 1976; Hoying and Croft 1977; Da Silva Noronha and de Moraes 2004; Nijhout 2003; Tixier et al. 2006a, 2008b) with negative consequences (Miller and Rossman 1995; Unruh and Woolley 1999).

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29 Following the taxonomic species concept, t he S. Florida and Maur itius populations belong to the species A. largoensis because specimens from both populations were identified as such by two taxonomists Dr. Cal Welbourn and Dr. H. A. Denmark. However, concerns arise when the char acters used to discern between the A. largoensis populations are few. In such sit uations, utilizing the biological and phylogenetic species concepts in addition to the taxonomic ma y help discriminate between the populations. Biological The biological species concept was pr oposed by Mayr (1942) and is based partially on work completed by Dobzhansky (1940) It states that a biological (genetic) species is a group of naturally interbreeding populations that are reproductively isolated from other such species, (King and Stansfield 1997). Reproductive isolation is the combined effect of all barriers to gene flow between divergent populations that are in contact and includes prezygotic and postzygot ic isolating mechanisms (Hoy and Cave 1988; Mallet 2006). Hybrid sterilit y, hybrid inviability, and asso rtative mating will result in partial reproductive isolation. Thus, the loss of interbreeding potential reproductively isolates clades, results in phylogenetical ly distinct taxa, and may lead to greater divergence (Mallet 2006). There are several caveats to the biological species concept. For example, there are vary ing degrees of reproductive isolation, and instances of hybridization and introgression do occur, es pecially under laborator y conditions in nochoice situations (Mantovani and Scali 1992; Salzburger et al. 2002). Also, there are limitations to testing reproductive compatibility in taxa with complex, irregular, or unknown interbreeding and mating behaviors (Adams 1998).

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30 Hybridization studies have been conducted to determine the species status of phytoseiid populations (Croft 1970; McMurtry et al. 1976; McMurtry 1980; McMurtry and Baddi 1989; Da Silva Noronha and de Mo raes 2004). Within the phytoseiids reproductive incompatibility can lead to shriveled eggs, reduced oviposition rates, increased mortality in immature stadia, or reproductive sterility of progeny (Amano and Chant 1978; Mahr and McMurtry 1979; Mc Murtry 1980; Hoy and Knop 1981; Hoy and Standow 1982; Hoy and Cave 1988). Incompatibility may vary within a population. For instance, some individuals may reproduce while others may not (Mahr and McMurtry 1979). In some instances unidirectional inco mpatibility may occur so that while one reciprocal cross (population 1 female x po pulation 2 male) produces viable progeny, the reverse (population 2 female x population 1 male) will not (Hoy and Knop 1981). Incompatibility between populations can develop rapidly, sometimes in as few as 1 or 2 years of reproductive isolation, among phyto seiids (Hoy and Knop 1981; Tanigoshi 1981). In addition to genetic isolation, reproducti ve incompatibility within the Acari can also be caused by the vertically and horizontally transmitted -proteobacteria Wobachia pipientis (A and B) (Hoy and Jeyaprakash 2005). Th is type of incompatibility is coined Cytoplasmic Incompatibility (CI) (Werren et al. 1995; Bandi et al. 1998) and can be unior bi-directional. Unidirectional CI occurs when a cross between an infected male and uninfected female result in mortality of t he embryos. Bidirecti onal CI occurs when a male and female of the same species are in compatible due to infection by different Wobachia strains (Stouthamer et al. 1999). In addition, the incompatibility phenotype will depend on the hosts genetic system. Within the parahaploid system found in most

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31 phytoseiids, Wobachia will cause reduced numbers of diploid females (Johanowicz and Hoy 1998). Returning to the biological species defin ition, if the Maur itius and S. Florida populations are found reproductive ly incompatible (do not mate or mate but do not produce viable progeny) and are not infected with CI-inducing Wobachia, they could be considered separate morphologi cally similar species (crypt ic species). If they are reproductively compatible or exhibit slight in compatibility, it is possible that they are biotypes within the same species and have not undergone complete biological speciation (reproductive isolation). For in stance, reproductive incompatibility in conjunction with differences in morphology developmental rate, and RH tolerance between two populations of Amblyseius addoensis van der Merwe and Ryke from South Africa (McMurty 1980) was cons idered sufficient to justify subspecific (biotype) names (Tanagoshi 1981). Phylogenetic With the advent of mol ecular systemati cs (phylogenetics) a new definition of species has evolved. A specie s will have distinguish able genotypic or genomic clusters (i.e. populations or genetic entities) that ar e stable in sympatry (spatially overlapping populations) or hybrid zones between parapatric forms (t wo geographic entities that abut at the boundaries to thei r range) (Packer and Taylor 1997; Williams et al. 2006; Mallet 2006). Phylogenetics has been used to i dentify cryptic species, used to avoid problems associated with hybridization and t he biological species concept, and used to expand our view of the level of taxonomic diversity present in nature (Williams et al. 2006; Condon et al. 2008). The phylogenetic species concept utilizes percentage sequence divergence to discriminate betw een species. The percentage sequence

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32 divergence calculated may vary between taxa and the genes examined and, therefore, must be examined on a case-by-case basis (H oy et al. 2000; Jeyaprakash et al. 2003; Hoy and Jeyaprakash 2005). Within bacteria, an arbitrary 3% sequence divergence in the 16S ribosomal RNA gene is considered su fficient to confirm species-level differences (Stackebrandt and Goebel 1994; Jeyaprakash et al. 2003). Within the Coleoptera, sequence variation of <0.8 % within the COI mitochondrial gene has been used to establish haplotype differences (E vans et al. 2000; Evans and Lopez 2002). There are several caveats to the phylogenetic species c oncept. The availability of sequence information and availability of primer s will limit the type and number of genes examined in a project. In addition, the cost of equipment and reagents, special training, and availability and quality of DNA can limit the feasibility of phylogenetic projects. However, the cost of analysis and speciali zed training may be just ified in biological control projects when identifying a potentia l classical biological control organism, discriminating biotypes or crypti c species, or identifying the origin of an invasive pest for natural enemy exploration (Dowling 2008). In this study, the phylogenetic species concept will be applied in addition to the taxonomic species concept. The phylogenetic relationship between the Mauritius and S. Florida A. largoensis populations will be evaluated using sequence divergence estimates and Bayesian analysis. Firstly, we will determine sequence divergence from taxa within the same genus or closely re lated genera of phytoseiids available in GenBank. Next, we will infer evolutionary relationships based on sequence homology using Bayesian phylogenetic analysis software.

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33 Molecular Markers To conduct a phylogenetic analy sis, molecular markers appropriate to the question posed must be determined. Molecular mark ers are useful for identifying or distinguishing taxa that are not well-studied are very small, ar e members of cryptic species complexes (Jeyaprakash and Hoy 2002; Ti xier et al. 2006a, 2008; Rowley et al. 2007; Smith et al. 2008), are different geographical populati ons of the same species (Croft 1970; McMurtry 1980; Abou-Setta et. al. 1991; Na vajas et al. 1992, 1994; Hoy et al. 2000; Navajas and Fenton 2000; Williams et al. 2006; Coleman 2009), or are present only in immature stages (Zahler et al. 1995) The molecular diagnostic tool used will depend on the type of question, the cost, ease of use, and the sample size (Hoy 2003). The genome and genome region to be analyzed wil l depend on the level of taxonomic resolution desired (Hillis and Moritz 1990; Avise 1994; Navajas and Fenton 2000; Cruickshank 2002). Caterino et al. (2000) suggested evaluat ing the most widely used molecular markers to create a standard compilation of taxa sequence information for insect systematics. These in clude mitochondrial genes: cytochrome oxidase subunit I ( COI ), the large mitochondrial ribosomal subunit (16S ); and nuclear genes: the small nuclear ribosomal subunit (18S ) and elongation factor-I alpha (EF-I) (Djernaes and Damgaard 2006). When approaching a taxonomic question it is important to choose the correct gene(s) or marker(s) for your taxa. There are several helpful reviews of molecular markers used in acarology (Navajas and Fenton 2000; Cruickshank 2002). Cruickshank (2002) identifies characteristics that s hould be considered when deciding what gene(s) or gene regions to use in a phylogenetic or taxonomic an alysis: rate of

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34 evolution/mutation, single-copy genes, ease of alignment, sufficient number of informative sites (points of mu tation, deletion, and inversions), availability of universal primers, and availability of sequence data from related taxa for comparison. At present, the number of useful genes is limited in the Acari but whole-genome sequencing of taxa currently in progress could change that. The Mitochondrial Genome Kocher et al. (1989) found that mi toc hondrial DNA (mtDNA) could be utilized in phylogenetic evolution/inference in anima ls. Since then, mtDNA has been used in taxonomic and population studies of mites and insects (Navajas et al. 1996; Navajas and Fenton 2000; Toda et al. 2000, 2001; Cruickshank 2002; Evans and Lopez 2002; Jeyaprakash and Hoy 2002; Brown 2004; Tixier et al. 2008c). There are several advantages to using mtDNA in taxonomic studi es: (1) high copy number within the cell makes mt genes easier to amplify by the PCR (Tixier et al. 2008c); (2) almost strict maternal inheritance (see Sunnucks and Hale s (1996) and Zhang and Hewitt (1996) for examples of nuclear copies of mitochondr ial genes) (Cummins et al. 1997; Schwartz and Vissing 2002; Cruickshank 2002); and (3) high levels of sequence divergence for species and biotype or population level resolution due to a high transitional mutation rate (Harrison 1989; Loxdale and Lushai 1998; Moriyama and Powell 1997; Norris et al. 1999; Cruickshank 2002). The mitochondrial genome base content in c helicerates tends to be A+T rich: 80% in the honeybee mite Varroa destructor (Navajas et al. 2002), 76.9% in the phytoseiid M. occidentalis (Jeyaprakash and Hoy 2007), and 64.5% in the scorpion Centruoides limpidus (Davila et al. 2005). The mitochondrial gen e order varies in arachnids (Qiu et al. 2005), Varroa mite (Navajas et al. 2002), in me tastriate and prostriate ticks (Black

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35 and Roehrdanz 1998), and in the phytoseiid M. occidentalis (Jeyaprakash and Hoy 2007). The mitochondrial genome of M. occidentalis is 25 Kb and has one unique region, one duplicated, and one triplicated re gion (Jeyaprakash and Hoy 2007). In addition, the genome may be missing two genes ( ND3 and ND6 ) when compared to other chelicerates (Jeyaprakash and Hoy 200 7). By contrast, the mitochondrial genome of Pseudocellus pearsei (Chelicerata: Ricinulei) is 15.1 Kb in size (Fahrein et al. 2007) and the oribatid mite Steganacarus magnus (Acari: Oribatida) is 13.8 Kb in size (Domes et al. 2008). The Nuclear Genome Nuclear genes may have slower mutation rates than mi tochondrial genes (Lin and Danforth 2003) due to selection for codon usage (Moriyama and Powell 1997). Nuclear protein-coding genes can have problems such as heterozygosity and low copy number (Djernaes and Damgaard 2006). To date seve ral nuclear genome sequencing projects are proposed or in various stages of comp letion, including several soft and hard ticks (Parasitiformes: Ixodidae and Ar gasidae) (Stuart et al. 2007), Tetranychus urticae (Acariformes) (Grb ic et al. 2007), M. occidentalis (Parasitiformes) (Jeyaprakash and Hoy 2009a), and Leptotrombidium spp. (Acariformes) (V an Zee et al. 2007). The nuclear genome of M. occidentalis is quite small at 88-90 5 Mb (Jeyaprakash and Hoy 2009a) and that of T. urticae at 75 Mbp, is even smaller (Dearden et al. 2002; Grbic et al. 2007). At the other end of the size spectrum, the haploid genome size of Ixodes scapularis was determined to be 2262 Mbp (Geraci et al. 2006) and is one-third to twice the size of the human genome (Venter et al. 2001). The Ixodida (soft and hard ticks) have interand intrafamilial variation in genome size and variation within species

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36 (Geraci et al. 2006). The large size of most tick genomes is considered a result of an excessive amount of duplicat ed and repetitive sequences, with 30 to 35% consisting of low-copy nuclear genes (Nene 2009; Van Zee et al. 2007). Until a phytoseiid nuclear genome is sequenced, only a complete inactive mariner transposable element (Jeyaprakash and Hoy 1995) and the ITSI, 5.8S, and ITS2 gene sequences (Navajas et al. 1999) are available from phytoseiids fo r phylogenetic studies. Sequencing acarine genomes, and especially species in the Parasitiformes, will provide the information necessary to produce new primers for phy toseiid nuclear molecular markers. Rate of Evolution Each gene or gene region evolv es at diffe rent rates that are dependent on the effect of changes to the function of the r egion (selection pressure) as well as the genome. For instance, non-coding regions will tolerate more transitional mutations and indels (insertions and deletions) than codi ng genes or rDNA stem regions (Hoy 2003) and insect mitochondrial genes are known to have a higher transitional mutation rate than nuclear genes (Moriyama and Powell 1997). With this in mind, it is important to consider how much variation occurs within a gene or gene region (slow or fast mutation) because this characteristic will affect the ability to produce accurate alignments among taxa and conduct phylogenetic analysis (Webber and Gunasekaran 1993; Hoy 2003). The rate of gene evolution/mu tation will determine the level of resolution obtained for a phylogeny. For example, a gene with a high er rate will be better for resolving genus, species, and biotype differences, while a gene with a slower rate will help resolve higher taxonomic questions (Kocher et al. 1989; Webber and Gunasekaran 1993; Cruickshank 2002; Hoy 2003). Therefore, variability in gene sequences can be a double-edged sword. If a very high level of sequence variab ility exists, an accurate alignment may not

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37 be feasible. If a gene has a low level of variabi lity, the level of taxonomic resolution will be low. The level of resolution a gene may provide will vary within genes, from gene to gene, and among taxa. The nuclear ribosomal RNA genes 18S, 5.8S, and 28S and their internal transcribed spacers ( ITS-1 and ITS-2 ) are commonly used for phylogenetic resolution at the genus, specie s, and biotype or population le vels (Hillis and Dixon 1991; Wesson et al. 1993; Vogler and DeSalle 1994; Fenton et al. 1998; Gotoh et al. 1998; Navajas and Fenton 2000; Santos et al. 200 7; Coleman et al. 2009). However, the phytoseiid nuclear ITS-1 5.8 and ITS-2 regions are much smaller (<0.6 kb) in size and have low nucleotide divergence at the specie s level in comparison to other arthropods (Navajas et al. 1999; Jeyaprakash and Hoy 2002) Therefore, this region cannot be used to answer questions requiring species and biotype resolution within phytoseiids. Single-Copy Genes In phylogenetic analy sis and genotyping studies it is important to know how many copies of a gene exist within the organism s examined. The goal is to compare orthologous (homologous sequenc es separated by a speciation event) genes between taxa and produce an accurate phylogeny based on gene-sequence homologies. However, paralogous genes occur when hom ologous sequences are separated by a duplication event within a genome. In population surveys, paralogous gene sequences can lead to an over estimation of spec ies diversity when the gene sequences in question are orthologous. They can also produce incongruent phylogenetic trees when two gene phylogenies are compared or concatenated (sequence data combined to produce a more robust analysis than the two genes alone). The presence and number of genome duplication events wil l vary between order and even genera (Geraci et al.

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38 2006). It is difficult to predict whether a gene will be single-copy or multi-copy in taxa when little is known about their genomes. Availability of Related Sequence Data and Primer Design Problems in phylogenetic analysis of the Phytoseiidae arise when searching for gene sequences of related taxa for comparis on or for primer design. Currently (Dec. 2009), there are a limited number of mito chondrial gene sequences in GenBank available for constructing phylogenies of the Phytoseiidae: one whole genome (Jeyaprakash and Hoy 2007), eight taxa for the 12S sequence (Jeyaprakash and Hoy 2002; Pham et al. unpub.; Tixier et al. unpub.; Xia et al. unpub.), one taxa for the 16S (Roy et al. 2009), 12 taxa for COI (Tixier et al. 2006b; Jeyaparakash and Hoy 2007; Tixier et al. 2008c; Roy 2009; Tixer et al unpub.), and three taxa for Cytb (Tixier et al. unpub.). Nuclear gene sequences availabl e (Dec. 2009) include: one taxa for Actin genes 1-4 (Jeyaprakash and Hoy 2009a), one taxa for 18S (Pham et al. unpub.; Xia et al. unpub.), one for 28S (Cruickshank and Thomas 1999), seven taxa for ITS-1 5.8S, and ITS-2 (Navajas et al. 1999; Ramadan et al. unpub.; Xia et al. unpub.), and one taxa for elongation factor-1 (EF-I) (Jeyaprakash and Hoy 2009a). Molecular Markers of Interest fo r This Study 12S rRNA The s mall mitochondrial ribosomal subunit ( 12S) has become an ideal tool for phylogenetic resolution of species within the ph ytoseiids. It is known to be fast evolving with higher sequence diver gence (9.5-45%) than the ITS-2 region (1.2-23%) (Navajas et al. 1999; Jeyaprakash and Hoy 2002, 2009b). Jeyaprakash and Hoy (2002) detected high sequence divergences in a portion of the mitochondrial 12S rRNA gene and subsequently utilized this diversity to create diagnostic pr imers for species

PAGE 39

39 discrimination of 6 commercially available phyt oseiids. Results of this study suggest the rRNA 12S gene may have great potential for species resolution. In 2009, Tixier et al. submitted partial 12S sequences to GenBank obtained from Phytoseiulus persimilis Athias-Henriot, P. macropilis (Banks), and P. fragariae Denmark & Schi cha populations found in the United Kingdom, Ar gentina, France, and Spain. For this thesis, these sequences were aligned, the 5 and 3 ends trimmed to a uniform length (Figure 1-3), and the percentage pairwise differences calcul ated (Table 1-5); species differences between P. fragariae and P. persimilis and P. macropilis ranged from 4 to 22%. The population pairwise differences ranged from none to 1% in P. persimilis zero in P. macropilis and 1 to 7% in P. fragariae (Table 1-5). Considering these pairwise difference scores, the mitochondrial 12S gene appears to be informative for species resolution within the phytoseiids (Norris et al. 1999; Murrell et al. 2000; Jeyaprakash and Hoy 2002). Phytoseiulus intraspecies population pair wise differences (Table 1-5) varied from species to species and it is unclear how valuable the 12S gene will be to discriminate between populations or biotypes. There are several advantages to utilizing the 12S rRNA gene in our study to discriminate between possible biotypes or cryp tic species: (1) availability of universal primers; (2) it may be possible to obtain resolution at the species, biotype or population levels; (3) availability of sequence data from other phytoseiid genera in GenBank for sequence comparisons and species divergence comparisons. Cytochrome Oxidase I ( COI ) The mitochondrial prot ein-coding gene cytochrome oxidase I (COI ) has been used in taxonomic and population studies (barcoding project) of arthropods (Simon et al. 1994; Roehrdanz and Degrugillie r 1998) as well as for genus and species resolution in

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40 spider mite, tick, and phytoseiid phylogenetic studies (Navajas et al. 1996; Navajas and Fenton 2000; Toda et al. 2000, 2001; Cruickshank 2002; Evans and Lopez 2002; Salomone et al. 2002; Walter and Campbell 2003; Tixier et al. 2006c). The universal COI primers designed for arthropods by Ka mbhampati and Smith ( 1995) do not anneal due to variability at the priming site in phytoseiids (Jeyaprakash and Hoy 2007; Tixier et al. 2008c). Interestingly, Tixier et al. (2006b, 2008c, 2009) utilized COI primers designed by Navajas et al. (1996) for Tetranychus urticae Koch as a genotyping tool for species within the phytoseiid genus Kampimodromus They obtained sequences approximately 430 bp in length that had averag e pairwise distances of 6, 18, and 20%. The usefulness of the primers designed by Navajas et al (1996) for this study was examined using high-fidelity PCR, but no COI amplification products were obtained from Neoseiulus californicus (McGregor), N. cucumeris Oudemans, and A. largoensis DNA, although an amplification product was obtained from A. swirskii (Athias-Henriot) (Bowman, unpub.). These results indicate that the COI primers designed by Navaja s et al. (1996) cannot be used for this study to survey the A. largoensis populations. Instead, if COI is to be used, new primers must be des igned from regions that are less variable or degenerate primers must be designed. Elongation Factor-I Alpha (EF-I) The nuclear gene encoding the elongation factor-I alpha (EF-I) protein is a likely candidate for phylogenetic analysis of phy toseiids (Klompen 2000; Cruickshank 2002). The EF-I protein is involved in the GTP-dependent binding of charged tRNAs to the acceptor site of the ribosome during trans lation (Webster 1985; Maroni 1993). It is known to be orthologus (single-copy) in some arthropods and sequences are easier to

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41 align among taxa because, as a protein-coding gene, it has a slower rate of mutation/evolution than rDNA or non-coding regions. Therefore, EF-I has been discussed as a possible tool for resolution at the genus level (Friedlander et al. 1992, 1994; Brower and DeSalle 1994, Mitchell et al. 1997; Cruickshank 2002; Arensburger et al. 2004). However, the use of EF-I for phylogenetic resolution is still novel in the Acari and has only been sequenced in a limited number of mite taxa. We are interested in exploring its usefulness for phylogenetic resolution within the phytoseiids. The possible advantages of utilizing EF-I are: (1) it is considered a single-copy nuclear gene in Acari; (2) it is possible to obtain resolution at the genus and species level; (3) there are degenerate primers available that may work for phytoseiids (Jeyaprakash and Hoy 2009a); (4) some sequence data are available from other phytoseiid genera in GenBank. One concern, however, is whether EF-I is actually a single-copy gene in phytoseiids. The genome of Drosophila melanogaster Meigen is known to have two EF-I genes (EF-I F1 and EF-I F2 ) with 93.3% amino acid and 90. 5% nucleotide similarity between the paralogs suggesting an ancient duplication (Hovem ann et al. 1988; Maroni 1993). The Drosophila EF-I F2 reading frame is interrupted by two introns while EF-I F1 has only one intron in the leader sequen ce and none in the coding sequence (Walldorf et al. 1985; Hovemann et al. 1988; Maroni 1993). Currently, other arthropod taxa known to have duplicated EF-I sequences include the honeybee ( Apis mellifera L.) with a 25% difference between paralogs and two introns in the F2 reading frame (Walldorf and Hovemann 1990; Danforth and Ji 1998), ambrosia beetles (Xyleborini) with a ~7% paralog sequence difference (Jordal 2002), and Scolytine weevils (Normark

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42 1996). Danforth and Ji (1998) consider EF-I to be a candidate model system for investigating gene duplication within insects. Within the Phytoseiidae, however, only one EF-I gene is known from Metaseiulus occidentalis (Nesbitt) thus far (GenBank accession no. FJ527739) (Jeyaprak ash and Hoy 2009a). In addition, EF-I paralogs have not been identified in the tick genome. Only one EF-I sequence has been deposited into GenBank (accessi on no. XM_002411102 ) from the Ixodes scapularis Say (black-legged tick) genome project (Jason Me yer, pers. comm.). It is important to note that some of the I. scapularis genome did not make it into the assembly and it is still in the process of annotati on. Therefore, no conclusions can be made at this time as to whether EF-I paralogous sequences exist in the I. scapularis genome. Further investigations into the utility of this gene for genotyping members of the Acari need to be conducted. Random Amplified Polymorphic DNA (RAPD) PCR In 1990, J. Welsh and M. McClelland devel oped Random Amplified Polymorphic DNA (RAPD) PCR as a technique to detect genom e-wide variabilit y. It can be used to determine biotypes or species or cryptic species identity, assess kinship, analyze paternity, estimate genetic variation wit hin populations, and monitor colonization (Hadrys et al. 1992, 1993; Landry et al 1993; Edwards and Hoy 1995a, 1995b; Edwards et al. 1997; Yli-Mattila et al. 2000; Hoy et al. 2000; Hoy 2003; Karam et al. 2008). The biggest advantage to RAPD-PCR is that no prior knowledge of sequence information is required and therefore, it can be used to develop markers for any species (Hoy 2003). RAPD-PCR is based on the ability of single primers (10-mers) to randomly prime and amplify throughout a genome (including both single-copy genes and

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43 repetitive DNA) resulting in several DNA fr agments, which are separated in an agarose gel. The banding pattern generated shows genetic differences and the presence or absence of specific amplified DNA fragments determines polymorphisms. Fifty-five RAPD primers were tested by Haymer (1994) and found to be informative when tested with insect DNA (Hoy 2003). Ed wards and Hoy (1993) found 92 out of 120 RAPD primers to be informative when test ing genetic variation in the parasitoids Trioxys pallidus (Haliday) and Diglyphus begini (Ashm.). Later, Edwards et al. (1997) were able to discriminate two morpholog ically similar phytoseiids Typhlodromalus limonicus (Garman and McGregor) and T. manihoti de Moraes, using RAPD banding patterns. The advantages of RAPDs are: (1) no pr ior sequence knowledge is needed; (2) small amounts of DNA (25 ng/reaction) are required; (3) the lab set-up is easy and cheap; (4) it does not require radioactive detection; and (5) there are many potential markers. There are also disadvantages to using RAPDs: (1) ther e are several possible mechanisms that may lead to an absence of a band (ranging from mutations to too high a concentration of DNA in the reaction); (2 ) there can be homology issues; (3) reliable bands are only from dominant Mendelian traits therefore, recessive traits are not considered except in haploid males (Peener et al. 1993; Edwards and Hoy 1993, MacPherson et al. 1993; Hoy 2003). Steps can be taken to avoid the RAPD-PCR concerns listed above. Firstly, replicate reactions should always be run to i dentify variations in band intensities due to concentration differences in the templa te DNA (Black 1993; Edwards et al. 1997). Secondly, consistent reaction concentrations of DNA, primers, MgCl2, and DNA polymerases must be maintained to avoi d variations in banding patterns (Black 1993;

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44 Ellesworth et al. 1993; MacPherson et al. 1993; Edwards et al. 1997). Inconsistency also can be caused by DNA degradation so fresh material should always be used (Edwards et al. 1997). Differences in banding patterns due to dominant genes can be avoided by using haploid males in a haploi d-diploid genetic system. Lastly, results will vary due to differences in thermocyclers and the brand of DNA pol ymerase used so results may not be fully comparable betw een laboratories (MacPherson et al. 1993; Schierwater and Ender 1993; Edwards et al. 1997). The use of the RAPD-PCR technique on single mites has been limited due to the quality and quantity of DNA extrac tions from single mite s pecimens (Edwards et al. 1997; Jeyaprakash and Hoy 2002). Severa l studies included DNA from several individuals, which can result in biased banding patterns (Mitchelmore et al. 1991; Hadrys et al. 1992; Edwards et al. 1997). Ho wever, Jeyaprakash and Hoy (in press) integrated the use of High-fi delity PCR (Hf-PCR) (Barnes 1994; Jeyaprakash and Hoy 2000; Hoy et al. 2001) and RAPD markers to amplify small amounts of DNA extracted from single mites using a soaking technique m odified from protocol outlined by Boom et al. (1990). Utilizing this technique, the Hf-RAPD PCR protocol could possibly be performed on as little as 10.9-20.0 ng/ l of nucleic acids of good quality. However, it is important to note that there is more mtDNA (high copy number in the cell) than nuclear DNA in the sample and ther efore, the nuclear genome may not be as well represented as in a cleaner and more highly concentrated DNA preparation. The phylogenetic species concept can enable discrimination between arthropod biotypes or cryptic species in biological c ontrol programs. Crypti c species or biotype identification through phylogenetic analysis may assist late r morphological analysis by

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45 increasing detection of slight, but significan t, character differences. A combination of phylogenetic analysis, whole genome analysi s (RAPD-PCR) (Edwards et al. 1997), reproductive compatibility, and intensive morphological study (taxonomic identification) (Tixier et al. 2006a) may be considered an e ffective approach to identify the Mauritius and S. Florida populations as cryptic spec ies or biotypes (Mendelson and Shaw 2002; Arensburger 2004). Research Objectives Research Aim The aim of this research was to dete rmine if the phytoseiids assoc iated with the RPM from Mauritius and S. Florida, morphologically identified as Amblyseius largoensis are biotypes of the same species or cryptic species. Main Objectives The main objective was to determine if the phytoseiid p opulations associated with the RPM from M auritius and South Florida coul d be discriminated using the mitochondrial 12S rRNA gene and the nuclear EF-I gene. Hf-PCR with populationspecific primers and random amplified polymorphic DNA (RAPD) primers are two methods that will be tested to determine if th ey can be used to discriminate between the Mauritius and South Florida populations. The main objective was tested through four experiments: 1. Determine if it is possible to discrim inate between phytoseiids associated with the RPM from Mauritius and S. Florida using the 12S mitochondrial rRNA gene. 2. Determine if the EF-I nuclear gene allows discr imination of phytoseiids associated with the RPM from Mauritius and S. Florida. 3. Evaluate five RAPD markers using Hf-P CR to discriminate between the Mauritius and S. Florida phytoseiid populations.

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46 4. Evaluate 12S rRNA sequences obtained from the Mauritius and S. Florida populations to develop populati on-specific primers.

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47 Table 1-1. A list of Raoiella indica hosts reported in Florida (Welbourn 2009). Family Genus and species Common name(s) Palmae Adonidia merrilli (Becc.) Becc. (=Veitchia H.A. Wendl .) Manila palm, Christmas palm Palmae Aiphanes caryotifolia (H.B.K.) H.A. Wendl. Coyure palm, Ruffle palm, Spine palm Palmae Archontophoenix alexandrae (F. Muell.) H.A. Wendl. & Dr ude Alexander palm, King palm Palmae Beccariophoenix madagascariensis Jum. & H. Perrier Giant windowpane palm Palmae Butia capitata (Mart) Becc. Pindo palm, Jelly palm Palmae Coccothrinax miraguama (H.B.K.) Becc. Miraguama palm Palmae Cocos nucifera L. Coconut palm Palmae Corypha umbraculifera L. Talipot palm Palmae Livistona chinensis (Jacq.) R. Br. ex Mart. Chinese fan palm Palmae Phoenix canariensis Hort. ex Chabaud Canary Islands date palm Palmae Phoenix dactylifera L. Date palm Palmae Phoenix reclinata Jacq. Senegal date palm Palmae Phoenix roebelenii OBrien Pygmy date palm, Roebelenii palm Palmae Pritchardia pacifica B.C. Seem. & H.A. Wendl. Fiji fan palm Palmae Pseudophoenix sargentii H.A. Wendl. ex Sarg. Buccaneer palm, Sargents cherry palm Palmae Ptychosperma elegans (R. Br.) Blume Solitaire palm, Alexander palm Palmae Ptychosperma macarthurii (H.A. Wendl.) Nichols Macarthur palm Palmae Schippia concolor Burret Silver pimento palm Palmae Syagrus romanzoffiana (Cham.) Glassman Queen palm Palmae Thrinax radiata Lodd. ex J.A. & J.H. Schultes Florida thatch palm Palmae Veitchia spp. H.A. Wendl. Manila palm Palmae Washingtonia robusta H.A. Wendl. Mexican fan palm Palmae Wodyetia bifurcata A.K. Irvine Foxtail palm Musaceae Heliconia spp. None Musaceae Musa spp. Banana, Plantain Zingiberaceae Alpinia zerumbet (Pers.) B.L. Burtt & R.M. Sm. Shell ginger, Pink porcelain lily Denotes hosts with observed high populations (Farzan Husin, unpub. data referenced in Roda et al. 2008).

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48 Table 1-2. A list of viruses transmitted by Brevipalpus mites to their host plants (Acari: Tenuipalpidae) (Kitajima et al. 2003). Host Virus Brevipalpus spp. Orange, mandarin ( Citrus spp.) Leprosis californicus obovatus phoenicis Coffee ( Coffea arabica ) L. Ringspot phoenicis Orchids Fleck, Ringspot californicus phoenicis Ligustrum sp. Ringspot obovatus Green spot phoenicis Passiflora edulis Sims, F. flavicarpa Degener Green spot phoenicis Hibiscus rosa-sinensis L. Green spot phoenicis Chlorotic spots phoenicis Hibiscus syriacus L. Green spot phoenicis Hibiscus schizopetalus Hook f. Green spot phoenicis Turks hat ( Malvaviscus arboreus Cavanilles) Ringspot phoenicis Solanum violaefolium Schott Ringspot phoenicis Solanum actinophylla Harms Ringspot phoenicis Ivy ( Hedera canariensis Willdenow) Green spot phoenicis Bleeding heart ( Clerodendrum speciosum Guerke)Chlorotic spots phoenicis Ball from dragon ( Clerodendrum thomsonae Balfour) Green spot phoenicis Brunfelsia sp. Green spot phoenicis Night jasmine ( Cestrum nocturnum L.) Chlorotic spots phoenicis Pelargonium ( Pelargonium sp.) Green spot phoenicis Kings mantle ( Thunbergia erecta (Bentham) T. Anderson ) Green spot phoenicis Mexican sage ( Salvia leucantha Cavanilles) Green spot phoenicis Pittosporum sp. Ringspot phoenicis

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49 Table 1-3. List of South Florida Amblyseius 'largoensis' collection sites. Date Plant host Location GPS coordinates Colony 01/ 2007 Banana Lake Worth 26.620764, -80.058891 Florida 1 03/ 2007 Coconut palm Lake Wo rth 26.569519, -80.050936 Florida 2 04/ 2007 Coconut palm Lake Wo rth 26.615649, -80.048417 Florida 3 04/ 2008 Coconut palm Hollyw ood 26.047855, -80.164667 Florida 4

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50 Table 1-4. The partial 12S rRNA sequences from Phytoseiulus spp. submitted by Tixier et al. (2009) to GenBank. Species include P persimilis Athias-Henriot, P. macropilis (Banks), and P. fragariae Denmark & Schicha individuals from populations in the United Kingdom (U K), Argentina (AR), France (FR), and Spain (SP) Phytoseiulus spp. Isolate GenBank accession fragariae Argentina (AR) 29 FJ985124 AR 30 FJ985125 AR 31 FJ985126 AR 32 FJ985127 AR 33 FJ985128 macropilis AR 34 FJ985129 AR 35 FJ985130 AR 36 FJ985131 AR 37 FJ985132 AR 38 FJ985133 persimilis France (FR) 10 FJ985106 FR 12 FJ985107 FR 13 FJ985108 FR 14 FJ985109 FR 15 FJ985110 FR 16 FJ985111 FR 17 FJ985112 FR 18 FJ985113 Spain (SP) 1 FJ985096 SP 2 FJ985097 SP 3 FJ985098 SP 4 FJ985099 SP 5 FJ985100 SP 6 FJ985101 SP 7 FJ985102 SP 8 FJ985103 SP 9 FJ985104 SP 11 FJ985105 United Kingdom (UK) 19 FJ985114 UK 20 FJ985115 UK 21 FJ985116 UK 22 FJ985117 UK 23 FJ985118 UK 24 FJ985119 UK 25 FJ985120 UK 26 FJ985121 UK 27 FJ985122 UK 28 FJ985123

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51 Table 1-5. Corrected pairwis e distances between the partial 12S rRNA sequences from Phytoseiulus spp. submitted by Tixier et al. (2009) to GenBank. Phytoseiulus persimilis P. macropilis and P. fragariae individuals from populations in the United Kingdom (U K), Argentina (AR), Franc e (FR), and Spain (SP) using PAUP 4.0b8 with Kimura 2-parameter and Among-site rate variation distance settings. Distance measured by using a scale of 0 1.

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52 Table 1-5. Continued.

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53 Figure 1-1. Map of current Raoiella indica range in Florida (Saeger 2010).

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54 Figure 1-2. M ap of Mauritius Amblyseius 'largoensis' collection sites ( http://www.bwinternships.com/jo/i mages/stories/mauritiu s%20map.jpg).

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55 10 20 30 40 50 60 70 80 90 . . P. fragariae AR29 CTTTTTAAATATCTTAGAGGAA-TTTATTCTG-TAAAGGATT-TAACACCAACAATCTTACTTTTGTTTGTATTTAAACAATTTACATAC AR30 CTTTTTAAATATCTTAGAGGAA-TTTATTCTG-TAAAGGATTCTAACACCAACAATCTTACTTTTGTTTGTATTTAAACAATTTACACAC AR31 CTTTTTAAATATTTTAGAGGAA-TTTATTCTG-TAAAGGATT-TAACACCAACAATCTTACTTTTGTTTGTATTTAAACAATTTACACAC AR32 CTTTTTAAATATTTTAGAGGAA-TTTATTCTG-TAAAGGATT-TAACACCAAAA-TCTTACTTTTGTTTGTATTTAAACAATTTACATAC AR33 CTTTTTAAATATTTTAGAGGAA-TTTATTCTG-TAAAGGATT-TAACACCAA-AATCTTACTTTTGTTTGTATTTAAACAATTTACATAC P. macropilis AR34 CTTTTTAAATATTTTAGAGGAA-TTTATTCTG-TAAAGGATT-TGACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC AR36 CTTTTTAAATATTTTAGAGGAA-TTTATTCTG-TAAAGGATT-TGACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC AR37 CTTTTTAAATATTTTAGAGGAA-TTTATTCTG-TAAAGGATT-TGACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC AR38 CTTTTTAAATATTTTAGAGGAA-TTTATTCTGGTAAAGGATT-TGACACCAAAAATCTTACTTTTATTTGTATTAAAACAGTTTACATAC P. persimilis FR10 CTTTTTAAATATTTTAGAGGAAATTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC FR12 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC FR13 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC FR14 CTTTTTAAATATTTTAGAAGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC FR15 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC FR16 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC FR17 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC FR18 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP1 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP2 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP3 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP4 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP5 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP6 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP7 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATCC SP8 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP9 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC SP11 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACAT-C UK19 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC UK20 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC UK21 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC UK22 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC UK23 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC UK25 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC UK26 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC UK27 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC UK28 CTTTTTAAATATTTTAGAGGAA-TTTATTCTA-TAAAGGATT-TAACACCAAAA-TCTTACTTTTATTTGTATTAAAACAGTTTACATAC Figure 1-3. CLUSTAL X DNA alignment for partial 12S rRNA sequences from Phytoseiulus spp. submitted by Tixier et al. (2009) to GenBank. Species include P persimilis Athias-Henriot, P. macropilis (Banks), and P. fragariae Denmark & Schicha individuals from populations in the United Kingdom (UK), Argentina (AR), France (FR), and Spain (SP).

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56 100 110 120 130 140 150 160 170 180 . . P. fragariae AR29 CTCTATTTTAAAATATCTTA-GATATATTTTAGCAATACTTTTATATGAAGTAGGTTTTTAGGTAAAGGTGTAGTTTATACAAAAAGAAT AR30 CTCTATTTTAAAATATt-TA-GATATATTTTAGCAATACTTTTATATGAAGTAGGTCTTTAGGTAAAGGTGTAGTTTATACAAAGAGAAT AR31 CTCTATTTTAAAATATt-TA-GATATATTTTAGCAATACTTTTATATGAAGTAGGTTTTTAGGTAAAGGTGTAGTTTATACAAAGAGAAT AR32 CTCTATTTTAAAATATT-TA-GATATATTTTAGCAATACTTTTATATGAAGTAGGTTTTTAGGTAAAGGTGTAGTTTATACAAAAGAA-T AR3 CTCTATTTTAAAATATt-TA-GATATATTTTAGCAATACTTTTATATGAAGTAGGTTTTTAGGTAAAGGTGTAGTTTATACAAA-AGAAT P. macropilis AR34 CTCTATTTTAGAATATT-TA-GATATATTCTAATAATACTATTATCTATAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T AR36 CTCTATTTTAGAATATT-TA-GATATATTCTAATAATACTATTATCTATAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T AR37 CTCTATTTTAGAATATTCTACGATATATTCTAATAATACTATTATCTATAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAAGT AR38 CTCTATTTTAGAATATT-TACGATATATTCTAATAATACTATTATCTATAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAAGT P. persimilis FR10 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T FR12 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T FR13 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T FR14 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T FR15 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T FR16 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T FR17 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T FR18 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP1 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP2 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP3 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP4 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP5 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP6 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP7 CTCTATTTTAAAATATT-CA-A-TATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA- SP8 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP9 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T SP11 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA- UK19 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T UK20 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T UK21 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T UK22 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T UK23 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T UK25 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T UK26 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T UK27 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T UK28 CTCTATTTTAAAATATT-TA-AATATATTTTATTAATACTATTATTT-TAGTAATTTTTTAGGTAAAGGTGTAGCTTATATAAAAGAA-T Figure 1-3. Continued.

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57 190 200 210 220 230 240 250 260 270 . . P. fragariae AR29 GAAATAAATGTATATTAATTATTATTTAGT--AACGAGATTTGAATGTAAACTAAACATTAAGTAAAATTTCAAA-GTAAAATTAGTTTA AR30 GAAATAAATGTATATTAATTATTATTTAGGTAAGCGAGATTTGAATGTAACCAAAACAGTAAGTAAAACTTCAAA-GTAAAATTAGTTTC AR31 GAAATAAATGTATATTAATTATTATTTAGT--AACGAGATTTGAATGTAACCTAAAAATTAAGTAAAACCTCAAAAGTAAAATTAGTTTA AR32 GAAATAAA-TTATATTAATTATTATTTAG--TAACGAGATTTGAATGTAAATAAAA-ATTAAGTAAAATTTAAAA-GTAAAATTAGTTTA AR33 GAAATAAA-TTATATTAATTATTATTTAGT--AACGAGATTTGAATGTAAATAAAA-ATTAAGTAAAATTTAAAA-GTAAAATTAGTTTA P. macropilis AR34 GAAATAAA-TTATAATAATTTTAATT-ATTTTAACAAAATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA AR36 GAAATAAA-TTATAATAATTTTAATT-ATTTTAACAAAATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTAGA AR37 GAAATAAACTTATAATAATTTTAATT-ATTTTAACAAAATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA AR38 GAAATAAACTTATAATAATTTTAATT-ATTTTAACAAAATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA P. persimilis FR10 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA FR12 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA FR13 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA FR14 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA FR15 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA FR16 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA FR17 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA FR18 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP1 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP2 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP3 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP4 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP5 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP6 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP7 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP8 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP9 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA SP11 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK19 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK20 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK21 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK22 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK23 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK25 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK26 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK27 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA UK28 GAAATAAA-TTATAATAATTTTTATT-ATTTTAACGAGATTTAAATGTAAATAATA-ATTTAGTAAAATTTAAAA-GTAAAATTAGTATA Figure 1-3. Continued.

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58 280 290 300 P. fragariae AR29 TC-CGGCGAATTTGAATATAATTTTAT-AAGTGTACATA AR30 CCACGGCGAATTTGAGTATAATTTTAT-AAGTGTACATA AR31 CCACGGCGAATTTGAATATAATTTTAT-AAGTGTACATA AR32 TC-AGGCGAATTTGAATATAATTTTAT-AAGTGTACATA AR33 TC-AGGCGAATTTGAATATAATTTTAT-AAGTGTACATA P. macropilis AR34 TT-TAGCTGATTTGAATTAGATTTTAT-AAGTGTACATA AR36 TT-TAGCTGATTTGAATTAGATTTTAT-AAGTGTACATA AR37 TT-TAGCTGATTTGAATTAGATTTTAT-AAGTGTACATA AR38 TT-TAGCTGATTTGAATTAGATTTTAT-AAGTGTACATA P. persimilis FR10 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA FR12 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA FR13 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA FR14 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACCTA FR15 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA FR16 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA FR17 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA FR18 TT-TAGCCAATTTGAATTAGATTTTAT-AAGTGTACATA SP1 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP2 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP3 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP4 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP5 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP6 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP7 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP8 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP9 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA SP11 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA UK19 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA UK20 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA UK21 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA UK22 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA UK23 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA UK25 TT-TAGCTAATTTGAATTAGATTTTATTAAGTGTACATA UK26 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA UK27 TT-TAGCTAATTTGAATTAGATTTTAT-AAGTGTACATA UK28 TT-TAGCTAATGTGAATTAGATTTTAT-AAGTGTACATA Figure 1-3. Continued.

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59 CHAPTER 2 MOLECULAR DISCRIMINATION OF PHYT OSEIIDS ASSOCIATED WITH THE RED PALM MITE FROM MAURITIUS AND SOUTH FLORIDA Introduction The red palm mite (RPM), Raoiella indica Hirst (Acari: Tenuipalpidae), is an invasive pest occurrin g mostly in tropical and subtropical climates. It was originally described from coconut palms in Coimbatore India (Hirst 1924) but is believed to have dispersed from the Middle East (Dowling et al. 2008). The RPM was first detected in the Western Hemisphere during survey work on t he Caribbean islands of Martinique and St. Lucia in 2004 (Kane et al. 2005) and by 200 7 it spread throughout the Caribbean and was detected in Floridas Palm Beach c ounty (Pea et al. 2006). As of 2009, the RPM has been detected in six South Florida count ies: Broward, Lee, Martin, Miami-Dade, Monroe, and Palm Beach (Welbourn 2009). A menace to banana and coconut production in the Caribbean and the ornamental horticulture and landscape industries in Florida, the RPM is known to attack 27 pal m hosts (Palmaceae) including the areca palm (Dypsis lutescens H. Wetland), coconut palm ( Cocos nucifera L.), canary island date palm ( Phoenix canariensis Chabaud), date palm ( Phoenix dactylifera L.), as well as bananas, plantains (Musaceae: Musa spp.), and gingers (Zingiberaceae: Zingiber spp.) (Welbourn 2009). The native natural enemies in Sout h Florida have not provided adequate suppression of the RPM (Pea et al. 2009). A classical biological c ontrol program could provide additional control. In 2007, Dr. M. A. Hoy colle cted phytoseiid populations preying on the RPM on coconuts from different climatic regions of Mauritius for importation and study in quarantine in the D epartment of Entomology and Nematology, University of Florida, Gainesville. The isla nd of Mauritius was chosen for natural enemy

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60 collection for several reasons, the RPM is not considered an economic pest, and the climactic conditions are similar to South Florida and the Caribbea n, and the cooperation by government officials. The import ed Mauritius predator populations were morphologically identified as Amblyseius largoensis Muma by taxonomists Dr. Cal Welbourn (FDACS-DPI, Gainesvill e, Florida) and Dr. H. A. D enmark. Native populations of phytoseiid mites associated with the RPM in South Florida, Tr inidad, Tobago, and Puerto Rico also have been mor phologically identified as A. largoensis (Pea et al. 2009) yet they are not considered to provide adequate control. Amblyseius largoensis is considered cosmopolitan in distribution (McMurtry and de Moraes 1984; de Moraes et al. 2004b) and is a type-III generalist predator (consumes prey, pollen, and nectar) (Yue and Tsai 1996; McMurtry and Croft 1997). The largoensis group consists of 11 closely related species distinguished morphologically by slight differences in setal lengths, shapes of spermathecal cervices, and the posterior margin of the sternal shield (McMurtry and de Moraes 1984; Denmark and Muma 1989). In biological control, correct identification is an essential step for conducting biological studies and selecting the most suitable natural enemy (Mahr and McMurtry 1979; de Len et al. 2006) and incorrect taxonomic ident ification of phytoseiid species is not uncommon (McMurtry et al. 1976; Hoying and Croft 1977; Miller and Rossman 1995; Unruh and Woolley 1999; Da Silva Noronha and de Moraes 2004; Nij hout 2003; Tixier et al. 2006a, 2008b). The populations of phytoseiids associated with the RPM from Mauritius and S. Florida were identified as A. largoensis using morphological characters. However, there are few taxonomic charac ters available to identify Amblyseius species (McMurtry

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61 and de Moraes 1984; Denmark and Muma 1989) and cryptic species are known to occur within the Acari (Tixier et al. 2006c ). In such instances, investigations incorporating informative molecular marker s with morphological taxonomy can provide additional characters for comparing taxa (Navajas and Fenton 200 0; Toda et al. 2000; Navajas et al. 2002; Walton et al. 2004; Tixier et al. 2006a, 2006b) In this investigation, sequence divergences and Baye sian analysis of partial 12S rRNA and EF-I gene sequences were used to infer phylogenetic relationships between the two populations (Jeyaprakash and Hoy 2002; Mallett et al. 2005; Dabert 2006; Laum ann et al. 2007). The 12S gene is known to be fast evolving with high sequence divergence (9.5-45%) compared to the commonly used ITS-2 (1.2-23%) mitochondrial gene (Navajas et al. 1999) and has potential for species resolution within the Phytoseiidae (Jeyaprakash and Hoy 2002, 2009b). The nuclear protein-coding gene EF-I is considered to be a singlecopy gene within the chelicerates and may be useful to resolve genusand specieslevel questions (Cruickshank, 2002; Jeyaprakash and Hoy, 2009a). In addition to sequence analysis, Neighbor-Joining analy sis of High-fidelity-RAPD-PCR was performed to characterize the whole genome of the Mauritius and the S. Florida populations (Edwards and Hoy 1993, 1995a, 1995b; Edwards et al. 1997). To evaluate the degree of divergence in 12S rRNA and EF-I sequences in other phytoseiid mites, sequence divergences were calculated using dat a available in GenBank. In addition, we utilized the variability between the 12S rRNA Mauritius and S. Florida colonies to develop population-specific primers to provide a rapid and easy method for evaluating the establishment, dispersal, and efficacy of the phytoseiid should it be released (de Len et al. 2006). Utilizing the quickly evolving 12S mitochondrial gene, the single-copy

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62 EF-I nuclear gene, and RAPD-PCR markers may provide strong speciesand genuslevel resolution to the phylogenetic com parison of the Maurit ius and S. Florida Amblyseius largoensis populations. Methods Phytoseiid Collection and Colony Maintenance Five Ambly seius populations found associated with the RPM were collected with a permit (#P526P-07-04232, USDA-APHIS-Plant Protection and Quarantine) by M. A. Hoy from three climactic regions within Mauritius in October 2007, including Flic en Flac (-20.270088, 57.375069), North of Port Louis (-20.144595, 57.504845), and three within Trou de Douce (-20.231597, 57.788429) (Figure 1-2). Amblyseius populations associated with the RPM also were collected from four loca tions within South Floridas Broward and Palm Beac h counties (Table 1-3) and were identified as A. largoensis In addition, A. swirskii Neoseiulus californicus N. cucumeris (Syngenta Bioline Inc., Oxnard, CA), and Metaseiulus occidentalis (a cultured colony originally from the western U.S.A., Dr. M. A. Hoy laboratory, Gainesville, Flor ida), were collected in 100% EtOH, and stored at -80C for DNA extraction and served as comparisons of phytoseiid genetic diversity. The Florida and Mauritius colonies were maintained on paraffin-coated arenas that were placed on wet cotton with a water moat in a plastic dish. Due to difficulties in rearing large populations of R. indica in quarantine Tetranychus urticae Koch nymphs and adults were used as a primary food source, but all stages of R. indica were provided periodically as well Cattail ( Typha latifolia L.) pollen and tissue paper strips (Kimwipes, Kimberly-Clark Corp., Irving, TX ) soaked with diluted honey were provided as alternative food sources. The T. urticae were cultured on pinto bean ( Phaseolus

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63 vulgaris L.) plants grown in a greenhouse ma intained at 28-30C at 30-40% RH. Raoiella indica mites were collected with a permit fr om the Florida Division of Plant Industry, Department of Agri culture and Consumer Services from four sites within Broward and Palm Beach countie s and reared in the University of Floridas Department of Entomology and Nematology quarantine facilities (Gainesville, Florida) on cut coconut palm leaves placed on wet co tton in plastic dishes at 23-28C and a 55-60% RH, with a 16L: 8D photoperiod. Excess RPM were provided to the Mauritius and S. Florida colonies when available. DNA Extractions Clean genomic DNA was extracted from a pool of 30 starved (Johanowicz and Hoy 1996) adult females from each of the fi ve Mauritius and the four S. Florida Amblyseius largoensis colonies separately, as well as A. swirskii Neoseiulus californicus N. cucumeris (obtained from Syngenta Bioline Inc., CA), and Metaseiulus occidentalis (cultured colony, Dr. M. A. Hoy labor atory, Gainesville, Florida). DNA was extracted using Puregene reagents (QIAGEN, Valencia, CA) and purified using a Tip-20 DNA Purification Kit (QIAGEN, Valencia, CA ) following the procedures outlined by the manufacturer. DNA was extracted from the Mauritius and S. Florida populations as needed over the course of 1 year and 10 mont hs, which translates to approximately 92 generations. The quantity and qualit y of purified extracted nucleic acids was examined using a BioPhotometer (Eppendo rf North America, Westbury, NY) by measuring the absorbance at 260 and 280 nM wavelengths.

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64 Amplification and Sequenci ng of Partial Mitochondrial 12S rR NA and Nuclear EFI Genes High-fidelity PCR was performed on each pooled DNA sample using universal 12S and EF-I primers. The primers us ed to amplify the partial 12S rRNA sequences were: SR-J-14199 5-TACTATGTTACGACTT AT-3 (18-mer) and SR-N-14194 5AAACTAGGATTAGATACCC-3 (19-mer) (Tabl e 2-1) (Kambhampati and Smith 1995). The degenerate primers used to amplify the partial EF-I gene sequences were forward 5-GAYTTYATHAARAAYATGAT-3 (20-mer) and reverse 5-GCYTCRTGRTGCATYTC3 (17-mer) (Table 2-1) (Jeyaprakash and Hoy 2009a). The EF-I degenerate primers used were chosen from several designed by Jeyaprakash and Hoy (2009a) after testing combinations of forward and reverse pr imers. The High-fide lity PCR protocol incorporated three linked profiles; (i) 1 cycle of denaturation at 94C for 2 min, (ii) 10 cycles of denaturation at 94C for 10 sec, an nealing at 43C for 30 sec, and extension at 68C for 1 min, and (iii) 25 cycles of denaturation at 94C fo r 10 sec, annealing at 43C for 30 sec, and extension at 68C for 1 min, plus an additional 20 sec added for every consecutive cycle. The EF-I PCR products (507 bp) were gel purified using the QIAGEN QIAquick Gel Extraction Kit (QIAGEN In c., Valencia, CA) to avoid preferential ligation of primer-dimers during cloning. The 12S PCR products (369 bp) were cleaned using a QIAGEN column following the pr ocedure suggested by the manufacturer (QIAGEN Inc., Valencia, CA). Next, A-tailin g reactions with purified PCR products as template were performed to increase the efficiency of cloning reactions. The A-tailed PCR products were cloned using the Invi trogen pCR2.1 TOPO F Cloning Kit (Invitrogen, Carlsbad, CA) following the protocol recommended by the manufacturer. Plasmid DNA was extracted using the QIAG EN Mini Plasmid Prep Kit according to

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65 procedures outlined by the manufacturer (Q IAGEN, Valencia, CA). Restriction digestions were performed on the pCR2.1 TOPO plasmids extracted to confirm positive transformation with the sequence of interest. Plasmids containing the correct inserts were submitted five at a time from each Mauritius and S. Florida location for Sanger sequencing by the University of Florida Interd isciplinary Core Faci lity for Biotechnology Research (ICBR) (Gainesville, Florida). A to tal of 20 plasmids containing the partial 12S rRNA gene and 10 plasmids for the partial EF-I gene were sequenced for each of the five Mauritius and four S. Florida colonies. Sequence Editing and Alignment The 12S and EF-I gene sequences obtained from t he ICBR were checked for errors by eye and by using the sequence analysis program MacDNASIS version 3.7 (Hitachi, San Bruno, CA, USA). The sequenc es were compared with similar sequences in the GenBank database usi ng the BLAST (Basic Alignment Search Tool) search program (Altschul et al. 1997). The most si milar sequences obtaine d from the GenBank BLAST results were included in the alignmen t for constructing the phylogenies (Tables 2-3, 2-4, 2-7, 2-8) (H all 2001; Hoy 2003). The percent age A-T content of sequences was calculated using the online Oligo Calc: Oligonucleotide pr operties calculator (Kibbe 2007). The partial EF-I DNA sequences were translated into amino acids to confirm the presence of the open r eading frame (ORF) and aligned (Figure 2-2) (to achieve maximum levels of conservation for asse ssing the degree of similarity and homology) after setting the parameters for pairwise alignment (gap ope ning = 15, gap extension = 6.66) and multiple alignment s (gap opening = 15, gap extens ion = 6.66) and protein weight matrix (Gonnet) (Hall 2001). The resulting alignment was checked for deletions

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66 using CLUSTALX multi-sequence alignmen t software (Larkin et al. 2007). The 12S and EF-I nucleotide data sets were aligned using CLUSTAL X v. 1.83 (Thompson et al. 1997) (Figures 2-1 and 2-3) to achieve maximum levels of identity for assessing the degree of similarity and the possibility of homology after setting the parameters for pairwise alignment (gap opening = 15, gap ext ension = 6.66) and multiple alignments (gap opening = 15, gap extension = 6.66) (H all 2001). To ensure a clearly resolved phylogenetic tree, the alig nments created by CLUSTALX were examined and, if necessary, edited manually using the MacClade software package. Phylogenetic Analysis Phylogenet ic analysis to obtain posteri or probabilities was performed using MrBayes 3.1.2 software package for Baye sian Markov chain Monte Carlo analysis (MCMC) (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). Modeltest 3.7 (Posada and Cran dall 1998) was used to select a best-fit model for alignments from gene data sets. The hierarchal Likeliho od Ratio Tests (hLRTs) and Akaike Information Criterion (AIC) parameters obtained for each dataset was used to preset the analyses in MrBayes. The hLR Ts and AIC parameter s generated for the 12S rRNA best-fit model (TVM + I + G) were [Base = (0.4212 0.1098 0.0851) Nst = 6 Rmat = (2.8564 10.5542 4.4554 1.2205 10.5542) Rates = gamma Shape = 1.3408 Pinvar = 0.0905]. The hLRTs and AIC param eters generated for the first EF-I best-fit model (TIMef + I + G) were [Bas e = equal Nst = 6, Rmat = (1.0000 2.3823 1.5549 1.5549 4.5442) Rates = gamma Shape = 1.4683 Pi nvar = 0.4559]. The hLRTs and AIC parameters generated for the second EF-I best-fit model (TrN + G) were [Base = (0.2375 0.2836 0.2641) Nst = 6 Rmat = (1 .0000 2.0910 1.0000 1.0000 7.1734) Rates =

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67 gamma Shape = 0.2752 Pinvar = 0] and the hLRTs and AIC parameters generated for the unknown EF best-fit model (TIMef + I + G) we re [Base = (equal) Nst = 6 Rmat = (1.0000 3.3897 1.9950 1.9950 6.6086) Rate s = gamma Shape = 0.7516 Pinvar = 0.3351]. The posterior probabi lities obtained from 1,000,000 generations were used to support each branch in the Bayesian trees. Pairwise Distance Analysis Pairwise distances were calculated for the 12S and EF-I sequence alignments using the PAUP 4.0b10 softwar e package (Swofford 2003). The 12S rRNA sequence pairwise distances were calculated with optim ality criteria set at [Rates = gamma, Shape = 1.3408, Kimura-2 parameters]. Pair wise distances were calculated for EF-I nucleic acid sequences with optimality criteria set at [R ates = gamma, Shape = 1.4683, Kimura-2 parameters] and amino acid pairwise distances were analyzed with distance settings at mean character difference and Am ong-site rate variation (Jeyaprakash and Hoy 2002). High-fidelity-RAPD-PCR A preliminary test of five random pr imers (UB C Biotechnology Laboratory, Vancouver, BC, Canada) (Table 2-1) for pr oduction of distinctive and consistent banding patterns was conducted on pooled extr acted DNA from each of the five Mauritius (25 mites total) and of each of the four S. Florida Amblyseius populations (20 mites total) using a High-fidelity RAPD-P CR (Hf-RAPD-PCR) protocol designed by Jeyaprakash and Hoy (in press). Three Hf -RAPD-PCRs of the fi ve primers were conducted on the 2 pooled DNA samples as well as DNA from M. occidentalis A. swirskii, N. californicus and N. cucumeris to evaluate whether the banding patterns

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68 were repeatable and consistent for each species or colony. Hf-RAPD-PCR was performed in 50 mM Tris (pH 9.2), 16 mM ammonium sulfate, 1.75 mM MgCl2, 350 M each of dATP, dGTP, dTTP, and dCTP, and 400 pM of the 10-mer primer, 1 U of Accuzyme, and 5 U of Taq DNA polymerase. The Hf-RAPD-PCR was performed incorporating three linked prof iles; (i) one cycle of denaturation at 94C for 2 min, (ii) 10 cycles of denaturation at 94C for 10 sec, an nealing at 36C for 30 sec, and extension at 68C for 2 min, and (iii) 25 cycles of denaturation at 94C fo r 10 sec, annealing at 36C for 30 sec, and extension at 68C for 2 min, plus an additional 20 sec added for every consecutive cycle. Electrophoresis wa s performed using a 2% TBE (Tris-borateEDTA) agarose gel (14 cm x 10.2 cm) and 1% TBE electrophoresis buffer containing ethidium bromide to separate and stain DN A amplicons. The DNA molecular weight marker XIV (Roche Diagnostics, Indianapolis IN) (100 bp to 2650 bp) was used to determine band size. Bands were visualized and photographed under UV illumination. The two informative primers that gave di stinctive and consistent banding patterns in the preliminary analysis were each employed in three independent replicated HfRAPD PCR reactions using three different freshly extracted DNA pools from 30 starved and pooled females from each of the five Maurit ius colonies and four S. Florida colonies (for a total of 10 reactions fr om the Mauritius colonies X 3 replicates and 8 reactions from the S. Florida colonies X 3 replicates). In addition, on each of the three days the replicates were conducted, duplicate reactions were conducted (pseudoreplication) to further confirm the consistency of the results. Only distinct brightly stained bands (400 to 2650 bp) were scored as 1 (present) or 0 (absent) for the analysis. The evolutionary history of taxa was inferred using the Nei ghbor-Joining method (Saitou and Nei 1987) in

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69 PAUP 4.0b10 (Swofford 2003). The posterior probabilities tree is inferred from 1,000,000 generations. Mitochondrial 12S rR NA Population-Specific Primers The mitochondrial 12S rRNA sequences obtained from the five Mauritius and four S. Florida populations were used to design population-specif ic forward primers (Table 2-1). The population-specific pr imers for Mauritius (SR-J-MU, 5 GCCTAATTAGCAAATATTACTTTT 3) and S. Florida (SR-J-S.FL, 5 GCTTAAAAATTTTACTTTTA 3) were designed in variable regions of the 12S gene. The specificity was tested with Amblyseius swirskii Neoseiulus californicus, N. cucumeris, and Metaseiulus occidentalis (Nesbitt) DNA. The population-specific primers in combination with the universal 12S reverse primer SR-N-14594 (Kambhampati and Smith 1995) should amplify a 406-bp fragment for the S. Fl orida populations and a 330bp fragment for the Maur itius populations. The specificity of the tw o population-specific primer s was tested using a Highfidelity PCR protocol with clean DNA extr acted by Puregene reagents (QIAGEN, Valencia, CA) from aliquots taken from pool s of DNA isolated from 30 starved females from each of the five Mauritiu s colonies and DNA pools from all four S. Florida colonies. DNA extracted from 30 pooled females from A. swirskii N. californicus N. cucumeris and M. occidentalis was tested as controls. To test t he consistency of these reactions, duplicate PCRs were run for each primer and colony source (pseudoreplication). The High-fidelity PCR protocol inco rporated three linked profiles; (i) 1 cycle of denaturation at 94C for 2 min, (ii) 10 cycl es of denaturation at 94C for 10 sec, annealing at 49C for 30 sec, and extension at 68C for 1 min, and (iii) 25 cycles of denaturation at 94C for 10 sec, annealing at 49C for 30 sec, and ex tension at 68C for 1 min, plus an

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70 additional 20 sec added for every consecutive cycle. Reaction products were separated using electrophoresis 2% TBE (Tris-borate-EDTA) agarose gel (14 cm x 10.2 cm) and 1% TBE electrophoresis buffer containing ethidi um bromide. Bands were visualized and photographed under UV illumination. The DNA mo lecular weight marker XIV (Roche Diagnostics, Indianapolis, IN) (100 bp to 2650 bp) was used to determine band size. The gels were scored for the presence or absence of products and product sizes were estimated for each of the reactions. Results Amblyseius largoensis Bay esian Analysis and Sequence Divergence A total of 20 clones each from the five Mauritius and four South Florida colonies (180 clones total) were sequenced for the 12S rRNA gene and 10 clones each were sequenced from the five Mauritiu s and four South Florida colo nies (90 clones total) for the EF-I gene. The 12S rRNA (Table 2-2) and EF-I (Table 2-3) unique gene sequences obtained from the Ma uritius and S. Florida colonies, including sequences from taxa in GenBank, were us ed in the Bayesian analysis. 12S rRNA Sequences All of the 12S rRNA s equences in this study aligned well (Figure 2-1). The 12S rRNA sequences obtained fr om the S. Florida A. largoensis populations had an A-T content of ~ 75% and were ~ 410 bp in length. The Mauritius A. largoensis populations had an A-T content of ~ 76% and were ~ 407 bp in length. Thes e are similar to the A-T content in the same region of DN A in other phytoseiids such as A. swirskii (68%, GenBank accession GU1284606), Neoseiulus californicus (78%, GenBank accession AY099367), N. cucumeris (72%, GenBank accession GU198153), N. fallacis (79%, GenBank accession AY099364), and M. occidentalis (77%, GenBank accession

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71 AY099363). Three clones from two Mauritius colonies, F lic en Flac 1 (FNF1), Trou dEau Douce 6 (TDD6) and Trou dEau Douce 7 (TDD7)) (Table 2-3, Figure 2-4), differed from the majority of sequences obtained, with an A-T content of 69% and length of 390 bp. A Bayesian consensus tree was created in wh ich the S. Florida and the Mauritius colony clones resolved into two distinctly separate clades (Figure 2-4). The S. Florida clade (clade-1) is supported by 99% posterior probability, and the Mauritius clade (clade-2) is supported by 94% posterior pr obability. Three Mauritius clones (FNF1, TDD6, and TDD7) separated out from the tw o main clades as a sister clade to N. cucumeris and were supported by 100% posterior probability scores. There is a 98.4% or higher DNA sequence si milarity within the S. Florida clade-1 clones and 99.2 to 100% DNA sequence similari ty within the Mauritius clade-2 clones. The 12S rRNA corrected nucleotide sequence si milarity between the Mauritius clade-2 clones and S. Florida clade-1 cl ones are 91 to 93% (Table 2-4), indicating that these two populations are more similar to each ot her than they are to the other phytoseiid species examined. The 12S rRNA sequences from Neoseiulus californicus are 62 to 64% similar to the Mauritiu s clade-2 clones and 61 to 62% similar to the S. Florida clade-1 clones. The 12S rRNA sequences from Neoseiulus fallacis are 63 to 64% similar to the Mauritius clade-2 clones and 63 to 64% similar to the S. Florida clade-1 clones; N. cucumeris sequences are only 18 to 22% si milar to the Mauritius clade-2 clones and 19 to 22% similar to the S. Florida clade-1 clones. Amblyseius swirskii 12S rRNA sequences are 64 to 66% similar to th e Mauritius clade-2 clones and 61 to 63% similar to the S. Florida clade-1 clones (Table 2-4).

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72 If we compare 12S rRNA sequences obtained from GenBank of three Neoseiulus spp., three Phytoseiulus spp., and three Amblyseius spp., we find 12S rRNA sequence similarities of 59 to 91% for the Neoseiulus spp., 78 to 96% sequence similarities for the Phytoseiulus spp. and 64 to 66% similarities for the Amblyseius sequences we examined (Jeyaprakash and Hoy 2002; Tixier et al. unpub.). The 91 to 93% sequence similarity observed in the Mauritius and S. Florida populations fa ll within the range of those observed between other phyto seiid species (Table 2-4). In comparing intra-species similarities (T able 1-5) the four S. Florida populations exhibit 98.4% 12S rRNA sequence similarities. The fi ve Mauritius populations exhibit 99.2% sequence similarity in the same gene. Sequence data from pop ulations of three Phytoseiulus spp. (Tixier et al. unpub.) indicate that there is 95 to 100% sequence similarity within each of the three P. persimilis populations, 96 to 100% sequence similarity within one P. macropilis population, and 93 to 99% sequence similarity within one P. fragariae population (Tixier et al. unpub.). Thus intaspecific variation in the Mauritius and S. Florida populations are similar to variation found in 12S rRNA sequences in GenBank from other phytoseiid species. Three clones from the Mauritius coloni es, FNF1, TDD6, and TDD7 (Table 2-3, Figure 2-4), were not consistent with the majority of 12S rRNA sequences obtained from Mauritius. In the Bayesian analysis, they resolved into a sister clade to N. cucumeris supported by 100% posterior probability score s (Figure 2-4). Further analysis of the sequences indicated they were only 29 to 34 % similar to the Mauritius and S. Florida A. largoensis clades and 62 to 63% similar to N. cucumeris These outlier sequences

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73 could not be identified, and could be the result of contamination or represent another phytoseiid species within the Flic en Flac and Trou dEau Douce colonies. Elongation Factor-I Alpha Seq uences None of the EF-I sequences obtained from the A. largoensis colonies and reference taxa ( A. swirskii N. californicus and N. cucumeris ) contained indels or stop codons (Figure 2-3). All nucleotide and inferred amino ac id (Figure 2-2) sequences aligned well with the acarine sequences, as well as the insect, spider, and Limulus sequences obtained from GenBank. All A. largoensis sequences had an open reading frame (ORF) that was 568 bp in l ength. The G-C content of A. largoensis sequences separated into two groups with 58% and 60% G-C ratio, which is similar to that of M. occidentalis (58%, GenBank accession FJ527739). With the assumption that EF-I is a single-copy gene, a Bayesian consensus tree was created (Figure 2-5). The resulting tr ee indicated two clades supported by 100% posterior probability scores with the S. Florida and Mauritius populations intermixed. All S. Florida colonies (1-4) are represented in the two clades. The Mauritius Trou d'Eau Douce colony was represented in both clades while clones obtained from the North of Port Louis colony were only found in clade-2 and Flic en Flac clones in clade-1. By contrast, within the 12S dataset, the S. Florida clones all fall within a single clade, which is distinctly separate fr om the Mauritius clones. EF-I is a protein-coding gene and considered to be more conserved than the 12S rRNA gene. Therefor e, the phylogenetic inference from EF-I Bayesian analysis should not be di vergent from the relationships inferred with the 12S rRNA gene. To explore the possibili ty that the putative EF-I sequence dataset represents two genes instead of one (as is the case with some insects), the EF-I sequences were

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74 reanalyzed. A set of BLAST searches were performed usi ng the discontiguous megablast algorithm (Ma et al. 2002) (more dissimilar sequences, i.e. deeper divergent taxa are compared) against the Drosophila melanogaster genome, which is known to have two copies of the EF-I gene, F1 and F2 (Hovemann 1988) (Table 2-5). The D. melanogaster EF-I F2 ORF is interrupted by two introns while the F1 copy has none (Hovemann 1988) and they share 90.5% nucleotide and 93.3% amino acid similarities. The sequences for the two A. largoensis clades are 82-38% and 95% similar in nucleotide and amino acid sequences, respec tively. The BLAST search comparisons are scored by E score and maximum percentage ident ity (sequence similarity). The E score is defined by GenBank as the num ber of different alignments with scores equivalent to or better than those that are expected to occur in a database search by chance; thus, the lower the E value, the more significant the score. The sequences in clade-1 had a 77% ( E = 2e-120) identity to the D. melanogaster EF-I F1 gene and the clade-2 sequences had a 76% ( E = 2e-101) identity to the EF-I F2 gene (Table 2-5). The primary reason why clade-2 and clade-1 cannot be considered equivalent to the D. melanogaster EF-I F1 and F2 is the lack of introns in the clade-1 sequences. However, it is possible that introns are lacking or lost in the F2 copy of EF-I in mites. An alternative hypothesis to ex plain the presence of two EF-I sequences is that the Mauritius and S. Florida coloni es each contain more than one species, but this is less likely. A second BLAST search using the megablast algorithm (Zang et al. 2000) (for highly similar sequences, i.e. closely rela ted taxa are compar ed) in the GenBank nucleotide database was performed for the clade-1 and clade-2 seque nces and yielded

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75 primarily acarine taxa (Table 2-5). Consist ently, the sequences in clade-2 had higher percentage (91 to 80%) maximum identity (similarity) and E scores with the acarine EFI sequences than the clade-1 sequences (85 to 79%) (Table 2-5), indicating the two sequences may represent two different genes. In addition to the Mauritius and S. Florida clones, the reference phytoseiid clones from A. swirskii, N. californicus, and N. cucumeris were examined using the megablast al gorithm and sorted according to their respective percentage maximum identity (Tabl es 2-6, 2-7). The five clones sequenced from A. swirskii had a 91% maximum identity ( E = 0) to the EF-I from M. occidentalis. Five clones were sequenced for N. californicus four of which had a 91% ( E = 0) maximum identity to M. occidentalis EF-I and the remaining clone had an 82% ( E = 1e129) maximum identity to the nest mite Dermanyssus hirundinis putative EF-I sequence (GenBank accession AM930860). Of 15 clones sequenced for N. cucumeris only one clone with an ORF was obtained, and BLAST search of this sequence returned an 81% ( E = 1e-130) maximum identit y to the feather mite, Pterolichus obtusus Robin, putative EF-I sequence (GenBank accession, EU152765) With these scores we may infer that the sequences in clade-2 are putative EF-I sequences (Table 2-6) and the sequences in clade-1 are an unknown elongation factor (ukn EF ) or a second EF-I gene (Table 2-7). The reference phytose iids were sorted as follows: A. swirskii (GenBank accession GU198152) and N. californicus (to be submitted) were placed in the EF-I sequence group (Table 2-6), N. californicus (GenBank accession GU198153) and N. cucumeris (GenBank accession GU198151) in the ukn EF sequence group (Table 2-7). The putative EF-I (Table 2-6) sequences were aligned using CLUSTALX, and examined through a second EF-I Bayesian analy sis to obtain posterior

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76 probabilities for the true EF-I sequences in A. largoensis and related phytoseiids. The resulting consensus tree (Figur e 2-6) reveals that the true EF-I clones obtained from the Mauritius and S. Florida populations resolve into a single clade, which is to be expected if this gene is only informative at the genus level in phytoseiids. The corrected nucleotide and amino acid divergences for the A. largoensis EFI clade-2 and the ukn EF clade-1 (or potential second EF-I gene) sequences were calculated (Table 2-8). The EF-I nucleotide divergences bet ween the Mauritius and S. Florida populations and the reference phytoseiids Amblyseius swirskii and Neoseiulus californicus are 5 to 6%. The EF-I nucleotide divergences between Amblyseius swirskii and Neoseiulus cucumeris are 2%. An analysis of the same region of EF-I in Dermanyssus species (D. carpathicus D. gallinae and D. hirundinis ; GenBank accessions AM930872, AM930879, and AM930860) (T able 2-8) reveals 2, 3 and 4% nucleotide divergences. When compared to the nucleotide divergences calculated between species and genera of the Acari, the high degree of similarity between the Mauritius and S. Florid a populations true EF-I sequences (99.6 to 99.8%) suggests they are the same species. High-fidelity-RAPD-PCR Analysis of A. lar goensis Populations High-fidelity-RAPD-PCR was pe rformed on the five Maurit ius and four S. Florida colonies using two RAPD markers. Consistently, 19 bands were scored. A consensus tree was inferred using the Neighbor-Joi ning method. The unrooted consensus tree (Figure 2-7) indicates the Mauritius and S. Florida colonies fall within two distinctly separate groups. The S. Florida clade is supported by a high (100%) bootstrap value in comparison to the relatively low bootstrap values for th e Mauritius clade (67%). The least support was found for the Flic en Flac colony that fell below 50% resulting in a

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77 collapsed branch. The Neighbo r-joining analysis of High-fide lity-RAPD scores places the two populations into separate clades sug gesting that they are genetically distinct. Mitochondrial 12S rR NA Population-Specific Primers The 12S rRNA population-specific primers de signed for the Mauritius and for the S. Florida A. largoensis populations each resulted in a product of the expected size (Figure 2-8). No amplicons of the expected si ze were obtained from the control taxa ( A. swirskii, N. californicus N. cucumeris and M. occidentalis). Discussion Bayesian analysis of the Mauritius and S. Florida 12S rRNA sequences places the clones into two distinct clades (Figure 24) and is supported by 91 to 93% sequence similarities between the two populations (Table 2-4), comparable to sequenc e similarities between other phytoseiid species. Additional phytoseiid 12S rRNA sequences would confirm whether this gene is informative at the species level in all phytoseiid genera. The outlier sequences obtained from the Mauritius Flic en Flac and Trou dEau Douce colonies suggest that they are mixed populations. This may indicate that a cryptic species complex was collect ed from Mauritius with one species outcompeting the other for resources in cultur e or may be a product of contamination while in culture. Using the 12S rRNA sequence data obtained fr om the phylogenetic analysis, Mauritius A. largoensis population-specific primers we re designed to for a rapid method for detecting these predators in fieldcollected samples if it were to be released (Figure 2-8A). In addition, S. Florida population-specific prim ers were created to detect contamination by the Florida pr edators in the cultured Maurit ius colonies (Figure 2-8B).

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78 The use of population-specific primers designed from variable regions within the 12S rRNA gene may prove useful in other phy toseiid biological control projects. The incongruence between the initial EF-I (Figure 2-5) and the 12S rRNA (Figure 2-4) Bayesian analyses led us to question whether the partial EF-I sequences are representatives of one or two genes. Through exam ination of the sequence similarity between the cloned partial EF-I sequences with D. melanogaster and M. occidentalis EF-I sequences in GenBank (Table 2-5), we were able to separate the two clades into EF-I (clade-2) (Table 2-6) and an unknown elongation factor that may represent a second copy of EF-I (Table 2-7). A second analysis of the Mauritius and S. Florida putative EF-I clones resolved the two populations into a single clade supported by posterior probabilities score of 100 (F igure 2-6). When com pared to the sequence divergences observed between other phytoseiids and those between Dermanyssus species (Table 2-8), the high similarity between the sequences fr om Mauritius and S. Florida populations (99.6 to 99.8%) suggest that they are the same species. However, this is a highly conserved gene and may not be suitable for species discrimination in these mites. The presence of both the EF-I and the ukn elongation factor gene in the same genome was confirmed using EF-I and ukn elongation factor -specific primers designed by Dr. A. Jeyaprakash (unpub.) on superclean DNA extracted from one Mauritius and one S. Florida isoline. PCR products from both primer sets were obtained from each of the two isolines tested. This indicated that both copies are present in individuals of the Mauritius and the S. Florida phytoseiids tested and the two sequences are not indicative of contamination.

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79 Both the ukn elongation factor sequences and the EF-I sequences were the result of using degenerate primers to survey the two populations. In future work, care should be taken when using EF-I degenerate primers because this gene is not always a single-copy gene and confusion can occur if more than one EF gene is amplified. An investigation of acarine EF-I sequence data in GenBank revealed that the putative EFI sequences were also obtained from degenerate primers (Roy et al. unpub; Lekveishvili and Klompen 2004). It is possibl e that some of these sequences may be incorrectly identified as EF-I and, as a result, incorrect phylogenetic inferences may have been proposed. The results of the RAPD analysis (Figure 28) indicate the Mauritius and S. Florida populations are in two di stinct genetic groups. These results support the 12S rRNA Bayesian analysis that also places the tw o populations into two separate and distinct clades. Future analysis with additional RAPD primers could provid e additional support. In addition, it would be useful to evaluate ot her closely related taxa in order to produce a rooted Neighbor-joining analysis for RAPD markers. In this study, the 12S rRNA gene in phytoseiids has proven to be informative at the species level and may be useful in future phylogenetic studies that require species resolution. Although no firm conclusions c an be made on the inform ative nature of the EF-I gene, its use as a marker for species re solution is questionable. Unfortunately, prior to conducting a phylogenetic analysis in groups for which little genetic information is available, there is no way to predict w hat genes will be informative. The task of finding informative genes in phytoseiids is especially problematic when limited primers and so little sequence information is available. At present, only sequences for mitochondrial

PAGE 80

80 genes, a mariner transposable element, Actin genes 1-4 (Jeyaprakash and Hoy 2009a), 18S (Pham et al. unpub.; Xia et al. unpub.), 28S (Cruickshank and Thomas 1999), the ITS-1 and ITS-2 complex (Navajas et al. 1999; Ramadan et al. unpub.; Xia et al. unpub.), and the EF-I gene (Jeyaprakash and Hoy 2009a ; Bowman et al. unpub.) are known for a limited number of phytoseiid taxa. Genome sequencing of one or more phytoseiid species could provide more informative genetic markers and could make phylogenetic analyses more effective. As a result of the incongruence between the 12S, EF-I, and RAPD analyses, a conclusion c annot be made as to whether the Mauritius and S. Florida popul ations are cryptic species or biotypes of A. largoensis without weighing the results of one analysis as more significant than the other. The incongruent results do not provide enough information to determine whether the Mauritius phytoseiid should be released as a bi ological control agent of the RPM in S. Florida. In order to determine the specie s status of the Maur itius and S. Florida populations, additional genes could be analyz ed in conjunction with reproductive compatibility tests. If the two populations are reproductively incompatible, biological assays to determine whether t he Mauritius phytoseiid can f eed, survive, and reproduce on a diet consisting only of RPM should be conducted. In addition, tests should be conducted to determine whether the Maur itius populations are likely to be more efficacious predators of the RP M than the S. Florida phytose iids. If the Mauritius and S. Florida populations hybridize and produce viable progeny, releasing the Mauritius phytoseiid into the Florida environment as a biological control agent of the RPM may not be justified. However, release may be ju stified if the Maur itius phytoseiids are

PAGE 81

81 determined to be more effi cacious RPM predators and t he populations hybrids are considered equally efficacious.

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82 Table 2-1. List of molecular mark ers used and expected PCR product size. Marker Primer Sequence Expected product size (bp) Author(s) 12S SR-J-14199 5'-TACTATGTTACGACTTAT-3' 390 408 Kambhampati and Smith 1995 SR-N-14594 5'-AAACTAGGA TTAGATACCC-3' SR-J-MU 5-GCCTAATTAGCAAATATTACTTT-3 330 For this study SR-J-S.FL 5-GCTTAAAAATTTTACTTTTA-3 406 EF-I Deg-EF1a-F2 5'-GAYTTYATHAARAAYATG AT-3' 605 Jeyaprakash and Hoy 2009a Deg-EF1a-R1 5'-GCYTCRTGRTGCATYTC-3' Hf-RAPD 122 5'-GTAGACGAGC-3' 400 2650 UBC Biotechnology Laboratory (Vancouver, BC, Canada) 183 5'-CGTGATTGCT-3' 400 2650 188 5'-GCTGGACATC-3' 400 2650 196 5'-CTCCTCCCCC-3' 600 2650 199 5'-GCTCCCCCAC-3' 400 2650

PAGE 83

83 Table 2-2. List of GenBank accession num bers for clones and taxa included in the 12S rRNA Bayesian analysis. The clone source or GenBank sequence authors are included. The S. Flori da (FL) and Mauritius (MU) A. largoensis clones are from pooled colonies. Clone or taxa Colony source or author(s) GenBank accession FL 1 Hollywood, FL GU807437 FL 2 Lake Worth, FL GU807438 FL 3 Lake Worth, FL GU807439 FL 4 Lake Worth, FL GU807440 FL 5 Lake Worth, FL GU807441 FL 6 Lake Worth, FL GU807442 FL 7 Lake Worth, FL GU807443 FL 8 Lake Worth, FL GU807444 FL 9 Lake Worth, FL GU807445 FL 10 Hollywood, FL GU807446 FF 1 Flic en Flac, MU GU807447 FF 2 Flic en Flac, MU GU807448 FF 3 Flic en Flac, MU GU807449 FF 4 Flic en Flac, MU GU807450 FF 5 Flic en Flac, MU GU807451 NPL 1 North of Port Louis, MU GU807452 NPL 2 North of Port Louis, MU GU807453 NPL 3 North of Port Louis, MU GU807454 NPL 4 North of Port Louis, MU GU807455 NPL 5 North of Port Louis, MU GU807456 NPL 6 North of Port Louis, MU GU807457 NPL 7 North of Port Louis, MU GU807458 NPL 8 North of Port Louis, MU GU807459 TDD 1 Trou d'Eau Douce (Colony 1), MU GU807460 TDD 2 Trou d'Eau Douce (Colony 1), MU GU807461 Number following a colony source location indicates the clone number.

PAGE 84

84 Table 2-2. Continued. Clone or taxa Colony source or author(s) GenBank accession TDD 3 Trou d'Eau Douce (Colony 1), MU GU807462 TDD 4 Trou d'Eau Douce (Colony 1), MU GU807463 TDD 5 Trou d'Eau Douce (Colony 1), MU GU807464 TDD 6 Trou d'Eau Douce (Colony 1), MU GU807465 TDD 7 Trou d'Eau Douce (Colony 1), MU GU807466 TDD 8 Trou d'Eau Douce (Colony 1), MU GU807467 TDD 9 Trou d'Eau Douce (Colony 2), MU GU807468 TDD 10 Trou d'Eau Douce (Colony 2), MU GU807469 TDD 11 Trou d'Eau Douce (Colony 2), MU GU807470 TDD 12 Trou d'Eau Douce (Colony 2), MU GU807471 TDD 13 Trou d'Eau Douce (Colony 2), MU GU807472 TDD 14 Trou d'Eau Douce (Colony 2), MU GU807473 TDD 15 Trou d'Eau Douce (Colony 3), MU GU807474 TDD 16 Trou d'Eau Douce (Colony 3), MU GU807475 TDD 17 Trou d'Eau Douce (Colony 3), MU GU807476 TDD 18 Trou d'Eau Douce (Colony 3), MU GU807477 TDD 19 Trou d'Eau Douce (Colony 3), MU GU807478 TDD 20 Trou d'Eau Douce (Colony 3), MU GU807479 Drosophila melanogaster Meigen Lewis et al. 1995 NC_001709 Habronattus oregonensis (Peckham & Peckham) Hedin and Maddison 2001 AF359082 Iphiseius degenerans (Berlese) Jeyaprakash and Hoy 2002 AY099368 Ixodes hexagonus Leach Black and Roehrdanz 1998 NC_002010 Limulus polyphemus Latr. Lavrov et al. 2000 AF216203 Locusta migratoria (L.) Flook et al. 1995 NC_001712 Number following a colony source location indicates the clone number.

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85 Table 2-2. Continued. Clone or taxa Colony source or author(s) GenBank accession Metaseiulus occidentalis (Nesbitt) Jeyaprakash and Hoy 2002 AY099363 Neoseiulus californicus (McGregor) Jeyaprakash and Hoy 2002 AY099367 N. cucumeris Oud. Jeyaprakash and Hoy 2002 AY099366 N. fallacis (Garman) Jeyaprakash and Hoy 2002 AY099364 A. swirskii (Athias-Henriot) Syngenta Bioline Inc. CA GU1284606 Nephila clavata Koch Lee et al. unpub. NC_008063 Ornithoctonus huwena Wang et al. Qiu et al. 2005 NC_005925 Phytoseiulus fragariae Denmark and Schicha Tixier et al. unpub. FJ985128 P. longipes Evans Tixier et al. unpub. FJ952535 P. persimilis Athias-Henriot Tixier et al. unpub. FJ985122 Rhipicephalus sanguineus (Latreille) Black and Roehrdanz 1998 NC_002074 Tetranychus urticae Koch Jeyaprakash and Hoy 2002 AY099365 Varroa destructor Oud. Evans and Lopez 2002 AY163547 Number following a colony source location indicates the clone number.

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86 Table 2-3. List of GenBank accession num bers for clones and taxa included in the EFI Bayesian analysis. The clone source or GenBank sequence authors are included. The S. Florida (FL) and Mauritius (MU) A. largoensis clones are from pooled colonies. Clone or taxa Colony source or author(s) GenBank accession FL 1 Lake Worth, FL GU198155 FL 2 Lake Worth, FL GU198156 FL 3 Lake Worth, FL GU198157 FL 4 Lake Worth, FL GU198158 FL 5 Lake Worth, FL GU198163 FL 6 Lake Worth, FL GU198162 FL 7 Hollywood, FL GU198164 FL 8 Hollywood, FL GU198149 FF 2 Flic en Flac, MU GU198161 FF 3 Flic en Flac, MU GU198160 NPL 1 North of Port Louis, MU GU814633 TDD 1 Trou d'Eau Douce colony 1, MU GU198168 TDD 2 Trou d'Eau Douce colony 2, MU GU198167 TDD 3 Trou d'Eau Douce colony 2, MU GU198169 TDD 4 Trou d'Eau Douce colony 3, MU GU198166 Amblyomma sp. Shultz and Regier unpub. AF240836 Amblyseius swirskii Syngenta Bioline Inc. CA GU198152 Aphonopelma chalcodes Chamberlin Regier and Shultz 1997 ACU90045 Dermanyssus hirundinis (Hermann) Roy et al. unpub. AM930860 Drosophila melanogaster Hoskins et al. 2007 NM_206593 Dysdera crocata Koch Regier and Shultz 1997 DCU90047 Number following a colony source location indicates the clone number.

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87 Table 2-3. Continued. Clone or taxa Colony source or author(s) GenBank accession Euzercon latum Lekveishvili and Klompen unpub. AY624008 Limulus polyphemus Regier and Shultz 1997 LPU90051 Locusta migratoria Zhou et al. 2002 AY077627 Metaseiulus occidentalis Jeyaprakash and Hoy 2009a FJ527739 Neacarus texanus Chamberlin & Mulaik Shultz and Regier unpub. AF240849 Neoseiulus californicus Syngenta Bioline Inc. CA GU198153 N. cucumeris Syngenta Bioline Inc. CA GU198151 Raoiella indica Hollywood, Florida GU198150 Tetranychus urticae Cultured colony, Gainesville, FL GU198154 Locusta migratoria Zhou et al. 2002 AY077627 Number following a colony source location indicates the clone number.

PAGE 88

88 Table 2-4. Corrected pairwise distances between unique mitochondrial 12S rRNA sequences obtain ed from the Mauritius and S. Florida A. largoensis populations* with additional phytoseiid GenBank accessions (24-27) using PAUP 4.0b8 with Kimura 2-parameter and among-site rate variation di stance settings. Distance measured by using a scale of 0 1. Number following a colony source location indicates the clone number.

PAGE 89

89 Table 2-5. BLAST searches performed for the EF-I consensus tree clade-1 and clade-2 nucleotide sequences (Figure 25) using the discontiguous megablast (more dissimilar sequences) and the megablast (for highly similar sequences) algorithms in the GenBank database. Consistently, a higher % maximum identity (similarity) and a lower expectation value or E score were obtained for the clade-2 sequence group. Clade-1 (lacks intron) Clade-2 Taxa GenBank accession E value Maximum % identity E value Maximum % identity Discontiguous megablast search: Drosophila melanogaster EF-I F1 NM_165850.2 2e-120 77 7e-138 79 Drosophila melanogaster EF-I F2 (contains intron) X06870 2e-101 76 2e-94 74 Drosophila melanogaster EF-I F1 NM_165850.2 2e-138 79 Drosophila melanogaster EF-I F2 (contains intron)* X06870 2e-95 75 Megablast search: (Acari lack introns) Metaseiulus occidentalis FJ527739 4e-180 85 0 91 Androlaelaps casalis (Berlese) AM930875 1e-162 82 0 85 Dermanyssus longipes Berlese & Trouessart AM930873 5e-160 82 0 85 Macronyssidae sp. AM930881 2e-159 82 0 85 Dermanyssus gallinae (De Geer) AM930878 2e-158 82 0 85 Dermanyssus carpathicus Dugs AM930872 1e-155 81 6e-178 84 Dermanyssus hirundinis AM930860 1e-154 81 2e-172 83 Megisthanus floridanus AY624009 1e-154 81 2e-164 82 Euzercon latum AY624008 4e-142 80 1e-155 81 Ixodes scapularis Say XM_002411102 2e-134 79 2e-145 80 Clone NPL 1 has a 1% nucleotide sequence divergence from all other clade-2 clones (Table 2-8), and was compared to the D. melanogaster EF-I F1 and F2 sequences.

PAGE 90

90 Table 2-6. List of GenBank accession num bers for clones and taxa included in the putative EF-I Bayesian analysis. The clone* source or GenBank sequence authors are included. The S. Fl orida (FL) and Mauritius (MU) A. largoensis clones are from pooled colonies. Clone or taxa Colony source or author(s) GenBank accession FL 1 Lake Worth, FL GU198155 FL 4 Lake Worth, FL GU198158 FL 6 Lake Worth, FL GU198162 FL 7 Hollywood, FL GU198164 TDD 1 Trou d'Eau Douce colony 1, MU. GU198168 TDD 3 Trou d'Eau Douce colony 2, MU GU198169 NPL 1 North of Port Louis, MU GU814633 Amblyseius swirskii Syngenta Bioline Inc. CA GU198152 Apis mellifera Danforth and Ji 1998 NM_001014993 Dermanyssus hirundinis Roy et al. unpub. AM930860 Drosophila melanogaster Hovemann et al. 1988 X06869 Euzercon latum Lekveishvili and Klompen 2004 AY624008 Megasthanus floridanus Lekveishvili and Klompen 2004 AY624009 Metaseiulus occidentalis Jeyaprakash and Hoy 2009a FJ527739 Neoseiulus californicus Syngenta Bioline Inc. CA To be submitted Raoiella indica Hollywood, FL GU198150 Tetranychus urticae Cultured colony, Gain esville, FL GU198154 Number following a colony source location indicates the clone number.

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91 Table 2-7. List of GenBank accession num bers for clones and taxa included in the unknown elongation factor sequence group. The clone* source or GenBank sequence authors are included. The S. Florida (FL) and Mauritius (MU) A. largoensis clones are from pooled colonies. Clone or taxa Colony source or author(s) GenBank accession FL 2 Lake Worth, FL GU198156 FL 3 Lake Worth, FL GU198157 FL 5 Lake Worth, FL GU198163 FL 8 Hollywood, FL GU198149 TDD 2 Trou d'Eau Douce colony 2, MU GU198167 TDD 4 Trou d'Eau Douce colony 3, MU GU198166 FF 2 Flic en Flac, MU GU198161 FF 3 Flic en Flac, MU GU198160 Neoseiulus californicus Syngenta Bioline Inc. CA GU198153 N. cucumeris Syngenta Bioline Inc. CA GU198151 Number following a colony source location indicates the clone number.

PAGE 92

92 Table 2-8. Corrected pairwise distances between the Maurit ius and S. Florida A. largoensis clones* putative EF-I amino acid (above diagonal) and nucleot ide (below diagonal) sequences with additional phytoseiid GenBank accessions using PAUP 4.0b8. Distance m easured by using a scale of 0 1. No. Clone or Taxa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1. S. Florida 1 0 0 0 0 0 0 0.01 0.01 0.02 0.05 0.05 0.05 0.20 2. S. Florida 4 0 0 0 0 0 0 0.01 0.01 0.02 0.05 0.05 0.05 0.20 3. S. Florida 6 0 0 0 0 0 0 0.01 0.01 0.02 0.05 0.05 0.05 0.20 4. S. Florida 7 0 0 0 0 0 0 0.01 0.01 0.02 0.05 0.05 0.05 0.20 5. N. of Port Louis 1 0 0 0 0 0 0 0.01 0.01 0.02 0.05 0.05 0.05 0.20 6. Trou d'Eau Douce 1 0 0 0 0 0.01 0 0.01 0.01 0.02 0.05 0.05 0.05 0.20 7. Trou d'Eau Douce 3 0 0 0 0 0.01 0 0.01 0.01 0.02 0.05 0.05 0.05 0.20 8. Amblyseius swirskii 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.01 0.02 0.05 0.05 0.05 0.20 9. Neoseiulus californicus 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.02 0.02 0.06 0.06 0.06 0.21 10. Metaseiulus occidentalis 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.09 0.09 0.06 0.06 0.06 0.20 11. Dermanyssus carpathicus 0.17 0.17 0.17 0.17 0.17 0.17 0. 17 0.18 0.19 0.18 0.01 0 0.21 12. D. gallinae 0.17 0.17 0.17 0.17 0.17 0.17 0.17 0.18 0.18 0.18 0.03 0.01 0.21 13. D. hirundinis 0.18 0.18 0.18 0.18 0.19 0.18 0.18 0.19 0.2 0.17 0.02 0.04 0.21 14. Drosophila melanogaster 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.26 0.27 0.25 0.29 0.27 0.29 Number following a colony source location indicates the clone number.

PAGE 93

93 10 20 30 40 50 60 70 80 90 . . South Florida 1 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 2 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 3 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 4 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 5 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 6 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 7 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 8 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 9 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT 10 CT---TTGC------------------------TCAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATCTAAATAAGAAT Flic en Flac 1 CT-ATCAAAA---------------------------GAGTGACGGG-CGATATGTACTCCTGCTTAAA---AACTA-AC-CAGCAACAG 2 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 3 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 4 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 5 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT North Port Louis 1 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 2 CT---TTAC------------------------ACAAGAGTGGCGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 3 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 4 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 5 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 6 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 7 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 8 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ACAAA----TAAAATTTAAATAAGCCT Trou dEau Douce 1 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 2 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 3 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 4 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 5 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 6 CT-ATCAAAA---------------------------GAGTGACGGG-CGATATGTACTCCTGCTTAAA---AACTA-AC-CAGCAACAG 7 CT-ATCAAAA---------------------------GAGTGACGGG-CGATATGTACTCCTGCTTAAA---AACTA-AC-CAGCAACAG 8 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 9 CT---TTAC------------------------ACAGGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 10 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAGATAAGCCT 11 CT---T-AC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 12 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 13 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 14 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 15 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 16 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 17 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 18 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 19 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT 20 CT---TTAC------------------------ACAAGAGTGACGGG-CGATATGTACACAA-ATAAA----TAAAATTTAAATAAGCCT Iphiseius degenerans CT---CTAA------------------------AAGAGAGTGACGGG-CAATATGTACACAT-CTAAAA---TTTTATTCAAATAAACAA Amblyseius swirskii CT---C-AAA-----------------------AAGAGAGTGACGGG-CGATATGTACACATACTGAAA---AGAAATTCAAATTAACCT Neoseiulus californicus CT---CTAAT-----------------------AAGAGAGTGACGGG-CAATATGTACACTT-CTAAAA---ACTAATTCAAATTAGCAA Neoseiulus cucumeris CT-ATCAAAA---------------------------GAGTGACGGG-CGATATGTACTTTCACCAAAA---ATCCACAT-CAATAAGAA Neoseiulus fallacis CT---CTAAT-----------------------AAGAGAGTGACGGG-CAATATGTACACTT-CTAAAA---ACTAATTCAAATTAGAAT Phytoseiulus fragariae CT---CTAAA-----------------------AAGAGAGCGACGGG-CAATATGTACACTT-ATAAAA---TTATATTCAAATTCGCCT Phytoseiulu s longipes CT---CTAAT-----------------------AAGAGAGTGACGGG-CAATATGTACACTT-TTAAAA---TCACATTCAACCAAGCTT Phytoseiulus persimilis CT---CTAAA-----------------------AAGAGAGTGACGGGGCAATATGTACACTT-ATAAAA---TCTAATTCAAATTAGCTA Metaseiulus occidentalis CT---CTTA--------------------------GAGAATGACGGG-CAATATGTACACTTAAAATTT---TTTTATTCAAATTTATTT Varroa destructor TTCATTTTAC------------------------TGAAAGTGACGGG-CGATATGTACACATTTTAGAG---CTTATTTCAAATATTTAT Rhipicephalus sanguineus CT--TATAA--------------------------AAGAGTGACGGG-CGATATGTACATATTTTAGAG---CTTAATTCAAATTGACAT Iphiseius hexagonus CT--CAAAAT------------------------TGAGAGCGACGGG-CGATATGTGCATATTCTAGAG---CTTAATTCAATTATCCAT Tetranychus urticae TTCATTTTAAA---------------------AATGAAAGTGATGGG-CAATATGTACATAAAATAATTATTTCATAATCATTTTTATAA Habronattus oregonensis CTTATTTTGT----------------------AATAAGGGTGACGGG-CGATATGTGCACTTCCGTAAA----GAAATTCAAGTTAA-AA Ornithoctonus huwena CTCATCTTTG----------------------GACGAGGGTGACGGG-CGATATGTACACCTTT-TTAG----CTTTTTCATAAAAA-AT Nephila clavata CTTGCCTTAG----------------------GGAAAGGGTGACGGG-CGATATGTGCACATTTATTAT----CATGTTCAAATTTA-AA Limulus polyphemus CTCACCTAAAA----------------------GCGAGAGCGACGGG-CGATGTGTACATGCCTTAGAG---CCCTATTCACAATTACAT Locusta migratoria CTCATATTAAAAGATATAAAATTTTAATCAATAACGAGAGTGACGGG-CGATGTGTACACACTTCAGAG---CCAATATCAGTTAAATTA Drosophila melanogaster CTTACCTTAA---------------------TAATAAGAGCGACGGG-CGATGTGTACATATTTTAGAG---CTAAAATCAAATTATTAA Figure 2-1. CLUSTAL X DNA alig nment for partial mitochondrial 12S rRNA gene sequences from the S. Florida and Mauritius A. largoensis populations with additional phytoseiid GenBank accessions Number following a colony source location indicates the clone number. Hyphen denotes deletion.

PAGE 94

94 100 110 120 130 140 150 160 170 180 . . South Florida 1 AATTAGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTATATAAT 2 AATTAGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTATATAAT 3 AATTCGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTATATAAT 4 AATTCGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTATATAAT 5 AATTCGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTATATAAT 6 AATTAGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTACATAAT 7 AATTAGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTGTATAAT 8 AATTCGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTATATAAT 9 GATTCGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTATATAAT 10 AATTCGCTTAAAAATTTTACTTTTAAATTTTTCTTTAATTCC---TTATTAAAAAAAAACTATAGTTA---AATAATAAT-TTATATAAT Flic en Flac 1 ATTTAAAC--AACTGCTTACTTTCAAATCCATTTTTAT-------------TGAGAATTACTTCACAAA--AATAATAAA----AGTAGC 2 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 3 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 4 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT Flic en Flac 5 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT North Port Louis 1 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 2 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 3 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 4 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 5 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 6 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 7 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 8 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT Trou dEau Douce 1 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 2 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 3 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 4 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 5 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 6 ATTTAAAC--AACTGCTTACTTTCAAATCCATTTTTAT-------------TGAGAATTACTTCACAAA--AATAATAAA----AGTAGC 7 ATTTAAAC--AACTGCTTACTTTCAAATCCATTTTTAT-------------TGAGAATTACTTCACAAA--AATAATAAA----AGTAGC 8 AATTAGC----AAATATTACTTTTAAATTTCTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 9 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 10 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 11 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 12 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 13 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 14 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 15 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 16 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 17 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 18 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 19 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT 20 AATTAGC----AAATATTACTTTTAAATTTTTCTTTAATCTC---TTATTAAAAAGAAATTATAGTTA---AATAATAAT-TCATATAAT Iphiseius degenerans AATTTAT-----TATTTTACTTTTAAATTCTTCTATACTTATAA-TTATTACAATATAAAGACAGTTA---AATAAAAAT-TTATGTAAT Amblyseius swirskii AATAAACC----AATTTTACTTTTAAATTTTAAATATTAAAA---T-ATTACAAAAACTA-ATTTTTAA--AATAAAAAT-TAATAAAAC Neoseiulus californicus AATATACT----AATATTACTTTTAAATTCTATTTTTAAACT---TTATTACAAAAATAA-ATCATTAA--A-TACTAAT-TAATGAAAC Neoseiulus cucumeris ATTCAATT--TCTTATTTACTATCAAATCCAATTTCAA-------------ATAAAATTAATTTTATAC--AA----AAA----AGTAGC Neoseiulus fallacis AATATACT----AATATTACTTTTAAATTCTATTTTTTATTT---TTATTACAAAAATAA-ATTATTAA--A-TTTTAAT-TAATGAAAC Phyt oseiulus fragariae GATAAACT----AATTTTACTTTTAAATTTTACTTAATTTTTAT-TTACA-TTCAAATCTCGTTACTA---AATAATAAT-TAATATAAT Phytoseiulus longipes AATATACT----TAATTTACTTTTAAATTTTACTAAATTTTAGA-TTACAACTAAAATATTGTTATTA---A-TAATAAT-TAATATAAT Phytoseiulus persimilis AATATACT----AATTTTACTTTTAAATTTTACTAAATTATTAT-TTACA-TTTAAATCTCGTTAAAAT--AATAAAAAT-TATTATAAT Metaseiulus occidentalis TATAGAT----AAATTTTACTTTTAAATTTTTCTTATTTTGA---TTATTTCAAATTTTCAATTTTTGTGAGATTTTAATCTGGTATAAT Varroa destructor AATTTAA----ATATTTTACTTTTAAATCTTTCTTTATAAAA---TTATTTAAAAATTCATATTGTTA---AAAATTAAT-TAATATAAT Rhipicephalus sanguineus TCTATTTC----AATTTTACTTTCAAATCCTAAATGTTATTT-----------AAATTTCTCTCTCTA---AAAAGAAAT-----GTAAT Iphiseius hexagonus CATATTAA----TAATTTACTTTTAAATCCTAATCTCATCTT-----------TCAACATATTTAATC---AAAATCAAT-----GTAAT Tetranychus urticae TTCTATTT---A----TTACTATTAAATTCTTTTTTA---AA----ATATTTTTTTTTTGTGTCTAAAT-----------------TAAA Habronattus oregonensis TATTATTA---AGTTTATACTTATAAATCCTTTTTCTAAAAA----TCTTTAGATTATAATGTCCTTAAA---TTTGGTT-TGATGTAAC Ornithoctonus huwena TATTAATA---ATTTATTACTATTAAATCCTTGACTCTTGAA----TGTTTGGAAAGAAGAATG-TAAAT---AAAGAGT-ATAAGTAAC Nephila clavata TATTGTTT---AAATATTACTAATAAATCCTTTTTGTAAAAA----AAATTTTTTTTTT-TCTCCTTAAT---TTTTTTT-TAAAGTAAC Limulus polyphemus ATTTAA-T--AAAAAATTACTTTCAAATCCACCTTCAATTCTATCCTTTCAATAAAACATCCGTATTAA--TAAATTAAT----TGTAAT Locusta migratoria AATAATTT---AA--ATTACTATCAAATCCACCTTCATTAAA----ATATTACAAAATTAAATCCATAATAAAAAAAAAT-TATTGTAAC Drosophila melanogaster TCTTTATA---AT--TTTACTACTAAATCCACTTTCAA-AAA----TTTTTTCATAATTTTATTCATAT--AAATAAATT-TATTGTAAC Figure 2-1. Continued.

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95 190 200 210 220 230 240 250 260 270 . . South Florida 1 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 2 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 3 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 4 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 5 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 6 TTATTTCAATCTTTAAAATAAGCTGGACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 7 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 8 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 9 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAAT 10 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACACCACTA---ACCTTAGAGAAAA--AAA--TAAATTTACAC--ATAATFlic en Flac 1 TCAATTTATCCATTAG-ATATTCTACACATTGACCTGAATTAAGATAAAATTATTTTTAGGAA---TAAGCTGTATCTTAAA--ACTTA 2 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--A-AA-TAAACTTACAT--ATAGT 3 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAA-TAAACTTACAT--ATAGT 4 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAAATAAACTTACAT--ATAGT 5 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AGAA-TAAACTTACAT--ATAGTNorth Port Louis 1 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAA--TAAACTTACAT--ATAGT 2 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AA---TAAACTTACAT--ATAGT 3 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--A-AA-TAAACTTACAT--ATAGT 4 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAA--TAAACTTACAT--ATAGT 5 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--GAAA-TAAACTTACAT--ATAGT 6 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAAATAAACTTACAT--ATAGT 7 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAA-TAAACTTACAT--ATAGT 8 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAAATAAACTTACAT--ATAGTTrou dEau Douce 1 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAA--TAAACTTACAT--ATAGT 2 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA----AA-TAAACTTACAT--ATAGT 3 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAAATAAACTTACAT--ATAGT 4 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAA--TAAACTTACAT--ATAGT 5 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAA-TAAACTTACAT--ATAGT 6 TCAATTTATCCATTAG-ATTTTCTACACATTGACCTGAATTAAGATAAAATTATTTTTAGGAA---TAAGCTGTATCTTAAA--ACTTA 7 TCAATTTATCCATTAG-ATTTTCTACACATTGACCTGAATTAAGATAAAATTATTTTTAGGAA---TAAGCTGTATCTTAAA--ACTTA 8 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGACAAAA--AAAA-TAAACTTACAT--ATAGT 9 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAA--TAAACTTACAT--ATAGT 10 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAA--TAAACTTACAT--ATAGT 11 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--A-AA-TAAACTTACAT--ATAGT 12 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAA--TAAACTTACAT--ATAGT 13 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA----AA-TAAACTTACAT--ATAGT 14 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAA-TAAACTTACAT--ATAGT 15 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAAATAAACTTACAT--ATAGT 16 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAA--TAAACTTACAT--ATAGT 17 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAA-TAAACTTACAT--ATAGT 18 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAA-TAAACTTACAT--ATAGT 19 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AAAA-TAAACTTACAT--ATAGT 20 TTATTTCAATCTTTAAAATAAGCTGAACCTTAACCTAAAACATCACTA---ACTCTAGAAAAAA--AGAA-TAAACTTACAT--ATAGTIphiseius degenerans TTATCTCAATCTTTTCCATAAGCTGCATCTTTACCTAAAATATTATTA---TGTTTAAGCTAAA--TAAAATATATCTTTAC--ATTTTAmblyseius swirskii TTATTTCAAACTTTTTCATTAACCGCACCTTTCCCTAAAATTTAACTT--TCCAAAAAAAGTTA--AATACTATTTTTTATAAAATAATNeoseiulus californicus TTATTTCATCCTTTTTTATTATCCACACCTTTCCCTAAAATTTA------TCTAAAAAAAGCTA---AATTTATAATTAAACTAGTAATC Neoseiulus cucumeris TCATTAAAACCACTTT-ATGATTTACACATTGACCTGAATTAAAACACTAACGTAAAAAGCAA---TAACCTGTATGTTAAA--ACTTANeoseiulus fallacies TTATTTCATTCTTTTTTATAATCCACACCTTTCCCTAAAATTTA------TCTAAATAATTAGA--CAATTTGTAATTAAACTAATAATT Phyt oseiulus fragariae TTATTTCATTCTTTTGTATAAACTACACCTTTACCTAAAAACCTACTTCATATAAAAGTATTGC--TAAAATATATCTAAAT--ATTTTPhytoseiulus longipes TTATTTCATTCTTTTGTATAAACTACACCTTTACCTAAAATTTTACT----ATAAAAGTATTAT--TAAAATATATTTAAAT--ATTTTPhytoseiulus persimilis TTATTTCATTCTTTTATATAAGCTACACCTTTACCTAAAAAATTACTA-AAATAATAGTATTAA--TAAAATATATTTAAAT--ATTTTMetaseiulus occidentalis TTATTTCAGACTTGCGCATTAGCTGCACTTTGCCCTAAAAATTCTTTT----TAAAAACATTTA--AAAATTTTTTATAAA---ATTTTVarroa destructor TCATAATAACCTTAAATATCAACTATATCTTGATTTAAAATATTTTTT--TTAATTAAAATTTC--TATTTTATATCTAAATAAAATATRhipicephalus sanguineus TCACTTCATTCTTAAATTTTTACTGCACCTTGACTTAATATAACTTAA--TTTAATAAATTTTAACAATTGAAGTTATTAATT-GTTTTT Iphiseius hexagonus TCACTTCATTCATAATTTTATATTGCACCTTGACTTAATATAATTCTT--TATAAAAAAATCT--TAAATATAATTATTTAAT-ATAATT Tetranychus urticae CTTTGAT---TTTAATTATATCTTGACCTGTAATCTTTAA-AATTTTTTTTTAATTAAAATTAA--TATTTT------------ATTTTHabronattus oregonensis TCATTAGTTTCTTTAATATAGACTGCACCTTGACCTAACTTTTTA--TAATTTATTGAGAGAAA--TTTTTG-------AAAATAT-TTC Ornithoctonus huwena TCGGC--TCCCTTTTTCATTGGTTTTATCTCGACCTGACGTGAAAAGTTTTTTTATTTTGAAAT--GGAATT-------TTATCGATTTC Nephila clavata CCACTA-TAACTTTTATGTGGTCTACACCTTTACCTAACTTATTAGATGATCTTTTTAGAAAAA--AATATG-------AAAATAAATTC Limulus polyphemus CCACTTCAACCTTTACCATAAGCTGCACCTTGACCTGACATAAAAAATAAATTTTAAACGATAGCTTTAACTCTATAAAGGC--AATCALocusta migratoria CCATCATACACTTAACTATAAGCTGCACCTTGACCTGAAATAAATTTTAATAAAAAAACAAGAA--AATTTTTTCTCCAAAAAGATTTTC Drosophila melanogaster CCATTAT-TACTTAAATATAAGCTACACCTTGATCTGATATAAATTTTTATTAAAATTATTGAA--TATTATTATTCTTATAAAATATTC Figure 2-1. Continued.

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96 280 290 300 310 320 330 340 350 360 . . South Florida 1 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 2 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 3 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 4 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 5 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 6 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 7 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 8 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 9 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC 10 --TAAATAGCGGTAGACAAACTGT-----------AAATACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTTATAG-AATAAATTC Flic en Flac 1 --A---CAGCCGCACATAAA------------------AATACTA-AAGGTGAAGATAAAGGAGGGGTATCAGGTTATAAAATCAAGCTC 2 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 3 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 4 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 5 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC North Port Louis 1 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 2 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAGAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 3 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 4 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 5 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 6 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 7 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 8 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC Trou dEau Douce 1 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 2 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 3 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 4 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 5 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 6 --A---CAGCCGCACATAAA------------------AATACTA-AAGGTGAAGATAAAGGAGGGGTATCAGGTTATAAAATCAAGCTC 7 --A---CAGCCGCACATAAA------------------AATACTA-AAGGTGAAGATAAAGGAGGGGTATCAGGTTATAAAATCAAGCTC 8 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 9 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 10 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 11 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 12 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 13 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 14 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 15 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 16 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 17 --TAAATAGCGGTAGACAAACTGT-----------AGGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 18 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 19 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC 20 --TAAATAGCGGTAGACAAACTGT-----------AAGTACAATTTAAAGT-AAGATTTTGGTGTTTTATC-CTTAATAG-AATAAATTC Iphiseius degenerans --TCAATAGCGATATACAAACTAA-----------AATTACAAGAAAAAGT-AAGATTTTGGTGTTGCATC-CTTTATAG-GATAAATTC Amblyseius swirskii --AAAATAGAGGTATACGAGCTGAAAT----------TTACATTAAAAAGT-AAGATATTGGGGTCATATC-CTTTATAG-AATAAGTTT Neoseiulus californicus AAAAGATAGAGGTATATGAAATGTAATGTATTAAATTATACAAAAAAAAGT-AAGATTTTGGGGTTGACTC-CTTTAAAG-AATAAATTT Neoseiulus cucumeris --A---CAGCCGTACATAAA------------------AACAAAA-GTGGTGAAATTAAAAGGGGGTTATCAAATTAAATTAACAAGCTC Neoseiulus fallacies AAAAAATAGAGGTATATGAAATGTTAT-----AACTAATACAAAAAAAAGT-AAGATACTGGTGTCAACTC-CTTTAAAG-AATAAGTTT Phyt oseiulus fragariae --AAAATAGAGGTATGTAAATTGTTTA--------AA-TACAAACAAAAGT-AAGATTTTGGTGTTAAATC-CTTTACAG-AATAAATTC Phytoseiulus longipes --AAAATAGAGGTATGTAAATTGTTGA--------AAATACAAACAAAAGT-AAGATACTGGTGTCAAATC-CTTTATAG-AATAAATTC Phytoseiulus persimilis --AAAATAGAGGTATGTAAACTGTTTT--------AA-TACAAATAAAAGT-AAGATTTTGGTGTTAAATC-CTTTATAG-AATAAATTC Metaseiulus occidentalis --TAAATAGAGGTATACAAACTGTAGA-------AGTTTACTAACGTAAGT-AAGATTAAGGGGTTTTATC-CATTACAG-AATAAATTC Varroa destructor ---AAATAACGATATATAAATTGA------------CTTTCAAATTTAAGT-AAGATATCGGCGTTTTATC-CCTTACTT-TACAAATTC Rhipicephalus sanguineus -----TAGTGGTATACAAATTGA-----------ATTTACAAATTTAAGT-AAGATTAAG-TGTTTTATC-CATTAAAG-AACAAATTC Iphiseius degenerans A-AATATAGTGGTATACAAATTGA-----------TTTAACCAAATTAAGT-AAGATCAAGGCGTTTTATC-CATTACAG-AGCAAATTC Tetranychus urticae -----ACGGAGATAAATAAATTAAA----------------AATCTTAAGTTTTTTTTAATGTGTATTTTCT-ATAAATA-ATCAAGACC Habronattus oregonensis TTTAGATGGCGATATACAAATTTTC-----------------TTTTAAAGTGAAATTTATCGGGGGTTGTCG-ATTATAC-AACAAGTTC Ornithoctonus huwena TTTAGACGGCGATATAAAGGCTTTG-----------------AGAAAAAGATAAATTTAACGGGGATTATCGGATTAAGA-TACGAGTTC Nephila clavata TTAAGATGGAGGTATATAAAATTTA-----------------AATAAAAGTAAAAATTAACGTGGATTATCG-ATTATTT-AGCAGGTTC Limulus polyphemus --AAGACGGCGGTATACAAACTGT-----------AATAACAAAATAAAGTAAAATTAAACGAGGACCATC-GATTACAG-AGCAGATTC Locusta migratoria TGATAACGGAGATATACAAACAAAT-----------------AAATTAAGTAAAGTAAATCGTGTACTATCA-ATCATGA-GATAGGTTC Drosophila melanogaster TGATAACGACGGTATATAAACTGATT-------------ACAAATTTAAGTAAGGTCCATCGTGGATTATCG-ATTAAAA-AACAGGTTC Figure 2-1. Continued.

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97 370 380 390 400 410 420 430 440 450 . . South Florida 1 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA 2 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA 3 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA 4 CTCTGAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA 5 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA 6 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA 7 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA 8 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA 9 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACGCCTA--CTACCTCACATT-----------CGTACAC-CTAATA 10 CTCTAAAAAATTTAAAAAGCCGCCAATAAATT--TAAGTTTCATGCACAACACCTA--CTACCTAACATT-----------CGTACAC-CTAATA Flic en Flac 1 CTCTGA--AGAAAAACAGGCCGCCAGAAAGGT--TAAGTTTTTTAATTATTAATTACTACCTTAAAGTA------------TCCACATCATAATA 2 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 3 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 4 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 5 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA North Port Louis 1 CTCTAAAAAATTTATATAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 2 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 3 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 4 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACCTA--CTACCTAACATT-----------CATACAC-CTAATA 5 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 6 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 7 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 8 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA Trou dEau Douce 1 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 2 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 3 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 4 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCAAACATT-----------CATACAC-CTAATA 5 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 6 CTCTGA--AGAAAAACAGGCCGCCAGAAAGGT--TAAGTTTTTTAATTATTAATTACTACCTTAAAGTA------------TCCACATCATAATA 7 CTCTGA--AGAAAAACAGGCCGCCAGAAAGGT--TAAGTTTTTTAATTATTAATTACTACCTTAAAGTA------------TCCACATCATAGTA 8 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 9 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAGTA 10 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 11 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 12 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 13 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 14 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 15 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 16 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 17 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 18 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTCA--CTACCTAACATT-----------CATACAC-CTAATA 19 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA 20 CTCTAAAAAATTTATAAAGCCGCCAATAAATT--TAAGTTTCATGTATAACACTTA--CTACCTAACATT-----------CATACAC-CTAATA Iphiseius degenerans CTCTAAAAAATTTAAAAAGCCGCCAATTAATT--TTAGTTTCATGTTATTCAACTA--CTACTAAATGTT-----------ATTTAATACTAATA Amblyseius swirskii CTCTAAAAAATTCATATAGCCGCCAATTTATT--TTAGTTTCATGAAAATCACTTA--CTACTAAATATA-----------AAAAATTTCTAATA Neoseiulus californicus CTCTAAGATATTTAAAAAGCCGCCAATTAATT--TTAGTTTCATGTTTTTCACTTA--CTACTAAATTTT-----------CTAAATTTATAATA Neoseiulus cucumeris CTCTGCT-AGAGTAGTGAACCGCCAGAAGAAT--TAAGTTTAGAAAAAATAATTTACTACTTTAAAGCA------------ATCTTTAAATAGTA Neoseiulus fallacies CTCTAATATATTTAAAAAGCCGCCAATTAATT--TTAGTTTCATGCATTACACTTA--CTACTAAATATA-----------CTAATATTATAATA Phytoseiulus fragariae CTCTAAAATATTTAAAAAGCCGCCAATTAATT--TTAGTTTCATGAAAACCACTTA--CTACTAAATATT-----------AAATATGACTAATA Phytoseiulu s longipes CTCTAAAATATTTAAAAAGCCGCCAATTAATT--TTAGTTTCATGAATATCACTTA--CTACTAAATCTT-----------AAAATTATCTAATA Phytoseiulus persimilis CTCTAAAATATTTAAAAAGCCGCCAATTAATT--TTAGTTTCATGTTCAACACTTTACCTACTAAATTTT-----------AAATGTAACTAATA Metaseiulus occidentalis CTCTAAAATTATTTTA-AGCCGCCAATTTTTT--TTAGTTTCGTGATTTTCATTTA--CTACTAATTTTT-----------ATATTTT---AATA Varroa destructor CTCTAATAAGAATAAAATGCCGCCATTTGACT--TTAATTTCAAAAAATTC---TA--CTCCTAATCTTT-----------GTTTAAGTATAATA Rhipicephalus sanguineus CTCTGAAAAGCTTAAAATACCGCCATAATTTT--TTGCTTTCGTAATTTTTATTTA--CTAACAATATTTA----------CCTCTTAAATAATA Iphiseius hexagonus CTCTAAAAAGCTTAAAATACCGCCAAAATCTT--ATGATTTCATAATCATTATATA--CTAACAACATATA----------GCT-TAAAATAATA Tetranychus urticae CTTTAACTATAATATTTTACCGCCAAAAATTT--CTAGTTTAA----CTT---TATAAGTTTATTACTAAAAAA---TTT---TTT----TACTT Habronattus oregonensis CTCTAA-TGAAATGAAA-GCCGCCATTTTATAATTAGGTTTTAA-----TAATTATTACTTCC-TAAGATGAT----------TGTAGTATACTA Ornithoctonus huwena CTCTAA-TAAGATGTAAGGCCGCCAAAGGGCA--TGGGTTTTCA-----TAATTTGTAATTTC--CTTATAAT----------TCTAGAATAAAA Nephila clavata CTCTAA-TATGAAAAAATGCCGCCAAACTACA--TAAGTTTTGA-----TAAAAAGTTCTACTACTTTTTTAT----------TTAGATATAAAA Limulus polyphemus CTCTGAACAGCTTAAAGCACCGCCAAATTTTT--TAGGTTTCATGATCAACAATTACTACCCTAATTTCCTTTAC------ACCTTAAAATAACA Locusta migratoria CTCTGAATGGAATGAAATACCGCCAAATTCTT--TGGGTTTAAAGACCTTAACTAATAATACCCAGGTAAAACAAAATTTACATTT-AAATAATA Drosophila melanogaster CTCTAGATAGACTAAAATACCGCCAAATTTTT--TAAGTTTCAAGAACATAACTATTACTACTTTAGCAATTTA---TTTACATTTTAAATAATA Figure 2-1. Continued.

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98 10 20 30 40 50 60 70 80 90 . . South Florida 2 TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 3 TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 5 TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 8 TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI Flic en Flac 2 TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 3 TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI Trou DEau Douce 2 TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 4 TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI South Florida 1 TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 4 TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 6 TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 7 TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI North of Port Louis 1 TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI Trou dEau Douce 1 TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI 3 TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI Amblyseius swirskii TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI Neoseiulus californicus TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSESRYEEIKKEVSSYIKKIGYNPATVPFVPI Neoseiulus cucumeris TGTSQADCAILICPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEPRFEEIKKEVSSYIKKIGYNPATVPFVPI Metaseiulus occidentalis TGTSQADCAILVCPAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEARFEEIKKEVSSYIKKIGYNPATVPFVPI Dermanyssus hirundinis TGTSQADCAILVCPAGTGEFEAGISQNGQTREHALLAYTLGVKQMIVGVNKMDTSEPPYSEDRFEEIKKEVSLYIKKIGYNPNSVPFVPI Euzercon latum TGTSQADCAVLICAAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTTEPPFSQPRFEEIQKEVTSYIKKIGYNPATVPFVPI Amblyomma sp. TGTSQADCAVLIVAAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTTEPPFSQSRFEEIQKEVSAYIKKIGYNPATVPFVPI Neacarus texanus TGTSQADCAVLIVAAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTTEPPFSQARFEEIQKEVSAYIKKIGYNPATVPFVPI Tetranychus urticae TGTSQADCAVLICAAGVGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDSAEPKYSQARYEEITKEVSSYIKKIGYNPATVPFVPI Raoiella indica TGTSQADCAVLIVAAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTTEPKFSQARFEEISKEVSNYIKKIGYNPATVPFVPI Aphonopelma chalcodes TGTSQADCAVLVVAAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTTEPPFSESRFEEIKKEVSAYIKKIGYNPATVPFVPI Dysdera crocata TGTSQADCAVLIVAAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTTEPPFSESRFEEIKKEVSAYIKKIGYNPATVPFVPI Limulus polyphemus TGTSQADCAVLIVAAGTGEFEAGISKNGQTREHALLAYTLGVKQMIVGVNKMDTTEPPYSEKRFEEIQKEVSAYIKKIGYNPATVAFVPI Locusta migratoria TGTSQADCAVLIVAAGTGEFEAGISKNGQTREHALLAFTLGVKQLIVGVNKMDSTEPPYSEARFEEIKKEVSNYIKKIGYNPVAVAFVPI Drosophila melanogaster TGTSQADCAVLIVAAGTGEFEAGISKNGQTREHALLAFTLGVKQLIVGVNKMDSTEPPYSEARYEEIKKEVSSYIKKIGYNPASVAFVPI Figure 2-2. CLUSTAL X amino acid ali gnment translated from partial nuclear EF-I gene sequences from the S. Florida and Mauritius A. largoensis populations with additional phytoseiid GenBank accessions for the EF-I Bayesian analysis. Number following a colony source location in dicates the clone number. Hyphen denotes deletion.

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99 100 110 120 130 140 150 160 170 180 . . South Florida 2 SGWCGDNMLEPSPNMTWYKGWTIERGGTKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV 3 SGWCGDNMLEPSPNMTWYKGWTIERGGTKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV 5 SGWCGDNMLEPSPNMTWYKGWTIERGGTKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV 8 SGWCGDNMLEPSPNMTWYKGWTIERGGTKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV Flic en Flac 2 SGWCGDNMLEPSPNMTWYKGWTIERGGTKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV 3 SGWCGDNMLEPSPNMTWYKGWTIERGGTKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV Trou dEau Douce 2 SGWCGDNMLEPSPNMTWYKGWTIERGGTKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV 4 SGWCGDNMLEPSPNMTWYKGWTIERGGTKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV South Florida 1 SGWAGDNMLEPSPNMGWYKGWQIERKGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV 4 SGWAGDNMLEPSPNMGWYKGWQIERKGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV 6 SGWAGDNMLEPSPNMGWYKGWQIERKGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV 7 SGWAGDNMLEPSPNMGWYKGWQIERKGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV North of Port Louis 1 SGWAGDNMLEPSPNMGWYKGWQIERKGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV Trou dEau Douce 1 SGWAGDNMLEPSPNMGWYKGWQIERKGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV 3 SGWAGDNMLEPSPNMGWYKGWQIERKGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV Amblyseius swirskii SGWAGDNMLEPSPNMPWYKGWQIERKGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV Neoseiulus californicus SGWCGDNMLEPSPNMTWYKGWTIERSGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV Neoseiulus cucumeris SGWCGDNMLEPSPNMTWYKGWTIERQGQKFEGKTLLQALDVMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPAHITTEVKSV Metaseiulus occidentalis SGWAGDNMLEPSPNMTWYKGWQIERKNQKFEGKTLLQALDVMEPPTSPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV Dermanyssus hirundinis SGWAGDNMLEVSANMPWYKGWQIERKGSKFEGKTLLQALDVMEPPARPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPSNLTTEVKSV Euzercon latum SGWNGDNMLEPSTNMPWYKGWSIERKGAKFEGKTLLQALDVMEPPSRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPANLTTEVKSV Amblyomma sp. SGWNGDNMLEPSQNMPWYKGWSIERKSGKSEGKTLLQALDAMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPANLTTEVKSV Neacarus texanus SGWNGDNMLDASSNMPWFKGWSIERKSGKSEGKTLLQALDAMEPPTRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPCNLTTEVKSV Tetranychus urticae SGWHGDNMIEPSPNMPWYKGWSIEKKGAKLEGKTLLQALDAMDPPSRPVDKALRLPLQDVYKIGGIGTVPVGRVETGTIKPGMIVTFAPVNLTTEVKSV Raoiella indica SGWNGDNMLEPSDNMPWFKGWQIEKKGSKLEGKTLLQALDAMDPPSRPVDKPLRLPLQDVYKIGGIGTVPVGRVETGVIKPGMVVTFAPVGITTEVKSV Aphonopelma chalcodes SGWNGDNMLEPSTNMPWYKGWNIERKSSKSDGKTLLQALDAMEPPSRPLDRPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGMVVTFAPVNLTTEVKSV Dysdera crocata SGWNGDNMLEPSTNMPWYKGWNIERKSGKNDGKTLLQALDAMEPPSRPLDKPLRLPLQDVYKIGGIGTVPVGRVETGVMKPGMVVTFAPVNITTEVKSV Limulus polyphemus SGWNGDNMLEASPNTPWFKGFKIERKGQTTEGKTLLQALDCAEPPSRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGVLKPGTVVTFAPAMITTEVKSV Locusta migratoria SGWHGDNMLEHSDKMSWFKGWSIERNEGKAEGKTLIEALDAILPPNRPTEKPLRLPLQDVYKIGGIGTVPVGRVETGILKPGMVVTFAPANLTTEVKSV Drosophila melanogaster SGWHGDNMLEPSEKMPWFKGWSVERKEGKAEGKCLIDALDAILPPQRPTDKPLRLPLQDVYKIGGIGTVPVGRVETGLLKPGMVVNFAPVNLVTEVKSV Figure 2-2. Continued.

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100 10 20 30 40 50 60 70 80 90 . . South Florida 1 CACCGGAACTTCGCAGGCCGATTGTGCCATCCTCGTCTGCCCCGCCGGTACCGGTGAATTCGAAGCCGGTATCTCGAAGAACGGCCAGAC 2 CACCGGAACTTCTCAAGCCGACTGCGCTATTCTCATCTGCCCTGCGGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAAAACGGCCAAAC 3 CACCGGAACTTCTCAAGCCGACTGCGCTATTCTCATCTGCCCTGCGGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAAAACGGCCAAAC 4 CACCGGAACTTCGCAGGCCGATTGTGCCATCCTCGTCTGCCCCGCCGGTACCGGTGAATTCGAAGCCGGTATCTCGAAGAACGGCCAGAC 5 CACCGGAACTTCTCAAGCCGACTGCGCTATTCTCATCTGCCCTGCGGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAAAACGGCCAAAC 6 CACCGGAACTTCGCAGGCCGATTGTGCCATCCTCGTCTGCCCCGCCGGTACCGGTGAATTCGAAGCCGGTATCTCGAAGAACGGCCAGAC 7 CACCGGAACTTCGCAGGCCGATTGTGCCATCCTCGTCTGCCCCGCCGGTACCGGTGAATTCGAAGCCGGTATCTCGAAGAACGGCCAGAC 8 CACCGGAACTTCTCAAGCCGACTGCGCTATTCTCATCTGCCCTGCGGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAAAACGGCCAAAC Flic en Flac 2 CACCGGAACTTCTCAAGCCGACTGCGCTATTCTCATCTGCCCTGCGGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAAAACGGCCAAAC 3 CACCGGAACTTCTCAAGCCGACTGCGCTATTCTCATCTGCCCTGCGGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAAAACGGCCAAAC North of Port Louis 1 CACCGGAACTTCGCAGGCCGATTGTGCCATCCTCGTCTGCCCCGCCGGTACCGGTGAATTCGAAGCCGGTATCTCGAAGAACGGCCAGAC Trou dEau Douce 1 CACCGGAACTTCGCAGGCCGATTGTGCCATCCTCGTCTGCCCCGCCGGTACCGGTGAATTCGAAGCCGGTATCTCGAAGAACGGCCAGAC 2 CACCGGAACTTCTCAAGCCGACTGCGCTATTCTCATCTGCCCTGCGGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAAAACGGCCAAAC 3 CACCGGAACTTCGCAGGCCGATTGTGCCATCCTCGTCTGCCCCGCCGGTACCGGTGAATTCGAAGCCGGTATCTCGAAGAACGGCCAGAC 4 CACCGGAACTTCTCAAGCCGACTGCGCTATTCTCATCTGCCCTGCGGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAAAACGGCCAAAC Amblyseius swirskii CACCGGTACTTCCCAGGCTGATTGTGCCATCCTTGTCTGCCCCGCCGGTACCGGAGAATTCGAAGCCGGTATCTCGAAGAACGGCCAGAC Neoseiulus californicus CACAGGAACTTCCCAAGCCGACTGCGCGATCCTCATCTGCCCCGCCGGAACCGGCGAGTTCGAGGCCGGAATCTCCAAGAACGGCCAAAC Neoseiulus cucumeris CACGGGAACTTCTCAGGCCGACTGCGCTATTCTCATCTGCCCTGCCGGAACCGGCGAGTTCGAAGCCGGCATTTCCAAGAATGGACAAAC Metaseiulus occidentalis CACCGGAACTTCCCAGGCCGATTGTGCCATCCTTGTCTGTCCCGCCGGTACCGGAGAATTCGAGGCCGGTATCTCCAAGAACGGTCAGAC Dermanyssus hirundinis CACGGGAACGTCGCAGGCCGACTGCGCCATCCTCGTCTGTCCGGCCGGTACCGGCGAATTCGAAGCTGGTATCTCGCAGAACGGCCAGAC Euzercon latum CACGGGAACATCTCAGGCTGATTGCGCTGTTCTCATCTGCGCCGCCGGTACCGGTGAATTCGAAGCCGGTATTTCTAAGAACGGCCAGAC Amblyomma sp CACTGGAACGTCGCAGGCTGACTGTGCTGTGCTGATTGTGGCTGCCGGTACCGGCGAGTTCGAGGCTGGTATCTCCAAGAACGGCCAGAC Neacarus texanus CACAGGAACATCACAGGCTGACTGTGCTGTCTTAATTGTTGCTGCTGGTACTGGTGAATTTGAAGCTGGTATCTCAAAGAACGGACAGAC Raoiella indica TACTGGAACTTCACAGGCCGACTGTGCAGTTCTGATCGTTGCTGCCGGTACTGGTGAATTTGAAGCTGGTATATCTAAGAACGGCCAGAC Tetranychus urticae TACTGGTACTTCTCAAGCTGATTGTGCTGTATTGATTTGTGCTGCTGGTGTTGGTGAATTCGAAGCTGGTATCTCTAAGAACGGTCAAAC Aphonopelma chalcodes TACGGGAACTTCACAAGCTGACTGTGCAGTCTTAGTAGTGGCAGCAGGAACAGGTGAATTTGAAGCAGGTATCTCAAAGAATGGACAAAC Dysdera crocata TACAGGAACCTCGCAGGCCGATTGTGCTGTCCTGATTGTGGCTGCAGGTACTGGTGAGTTTGAGGCTGGTATCTCCAAGAACGGACAGAC Limulus polyphemus TACTGGAACATCCCAGGCTGATTGTGCTGTTCTGATTGTGGCTGCTGGCACTGGTGAATTTGAAGCTGGAATTTCCAAAAATGGCCAGAC Locusta migratoria TACAGGAACGTCACAGGCTGACTGTGCAGTGTTGATCGTAGCAGCTGGTACAGGTGAATTTGAAGCCGGTATTTCTAAGAACGGACAAAC Drosophila melanogaster TACCGGTACCTCTCAGGCCGATTGTGCGGTGCTGATCGTCGCCGCCGGAACTGGAGAGTTCGAGGCCGGGATCTCGAAGAACGGCCAGAC Figure 2-3. CLUSTAL X DNA al ignment for partial nuclear EF-I gene sequences from the S. Florida and Mauritius A. largoensis populations with additional phytoseiid GenBank accessions for the EF-I Bayesian analysis. Number following a colony source location indicate s the clone number. Hyphen denotes deletion.

PAGE 101

101 100 110 120 130 140 150 160 170 180 . . South Florida 1 TCGTGAACACGCTCTGCTCGCCTACACCCTCGGTGTGAAGCAGATGATCGTCGGTGTCAACAAGATGGACACTTCGGAGCCTCCCTACTC 2 GCGTGAGCACGCTCTTCTCGCTTACACTCTCGGTGTCAAGCAGATGATCGTCGGTGTCAACAAGATGGACACCTCCGAGCCTCCGTACTC 3 GCGTGAGCACGCTCTTCTCGCTTACACTCTCGGTGTCAAGCAGATGATCGTCGGTGTCAACAAGATGGACACCTCCGAGCCTCCGTACTC 4 TCGTGAACACGCTCTGCTCGCCTACACCCTCGGTGTGAAGCAGATGATCGTCGGTGTCAACAAGATGGACACTTCGGAGCCTCCCTACTC 5 GCGTGAGCACGCTCTTCTCGCTTACACTCTCGGTGTCAAGCAGATGATCGTCGGTGTCAACAAGATGGACACCTCCGAGCCTCCGTACTC 6 TCGTGAACACGCTCTGCTCGCCTACACCCTCGGTGTGAAGCAGATGATCGTCGGTGTCAACAAGATGGACACTTCGGAGCCTCCCTACTC 7 TCGTGAACACGCTCTGCTCGCCTACACCCTCGGTGTGAAGCAGATGATCGTCGGTGTCAACAAGATGGACACTTCGGAGCCTCCCTACTC 8 GCGTGAGCACGCTCTTCTCGCTTACACTCTCGGTGTCAAGCAGATGATCGTCGGTGTCAACAAGATGGACACCTCCGAGCCTCCGTACTC Flic en Flac 2 GCGTGAGCACGCTCTCCTCGCTTACACTCTCGGTGTCAAGCAGATGATCGTCGGTGTCAACAAGATGGACACCTCCGAGCCTCCGTACTC 3 GCGTGAGCACGCTCTTCTCGCTTACACTCTCGGTGTCAAGCAGATGATCGTCGGTGTCAACAAGATGGACACCTCCGAGCCTCCGTACTC North of Port Louis 1 TCGTGAACACGCTCTGCTCGCCTACACCCTCGGTGTGAAGCAGATGATCGTCGGTGTCAACAAGATGGACACTTCGGAGCCTCCGTACTC Trou dEau Douce 1 TCGTGAACACGCTCTGCTCGCCTACACCCTCGGTGTGAAGCAGATGATCGTCGGTGTCAACAAGATGGACACTTCGGAGCCTCCCTACTC 2 GCGTGAGCACGCTCTTCTCGCTTACACTCTCGGTGTCAAGCAGATGATCGTCGGTGTCAACAAGATGGACACCTCCGAGCCTCCGTACTC 3 TCGTGAACACGCTCTGCTCGCCTACACCCTCGGTGTGAAGCAGATGATCGTCGGTGTCAACAAGATGGACACTTCGGAGCCTCCCTACTC 4 GCGTGAGCACGCTCTTCTCGCTTACACTCTCGGTGTCAAGCAGATGATCGTCGGTGTCAACAAGATGGACACCTCCGAGCCTCCGTACTC Amblyseius swirskii TCGTGAACACGCTCTTCTCGCCTACACCCTTGGTGTGAAGCAGATGATCGTTGGTGTCAACAAGATGGACACTTCTGAGCCTCCTTACTC Neoseiulus californicus GCGTGAACACGCCCTGCTCGCTTACACTCTCGGTGTGAAGCAAATGATTGTCGGTGTCAACAAGATGGACACTTCCGAGCCTCCGTACTC Neoseiulus cucumeris GCGTGAACACGCTCTGCTCGCGTACACTCTCGGAGTGAAGCAAATGATTGTCGGTGTCAACAAGATGGACACTTCCGAGCCCCCGTACTC Metaseiulus occidentalis TCGTGAGCACGCTCTTCTCGCCTACACCCTTGGTGTGAAGCAGATGATCGTCGGTGTGAACAAGATGGACACCTCTGAGCCGCCGTACTC Dermanyssus hirundinis TCGTGAGCACGCCCTGCTCGCGTACACGCTCGGCGTGAAGCAAATGATTGTCGGCGTCAACAAGATGGACACCTCGGAGCCGCCCTACTC Euzercon latum TAGAGAACACGCTCTTCTTGCCTACACTCTCGGTGTGAAGCAAATGATCGTTGGCGTCAACAAGATGGACACCACCGAGCCTCCTTTCAG Amblyomma sp CCGAGAGCACGCCCTGCTGGCTTACACCCTTGGCGTGAAGCAGATGATTGTCGGCGTGAACAAGATGGATACCACCGAGCCTCCCTTCTC Neacarus texanus CAGAGAACATGCCCTTCTGGCTTACACTTTGGGTGTGAAGCAGATGATTGTGGGTGTTAACAAGATGGACACTACTGAGCCTCCTTTCAG Raoiella indica TCGAGAACATGCTTTGTTGGCATATACCTTGGGCGTAAAGCAAATGATCGTTGGTGTCAACAAGATGGACACCACTGAGCCAAAATTTAG Tetranychus urticae TCGAGAACATGCTTTGTTGGCATACACCTTGGGTGTAAAACAAATGATTGTAGGTGTTAACAAAATGGATTCAGCTGAGCCAAAATATTC Aphonopelma chalcodes CAGAGAACATGCTTTACTTGCATATACCTTAGGAGTAAAACAAATGATTGTAGGTGTGAACAAGATGGATACCACTGAACCACCCTTCAG Dysdera crocata CAGAGAACACGCTCTGCTTGCCTACACCTTGGGTGTCAAGCAGATGATTGTTGGTGTCAACAAGATGGACACCACTGAACCACCATTCAG Limulus polyphemus CCGTGAACACGCCTTGTTGGCCTACACCCTGGGTGTGAAGCAGATGATTGTGGGAGTGAACAAGATGGACACAACTGAACCTCCTTATAG Locusta migratoria CCGTGAGCATGCCTTGTTGGCTTTCACTTTGGGTGTCAAGCAACTGATTGTGGGTGTGAACAAAATGGATTCGACTGAGCCACCATACAG Drosophila melanogaster CCGCGAGCACGCCCTTCTGGCATTCACGCTGGGCGTGAAGCAGCTTATTGTGGGCGTCAACAAGATGGACTCCACTGAGCCGCCGTACAG Figure 2-3. Continued.

PAGE 102

102 190 200 210 220 230 240 250 260 270 . . South Florida 1 CGAGCCTCGTTTCGAGGAAATCAAGAAGGAAGTGTCTTCCTACATCAAGAAGATCGGATACAACCCCGCCACCGTTCCGTTCGTCCCGAT 2 GGAGCCTCGTTTCGAGGAAATCAAGAAGGAGGTGTCCTCCTACATCAAGAAGATCGGTTACAACCCCGCGACTGTGCCATTCGTTCCCAT 3 GGAGCCTCGTTTCGAGGAAATCAAGAAGGAGGTGTCCTCCTACATCAAGAAGATCGGTTACAACCCCGCGACTGTGCCATTCGTTCCCAT 4 CGAGCCTCGTTTCGAGGAAATCAAGAAGGAAGTGTCTTCCTACATCAAGAAGATCGGATACAACCCCGCCACCGTTCCGTTCGTCCCGAT 5 GGAGCCTCGTTTCGAGGAAATCAAGAAGGAGGTGTCCTCCTACATCAAGAAGATCGGTTACAACCCCGCGACTGTGCCATTCGTTCCCAT 6 CGAGCCTCGTTTCGAGGAAATCAAGAAGGAAGTGTCTTCCTACATCAAGAAGATCGGATACAACCCCGCCACCGTTCCGTTCGTCCCGAT 7 CGAGCCTCGTTTCGAGGAAATCAAGAAGGAAGTGTCTTCCTACATCAAGAAGATCGGATACAACCCCGCCACCGTTCCGTTCGTCCCGAT South Florida 8 GGAGCCTCGTTTCGAGGAAATCAAGAAGGAGGTGTCCTCCTACATCAAGAAGATCGGTTACAACCCCGCGACTGTGCCATTCGTTCCCAT Flic en Flac 2 GGAGCCTCGTTTCGAGGAAATCAAGAAGGAGGTGTCCTCCTACATCAAGAAGATCGGTTACAACCCCGCGACTGTGCCATTCGTTCCCAT 3 GGAGCCTCGTTTCGAGGAAATCAAGAAGGAGGTGTCCTCCTACATCAAGAAGATCGGTTACAACCCCGCGACTGTGCCATTCGTTCCCAT North of Port Louis 1 CGAGCCTCGTTTCGAGGAAATCAAGAAGGAAGTGTCTTCCTACATCAAGAAGATCGGATACAACCCCGCCACCGTTCCGTTCGTCCCGAT Trou dEau Douce 1 CGAGCCTCGTTTCGAGGAAATCAAGAAGGAAGTGTCTTCCTACATCAAGAAGATCGGTTACAACCCCGCCACCGTTCCGTTCGTCCCGAT 2 GGAGCCTCGTTTCGAGGAAATCAAGAAGGAGGTGTCCTCCTACATCAAGAAGATCGGTTACAACCCCGCGACTGTGCCATTCGTTCCCAT 3 CGAGCCTCGTTTCGAGGAAATCAAGAAGGAAGTGTCTTCCTACATCAAGAAGATCGGTTACAACCCCGCCACCGTTCCGTTCGTCCCGAT 4 GGAGCCTCGTTTCGAGGAAATCAAGAAGGAGGTGTCCTCCTACATCAAGAAGATCGGTTACAACCCCGCGACTGTGCCATTCGTTCCCAT Amblyseius swirskii CGAGCCTCGATTCGAGGAAATCAAGAAGGAAGTGTCTTCCTACATCAAGAAGATCGGATACAACCCCGCCACTGTGCCTTTCGTTCCGAT Neoseiulus californicus GGAGTCCCGATACGAGGAAATCAAGAAGGAGGTGTCTTCTTACATCAAGAAGATCGGCTACAATCCCGCAACCGTGCCATTCGTACCCAT Neoseiulus cucumeris AGAGCCTCGCTTCGAGGAAATCAAGAAGGAAGTGTCCTCTTACATCAAGAAGATCGGTTACAACCCCGCGACCGTGCCATTCGTGCCCAT Metaseiulus occidentalis CGAGGCTCGATTCGAGGAAATCAAGAAGGAGGTCTCGTCGTACATCAAGAAGATCGGATACAACCCCGCCACGGTTCCCTTCGTCCCCAT Dermanyssus hirundinis TGAGGACCGGTTCGAGGAGATCAAGAAAGAGGTGTCGCTGTACATCAAGAAGATCGGCTACAACCCGAACTCTGTGCCGTTTGTGCCCAT Euzercon latum CCAACCTCGCTTTGAAGAAATCCAGAAGGAAGTTACCTCCTACATCAAAAAGATTGGTTACAACCCCGCAACCGTACCCTTTGTGCCAAT Amblyomma sp TCAGAGCCGTTTCGAGGAAATCCAGAAGGAAGTGTCCGCCTACATCAAGAAGATTGGCTACAACCCTGCTACTGTTCCGTTTGTGCCCAT Neacarus texanus CCAGGCCAGGTTTGAGGAAATCCAGAAGGAAGTGTCTGCCTACATCAAGAAGATTGGATACAACCCTGCCACTGTACCCTTCGTTCCCAT Raoiella indica CCAAGCTAGGTTTGAGGAGATATCTAAAGAAGTAAGCAACTATATTAAAAAAATTGGGTACAACCCTGCCACAGTACCATTTGTGCCCAT Tetranychus urticae TCAAGCTCGTTATGAAGAAATTACCAAGGAAGTTAGCAGTTACATTAAGAAGATTGGTTACAATCCAGCAACTGTACCATTTGTACCAAT Aphonopelma chalcodes TGAGTCAAGATTTGAAGAAATCAAGAAAGAAGTATCTGCTTATATCAAAAAAATTGGCTACAATCCAGCAACTGTACCATTTGTTCCAAT Dysdera crocata TGAGTCTCGATTTGAGGAAATCAAGAAGGAAGTATCCGCTTACATCAAGAAGATTGGTTACAACCCTGCCACCGTACCTTTTGTTCCCAT Limulus polyphemus TGAGAAACGTTTTGAAGAAATCCAGAAGGAAGTTAGTGCCTACATTAAGAAGATAGGCTACAATCCTGCCACTGTTGCCTTTGTGCCAAT Locusta migratoria TGAGGCTCGTTTTGAGGAAATTAAGAAGGAAGTCAGTAACTACATTAAGAAGATTGGTTACAATCCAGTAGCTGTTGCCTTTGTTCCTAT Drosophila melanogaster CGAGGCCCGCTACGAGGAGATCAAGAAGGAGGTGTCCTCGTACATCAAGAAGATCGGCTACAATCCGGCCTCGGTGGCCTTCGTGCCCAT Figure 2-3. Continued.

PAGE 103

103 280 290 300 310 320 330 340 350 360 . . South Florida 1 TTCCGGATGGGCCGGAGACAACATGCTCGAGCCCTCTCCCAACATGGGATGGTACAAGGGATGGCAGATCGAGCGCAAGGGCCAGAAGTT 2 CTCGGGATGGTGTGGAGACAACATGCTGGAGCCCTCGCCAAACATGACCTGGTACAAAGGATGGACCATCGAGCGCGGAGGCACGAAATT 3 CTCGGGATGGTGTGGAGACAACATGCTGGAGCCCTCGCCAAACATGACCTGGTACAAAGGATGGACCATCGAGCGCGGAGGCACGAAATT 4 TTCCGGATGGGCCGGAGACAACATGCTCGAGCCCTCTCCCAACATGGGATGGTACAAGGGATGGCAGATCGAGCGCAAGGGCCAGAAGTT 5 CTCGGGATGGTGTGGAGACAACATGCTGGAGCCCTCGCCAAACATGACCTGGTACAAAGGATGGACCATCGAGCGCGGAGGCACGAAATT 6 TTCCGGATGGGCCGGAGACAACATGCTCGAGCCCTCTCCCAACATGGGATGGTACAAGGGATGGCAGATCGAGCGCAAGGGCCAGAAGTT 7 TTCCGGATGGGCCGGAGACAACATGCTCGAGCCCTCTCCCAACATGGGATGGTACAAGGGATGGCAGATCGAGCGCAAGGGCCAGAAGTT 8 CTCGGGATGGTGTGGAGACAACATGCTGGAGCCCTCGCCAAACATGACCTGGTACAAAGGATGGACCATCGAGCGCGGAGGCACGAAATT Flic en Flac 2 CTCGGGATGGTGTGGAGACAACATGCTGGAGCCCTCGCCAAACATGACCTGGTACAAAGGATGGACCATCGAGCGCGGAGGCACGAAATT 3 CTCGGGGTGGTGTGGAGACAACATGCTGGAGCCCTCGCCAAACATGACCTGGTACAAAGGATGGACCATCGAGCGCGGAGGCACGAAATT North of Port Louis 1 TTCCGGATGGGCCGGAGACAACATGCTCGAGCCCTCTCCCAACATGGGATGGTACAAGGGATGGCAGATCGAGCGCAAGGGCCAGAAGTT Trou dEau Douce 1 TTCCGGATGGGCCGGAGACAACATGCTCGAGCCCTCTCCCAACATGGGATGGTACAAGGGATGGCAGATCGAGCGCAAGGGCCAGAAGTT 2 CTCGGGATGGTGTGGAGACAACATGCTGGAGCCCTCGCCAAACATGACCTGGTACAAAGGATGGACCATCGAGCGCGGAGGCACGAAATT 3 TTCCGGATGGGCCGGAGACAACATGCTCGAGCCCTCTCCCAACATGGGATGGTACAAGGGATGGCAGATCGAGCGCAAGGGCCAGAAGTT 4 CTCGGGATGGTGTGGAGACAACATGCTGGAGCCCTCGCCAAACATGACCTGGTACAAAGGATGGACCATCGAGCGCGGAGGCACGAAATT Amblyseius swirskii TTCCGGATGGGCCGGAGACAACATGCTCGAGCCCTCTCCCAACATGCCCTGGTACAAGGGATGGCAGATTGAGCGCAAGGGTCAGAAGTT Neoseiulus californicus CTCAGGATGGTGTGGAGACAACATGCTGGAACCTTCGCCAAACATGACCTGGTACAAGGGATGGACCATCGAGCGTTCCGGCCAGAAATT Neoseiulus cucumeris TTCGGGATGGTGTGGAGACAACATGCTGGAACCTTCGCCAAACATGACCTGGTACAAGGGATGGACCATCGAGCGTCAGGGCCAGAAATT Metaseiulus occidentalis TTCTGGATGGGCTGGAGACAACATGCTTGAGCCCTCTCCCAACATGACTTGGTACAAGGGATGGCAGATCGAGCGAAAAAATCAGAAGTT Dermanyssus hirundinis CTCCGGCTGGGCTGGCGACAACATGCTTGAGGTGTCGGCCAACATGCCCTGGTACAAGGGATGGCAGATCGAACGAAAGGGCAGCAAGTT Euzercon latum TTCTGGCTGGAATGGAGACAACATGCTCGAGCCGTCTACCAACATGCCGTGGTACAAGGGATGGAGTATTGAACGTAAGGGAGCCAAGTT Amblyomma sp CTCTGGCTGGAACGGCGACAACATGCTCGAGCCTAGCCAGAACATGCCCTGGTACAAGGGGTGGTCTATTGAGCGCAAGTCTGGCAAGTC Neacarus texanus TTCTGGCTGGAATGGAGACAACATGCTGGATGCCTCTTCCAACATGCCCTGGTTTAAGGGATGGTCTATCGAGAGGAAGTCTGGCAAGTC Raoiella indica CTCCGGCTGGAACGGTGACAACATGCTTGAACCAAGTGATAACATGCCCTGGTTCAAGGGATGGCAAATTGAGAAGAAAGGATCTAAACT Tetranychus urticae TTCTGGATGGCATGGTGACAACATGATTGAACCATCACCTAACATGCCTTGGTATAAGGGATGGTCAATTGAAAAGAAGGGAGCTAAATT Aphonopelma chalcodes TTCTGGCTGGAATGGTGACAACATGTTGGAACCCAGCACAAACATGCCATGGTACAAGGGATGGAACATTGAACGCAAGAGCTCAAAATC Dysdera crocata TTCCGGCTGGAACGGTGACAACATGTTGGAGCCCAGCACCAACATGCCGTGGTACAAGGGATGGAACATCGAACGCAAGAGTGGAAAGAA Limulus polyphemus CTCTGGGTGGAATGGTGACAATATGCTGGAAGCCAGCCCTAACACTCCATGGTTTAAGGGGTTTAAAATTGAACGCAAGGGTCAAACAAC Locusta migratoria TTCTGGATGGCATGGTGACAACATGTTGGAGCATTCTGACAAGATGAGCTGGTTCAAGGGATGGTCTATTGAACGTAACGAAGGAAAGGC Drosophila melanogaster CTCCGGATGGCACGGCGACAATATGCTGGAGCCGTCCGAGAAGATGCCCTGGTTCAAGGGATGGTCCGTGGAGCGCAAGGAAGGCAAGGC Figure 2-3. Continued.

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104 370 380 390 400 410 420 430 440 450 . . South Florida 1 CGAAGGAAAGACCCTCCTCCAGGCCCTCGACGTCATGGAGCCTCCCACCAGGCCCACCGACAAGCCTCTCCGTCTTCCCCTCCAGGACGT 2 CGAGGGCAAGACGCTTCTGCAGGCCCTCGATGTCATGGAGCCCCCGACCAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTCCAGGACGT 3 CGAGGGCAAGACGCTTCTGCAGGCCCTCGATGTCATGGAGCCCCCGACCAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTCCAGGACGT 4 CGAAGGAAAGACCCTCCTCCAGGCCCTCGACGTCATGGAGCCTCCCACCAGGCCCACCGACAAGCCTCTCCGTCTTCCCCTCCAGGACGT 5 CGAGGGCAAGACGCTTCTGCAGGCCCTCGATGTCATGGAGCCCCCGACCAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTCCAGGACGT 6 CGAAGGAAAGACCCTCCTCCAGGCCCTCGACGTCATGGAGCCTCCCACCAGGCCCACCGACAAGCCTCTCCGTCTTCCCCTCCAGGACGT 7 CGAAGGAAAGACCCTCCTCCAGGCCCTCGACGTCATGGAGCCTCCCACCAGGCCCACCGACAAGCCTCTCCGTCTTCCCCTCCAGGACGT 8 CGAGGGCAAGACGCTTCTGCAGGCCCTCGATGTCATGGAGCCCCCGACCAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTCCAGGACGT Flic en Flac 2 CGAGGGCAAGACGCTTCTGCAGGCCCTCGATGTCATGGAGCCCCCGACCAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTCCAGGACGT 3 CGAGGGCAAGACGCTTCTGCAGGCCCTCGATGTCATGGAGCCCCCGACCAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTCCAGGACGT North of Port Louis 1 CGAAGGAAAGACCCTCCTCCAGGCCCTCGACGTCATGGAGCCTCCCACCAGGCCCACCGACAAGCCTCTCCGTCTTCCCCTCCAGGACGT Trou dEau Douce 1 CGAAGGAAAGACCCTCCTCCAGGCCCTCGACGTCATGGAGCCTCCCACCAGGCCCACCGACAAGCCTCTCCGTCTTCCCCTCCAGGACGT 2 CGAGGGCAAGACGCTTCTGCAGGCCCTCGATGTCATGGAGCCCCCGACCAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTCCAGGACGT 3 CGAAGGAAAGACCCTCCTCCAGGCCCTCGACGTCATGGAGCCTCCCACCAGGCCCACCGACAAGCCTCTCCGTCTTCCCCTCCAGGACGT 4 CGAGGGCAAGACGCTTCTGCAGGCCCTCGATGTCATGGAGCCCCCGACCAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTCCAGGACGT Amblyseius swirskii CGAGGGCAAGACCCTTCTCCAAGCTCTCGACGTCATGGAGCCTCCCACCAGGCCCACCGACAAGCCCCTTCGTCTTCCCCTCCAGGACGT Neoseiulus californicus CGAGGGCAAAACCCTCCTGCAGGCTCTTGATGTAATGGAGCCTCCCACCAGGCCCACCGACAAGCCTCTGCGTCTGCCACTCCAGGATGT Neoseiulus cucumeris CGAGGGCAAAACCCTCCTCCAGGCTCTCGATGTCATGGAACCGCCTACCAGGCCCACCGACAAGCCTCTCCGTCTGCCTCTGCAGGACGT Metaseiulus occidentalis TGAAGGCAAGACCCTTCTCCAGGCCCTCGATGTCATGGAGCCGCCCACCAGGCCCACCGACAAGCCGCTTCGTCTTCCCCTCCAGGACGT Dermanyssus hirundinis TGAGGGCAAGACGCTGCTGCAGGCCCTCGACGTCATGGAGCCGCCCGCGAGGCCCACCGACAAGCCTCTGCGTCTGCCCCTGCAGGACGT Euzercon latum CGAGGGAAAGACCCTCTTGCAGGCCCTCGATGTCATGGAGCCACCGAGCAGGCCTACCGACAAGCCTCTTCGATTGCCTCTGCAGGATGT Amblyomma sp. TGAGGGCAAGACCCTTCTTCAGGCTCTCGACGCGATGGAGCCCCCGACCCGGCCCACGGACAAGCCCCTCCGACTTCCCCTGCAGGACGT Neacarus texanus TGAAGGCAAGACACTTCTGCAGGCTCTGGATGCCATGGAGCCCCCCACTAGGCCAACTGACAAACCCCTTAGGCTTCCCCTTCAGGATGT Raoiella indica TGAGGGGAAAACTTTGCTCCAAGCTCTTGATGCCATGGACCCACCATCCAGACCGGTTGACAAGCCTCTACGTCTACCACTCCAGGATGT Tetranychus urticae GGAAGGTAAAACATTGTTACAAGCCTTAGATGCTATGGATCCTCCATCTCGACCAGTTGATAAGGCACTTCGACTTCCACTTCAAGATGT Aphonopelma chalcodes TGATGGCAAGACATTATTGCAAGCATTGGATGCTATGGAGCCACCATCTCGACCTCTGGACAGGCCACTCAGGTTGCCTCTTCAGGATGT Dysdera crocata TGACGGCAAGACCTTGTTGCAAGCTTTGGATGCCATGGAGCCACCCTCCAGGCCTTTGGACAAACCTCTTAGATTGCCCCTCCAGGATGT Limulus polyphemus TGAAGGCAAGACTCTCTTGCAAGCTTTGGACTGTGCTGAACCTCCATCTCGTCCCACTGACAAGCCTCTTCGTCTGCCTCTGCAGGATGT Locusta migratoria TGAGGGAAAGACTTTAATTGAAGCTCTCGATGCCATCCTCCCTCCCAACAGGCCAACTGAGAAGCCTCTTAGGCTTCCTCTTCAGGATGT Drosophila melanogaster AGAGGGCAAGTGCTTGATCGACGCGCTGGACGCGATCCTTCCACCCCAGCGTCCCACCGACAAGCCGCTGCGCCTGCCGCTCCAGGACGT Figure 2-3. Continued.

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105 460 470 480 490 500 510 520 530 540 . . South Florida 1 TTACAAGATCGGTGGTATCGGCACAGTGCCCGTCGGTCGTGTCGAAACCGGTGTCATCAAGCCTGGTATGGTGGTCACTTTCGCTCCTTC 2 CTACAAGATCGGAGGTATCGGAACGGTCCCCGTGGGCCGTGTGGAAACCGGCGTCCTCAAGCCCGGCATGGTGGTCACCTTCGCCCCGGC 3 CTACAAGATCGGAGGTATCGGAACGGTCCCCGTGGGCCGTGTGGAAACCGGCGTCCTCAAGCCCGGCATGGTGGTCACCTTCGCCCCGGC 4 TTACAAGATCGGTGGTATCGGCACAGTGCCCGTCGGTCGTGTCGAAACCGGTGTCATCAAGCCTGGTATGGTGGTCACTTTCGCTCCTTC 5 CTACAAGATCGGAGGTATCGGAACGGTCCCCGTGGGCCGTGTGGAAACCGGCGTCCTCAAGCCCGGCATGGTGGTCACCTTCGCCCCGGC 6 TTACAAGATCGGTGGTATCGGCACAGTGCCCGTCGGTCGTGTCGAAACCGGTGTCATCAAGCCTGGTATGGTGGTCACTTTCGCTCCTTC 7 TTACAAGATCGGTGGTATCGGCACAGTGCCCGTCGGTCGTGTCGAAACCGGTGTCATCAAGCCTGGTATGGTGGTCACTTTCGCTCCTTC 8 CTACAAGATCGGAGGTATCGGAACGGTCCCCGTGGGCCGTGTGGAAACCGGCGTCCTCAAGCCCGGCATGGTGGTCACCTTCGCCCCGGC Flic en Flac 2 CTACAAGATCGGAGGTATCGGAACGGTCCCCGTGGGCCGTGTGGAAACCGGCGTCCTCAAGCCCGGCATGGTGGTCACCTTCGCCCCGGC 3 CTACAAGATCGGAGGTATCGGAACGGTCCCCGTGGGCCGTGTGGAAACCGGCGTCCTCAAGCCCGGCATGGTGGTCACCTTCGCCCCGGC North of Port Louis 1 TTACAAGATCGGTGGTATCGGCACAGTGCCCGTCGGTCGTGTCGAAACCGGTGTCATCAAGCCTGGTATGGTGGTCACTTTCGCTCCTTC Trou dEau Douce 1 TTACAAGATCGGTGGTATCGGCACAGTGCCCGTCGGTCGTGTCGAAACCGGTGTCATCAAGCCTGGTATGGTGGTCACTTTCGCTCCTTC 2 CTACAAGATCGGAGGTATCGGAACGGTCCCCGTGGGCCGTGTGGAAACCGGCGTCCTCAAGCCCGGCATGGTGGTCACCTTCGCCCCGGC 3 TTACAAGATCGGTGGTATCGGCACAGTGCCCGTCGGTCGTGTCGAAACCGGTGTCATCAAGCCTGGTATGGTGGTCACTTTCGCTCCTTC 4 CTACAAGATCGGAGGTATCGGAACGGTCCCCGTGGGCCGTGTGGAAACCGGCGTCCTCAAGCCCGGCATGGTGGTCACCTTCGCCCCGGC Amblyseius swirskii TTACAAGATCGGAGGTATTGGCACAGTGCCCGTAGGTCGTGTCGAAACCGGTGTCATCAAGCCTGGTATGGTCGTCACTTTCGCTCCGTC Neoseiulus californicus TTACAAGATCGGAGGAATCGGAACAGTCCCTGTAGGCCGTGTGGAAACCGGAGTTCTGAAACCCGGCATGGTCGTCACCTTCGCCCCGGC Neoseiulus cucumeris TTACAAGATCGGAGGAATCGGAACAGTTCCTGTGGGCCGTGTTGAAACCGGAGTTCTCAAACCCGGCATGGTGGTCACCTTCGCTCCGGC Metaseiulus occidentalis CTACAAGATCGGCGGTATCGGAACAGTGCCCGTGGGCCGTGTCGAAACCGGTGTCATCAAGCCCGGTATGGTGGTCACCTTCGCGCCGTC Dermanyssus hirundinis CTACAAGATCGGCGGTATTGGTACGGTGCCCGTAGGCCGTGTCGAGACTGGCGTCATCAAGCCCGGCATGGTCGTCACGTTCGCGCCGTC Euzercon latum CTACAAAATTGGAGGTATTGGCACAGTACCCGTGGGTCGTGTTGAAACTGGTGTGCTCAAGCCCGGCATGGTTGTCACGTTTGCACCTGC Amblyomma sp CTACAAGATTGGTGGCATTGGCACGGTGCCCGTCGGCCGTGTGGAGACCGGCGTTCTCAAGCCCGGCATGGTCGTCACCTTTGCCCCTGC Neacarus texanus GTATAAAATTGGAGGTATTGGAACTGTGCCAGTTGGTAGAGTTGAAACTGGTGTTCTTAAGCCGGGTATGGTGGTTACCTTTGCTCCATG Raoiella indica CTACAAGATTGGTGGTATTGGTACAGTACCTGTTGGTCGTGTCGAAACTGGTGTTATTAAGCCTGGTATGGTCGTTACGTTCGCTCCTGT Tetranychus urticae CTACAAAATCGGTGGTATTGGTACTGTACCAGTTGGTAGAGTTGAAACTGGTACAATTAAGCCAGGTATGATTGTTACATTTGCACCAGT Aphonopelma chalcodes CTACAAAATTGGAGGTATTGGTACTGTTCCTGTTGGCAGAGTTGAAACTGGAGTGTTGAAACCTGGAATGGTTGTTACTTTTGCTCCTGT Dysdera crocata CTACAAAATCGGAGGTATTGGAACTGTCCCAGTCGGCAGAGTGGAAACTGGTGTCATGAAACCTGGTATGGTCGTCACCTTTGCTCCAGT Limulus polyphemus CTACAAAATTGGAGGTATTGGTACTGTACCTGTTGGTAGAGTTGAAACTGGTGTCTTGAAACCTGGCACCGTGGTTACCTTTGCCCCTGC Locusta migratoria GTACAAAATTGGTGGTATTGGAACAGTACCTGTGGGCCGAGTAGAAACAGGTATTCTCAAACCTGGTATGGTTGTGACATTTGCTCCAGC Drosophila melanogaster CTACAAGATCGGAGGCATCGGAACCGTACCAGTAGGTCGTGTGGAGACTGGTCTCCTCAAGCCAGGCATGGTCGTCAACTTTGCGCCGGT Figure 2-3. Continued.

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106 550 560 South Florida 1 CAACCTCACCACTGAAGTCAAGTCCGTC 2 CCACATCACCACCGAGGTGAAGTCCGTG 3 CCACATCACCACCGAGGTGAAGTCCGTG 4 CAACCTCACCACTGAAGTCAAGTCCGTC 5 CCACATCACCACCGAGGTGAAGTCCGTG 6 CAACCTCACCACTGAAGTCAAGTCCGTC 7 CAACCTCACCACTGAAGTCAAGTCCGTC 8 CCACATCACCACCGAGGTGAAGTCCGTG Flic en Flac 2 CCACATCACCACCGAGGTGAAGTCCGTG 3 CCACATCACCACCGAGGTGAAGTCCGTG North of Port Louis 1 CAACCTCACCACTGAAGTCAAGTCCGTG Trou dEau Douce 1 CAACCTCACCACTGAAGTCAAGTCCGTC 2 CCACATCACCACCGAGGTGAAGTCCGTG 3 CAACCTCACCACTGAAGTCAAGTCCGTC 4 CCACATCACCACCGAGGTGAAGTCCGTG Amblyseius swirskii CAACCTCACCACTGAAGTCAAGTCCGTC Neoseiulus californicus TCACATCACCACTGAAGTGAAGTCCGTG Neoseiulus cucumeris TCACATCACTACCGAAGTGAAGTCCGTG Metaseiulus occidentalis CAACCTCACCACTGAAGTCAAGTCCGTC Dermanyssus hirundinis TAACCTCACCACTGAGGTGAAGTCGGTC Euzercon latum CAACTTGACTACTGAAGTAAAGTCTGTT Amblyomma sp. CAACCTGACCACTGAGGTCAAGTCCGTG Neacarus texanus CAACCTTACAACTGAAGTCAAGTCTGTT Raoiella indica CGGTATTACCACTGAAGTTAAATCAGTC Tetranychus urticae TAACTTGACAACTGAAGTAAAATCAGTT Aphonopelma chalcodes CAACTTAACTACTGAAGTGAAGTCTGTG Dysdera crocata TAACATCACCACTGAAGTAAAATCTGTG Limulus polyphemus TATGATCACCACCGAAGTAAAGTCTGTT Locusta migratoria TAATTTGACGACTGAAGTAAAATCTGTA Drosophila melanogaster CAACCTGGTCACCGAAGTAAAGTCTGTG Figure 2-3. Continued.

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107 Figure 2-4. The 12S rRNA concensus tree was inferr ed using the MCMC method in MrBayes (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003). The Mauritius and S. Florida 12S clones are in two distinct sister clades. Number following a colony source location indicates the clone number. The posterior probabi lities tree inferred from 1,000,000 generations is taken to represent the evolutionary history of t he taxa analyzed. Branches corresponding to partitions with less than a posterior probability of 50 are collapsed.

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108 Figure 2-5. The EF-I concensus tree was inferred using the MCMC method in MrBayes (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) with the assumption that all sequ ences represent a single gene. The Mauritius and S. Florida clones are inte rmingled between two clades. Number following a colony source location indi cates the clone number. The posterior probabilities tree inferred fr om 1,000,000 generations is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions with less than a posterior probability of 50 are collapsed.

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109 Figure 2-6. The putative EF-I concensus tree was inferred using the MCMC method in MrBayes (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003) assuming two different genes are present. Both the Mauritius and S. Florida clones are grouped together in the same clade when a single putative EF-I gene is used. Number following a colony source location indicates the clone number. The posterior probabilities tree was in ferred from 1,000,000 generations is taken to represent t he evolutionary hist ory of the taxa analyzed. Branches corresponding to par titions with less than a posterior probability of 50 are collapsed.

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110 Figure 2-7. The evolutionary history obtained from the high-fidelity-RAPD-PCR markers 196 and 199 for S. Florida (colonies 1-4) and Mauritius (colonies Flic en Flac, Trou dEau Douce (1-3), and North of Port Louis) Amblyseius largoensis populations was inferred using the Neighbor-Joining method (Saitou and Nei 1987) in PAUP 4.0b10 (Swofford 2003). The bootstrap consensus tree inferred from 100,000 replicates is taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). Branches corresponding to partitions reproduced in less than 50% bootstr ap replicates are collapsed.

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111 Figure 2-8. High-fidelity PCR products obt ained from the Maurit ius and S. Florida colonies using 12S rRNA population-specific primers. PCR products obtained using the Mauritius forward primer (A) and S. Florida population-specific forward primer (B) indicate the appropr iate band size for the Mauritius (330 bp) and the S. Florida (406 bp) populations while products of that band size were not produced from Amblyseius swirskii, Neoseiulus californicus, N. cucumeris, and Metaseiulus occidentalis

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112 APPENDIX PERSPECTIVES First, I would like to express how happy and privileged I feel to be involved with the Entomology and Nematology department at the University of Florida. It is a w onderful department composed of insightful faculty me mbers, supportive staff and a vibrant student body excited about t he study of acarology, ent omology, and nematology. The past two years as an M.S. student have gr eatly enriched my life. I have gained confidence as a scientist had the opportunity to learn innumerable professional and life lessons. Thank you. If you can't ride two horses at on ce, you shouldn't be in the circus James Maxton. This quote by James Maxton perfectly embodies the expectations placed on graduate students and scientists as a whole. To be successful, I have learned that I must be able to multi-task, efficiently super vise others, work well with a variety of personalities and work styles, be able to clear ly communicate ideas to the scientific community and the layman, and maintain a dynamic approach to my research. Firstly, I learned to work harder and sm arter than I thought possible before this experience. I learned to multi-task many layers of my daily life: mu ltiple lab protocol, research and study, writing and lab work, and student life and personal life. Dr. Hoy taught me that the best way to learn a techni que is to be taught, to practice, and then to teach someone else. This proved true when I had the opportunity to teach a visiting scientist from Egypt how to set-up a high-fide lity PCR and how to visualize your product. I was given the opportunity to train and m anage two OPS workers while a student in Dr. M. A. Hoys lab. This was a wonderful opportunity to learn management tactics for

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113 different employee personalities, to observe different working habits between the artisan and time management personalities, and to gain r ealistic work expectations. During my experience as an M.S. student, I learned that communication is critical to the success of any research project. I had a di fficult time communi cating my thoughts clearly and concisely when I joined the lab. I was able to improve my communication skills and confidence though conversations with Dr. Hoy, Dr. Jeyaprakash, and my fellow students. Through my learning experi ences, I have observed that often people enter a scientific discussion with assumpti ons that may skew what is being said. Therefore, in order to gain the most from the experience, it is important to approach every conversation with an open mind as a speaker and as a listener. In addition, when there is a breakdown in communication due to poor skills or personality conflict, the research may still be successful but not to the level of achievement possible. I learned that in science, it is essent ial to maintain a dynamic approach to research. It is important to discover to new and creative ways to solve problems in the lab and to answer scientific questions. I have al so learned that a hypothesis can be ever changing. If an experiment indica tes an original hypothesis incorrect, it is important to adjust accordingly so that the proceeding series of experiments will produce valuable information. As a M.S. student of Dr. M. A. Hoys I l earned many valuable ski lls that I can apply to my personal and future professional life. I am very grateful fo r the experiences and opportunities to learn while under her tutelage.

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114 LIST OF REFERENCES Abou-Setta MM, Childers CC, Denmark HA Browning, H W (1991) Comparative morphology and reproductive compat ibility between populations of Euseius mese mbrinus (Acari: Phytoseiidae) from Flor ida and Texas. Exp Appl Acarol, 10:213-220 Adams BJ (1998) Species concepts and t he evolutionary paradigm in modern nematology. J Nematol, 30:1-21 Altschul SF, Stephen F, Maden TL, Schffer A A, Zhang J, Zang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 25:3389-3402 Altschul SF, Wootton JC, Gertz EM, Agarwala R, Morgulis A, Schffer A A, Yu Y-K (2005) Protein database searches using compositionally adjusted substitution matrices. FEBS J, 272:5101-09 Amano H, Chant DA (1978) Some factors affe cting reproduction and sex ratios in two species of predacious mites, Phytoseiulus persimilis Athias-Henriot and Amblyseius andersoni (Chant) (Acarina: Phytosei idae). Can J Zool, 56:15931607 Arensburger P, Buckley TR, Simon C, M oulds M, Holsinger KE (2004) Biogeography and phylogeny of the New Zealand cic ada genera (Hemiptera: Cicadidae) based on nuclear and mitochondrial DNA data. J Biogeogr, 31:557-569 Arthurs S, McKenzie CL, Chen J, Dogramaci M, Brennan M, Houben K, Osborne L (2009) Evaluation of Neoseiulus cucumeris and Amblyseius swirskii (Acari: Phytoseiidae) as biological control agents of chilli thrips, Scirtothrips dorsalis (Thysanoptera: Thripidae), on pepper. Biol Cont, 49:91-96 Avise J C (1994) Molecular Markers, Natu ral History and Evolution. Chapman and Hall, New York, NY, 684 pp Bandi C, Anderson TJC, Genchi C, Blaxter ML (1998) Phylogeny of Wolbachia -like bacteria in filarial nematodes. Proc R Soc London Ser. B, 265:2407-13 Barnes W (1994) PCR amplification of up to 35-kb DNA with high-fidelity and high yield from bacteriophage templates. Proc Natl Acad Sci, USA, 91:2216-2220 Bellows TS, Van Driesche RG, Elkinton JS (1992) Life-table construction and analysis in the evaluation of natural enemies. Annu Rev Entomol, 37:587-614 Black WC (1993) PCR with arbitr ary primers: approach with care Insect Mol Biol, 2:1-6

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115 Black WC IV, Roehrdanz RL (1998) Mitoc hondrial gene order is not conserved in arthropods: prostriate and metastriate ti ck mitochondrial genomes. Mol Biol Evol, 15:1772-85 Boom R, Sol CJA, Salimans MMM, Jans en CL, van Dillen PMEW, van der Noordaa J (1990) Rapid and simple method for purif ication of nucleic acids. J Clinical Micro, 28:495-503 Borchert D, Margosian M ( 2007) Risk analysis of potentia l consequences associated with the introduction of the red palm mite, Raoiella indica into the United States. USDA-APHIS-PPQ-CPHST-PERAL, 24 pp Bronson CH (2009) Nursery Industry Update. Florida Department of Agriculture and Consumer Services, Division of Plant Industry. [updated 15 May 2009; cited 15 July 2009]. Available from http://www.doacs.state.fl.us/ pi/enpp/ento/red_palm_mite.html Brower AVZ, DeSalle R (1994) Practical and theoretical cons iderations for choice of a DNA sequence region in insect molecula r systematics, with a short review of published studies using nuclear gene regi ons. Ann Entomol Soc Amer, 87:702716 Brown JK (2004) Tracing the origin of crypti c insect pests and vectors, and their natural enemies. In: Genetics, Evolution and Biol ogical Conrol. Ehler LE, Sforza R, Mateille T (eds), CABI Publishing, Cambridge, MA, 260 pp Bruce-Oliver SJ, Hoy MA ( 1990) Effect of prey stage on life-table attributes of a genetically manipul ated strain of Metaseiulus occidentalis (Acari: Phytoseiidae). Exp Appl Acarol, 9:201-217 Bryan RT (1999) Alien species and emerging infectious diseases: past lessons and future implications. In: Invasive Species and Biodiv ersity Management. Kluwer Academic, Dordrecht, Netherlands, pp 163-175 Buckley TR, Cordeiro M, Marshall DC, Simon C (2006) Diffe rentiating between hypotheses of lineage sorting and introgre ssion in New Zealan d alpine cicadas ( Maoricicada Dugdale). Syst Biol, 55:411-425 CAPS Surveys (2008) Current and Ongoing Su rvey Programs: Red Palm Mite. CAPS Surveys, Florida Dept. of Agric. and C onsumer Services, Div. of Plant Industry. [updated 20 May 2008; cited 29 A ug. 2008]. Available from http://www.doacs.state.fl.us/pi/caps/surveys.html CAPS Surveys (2010) Current and Ongoing Su rvey Programs: Red Palm Mite. CAPS Surveys, Florida Dept. of Agric. and C onsumer Services, Div. of Plant Industry. [updated 11 Feb. 2010; cited 10 March 2010]. Available from http://www.doacs.state.fl.us/pi/caps/surveys.html

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136 BIOGRAPHICAL SKETCH Heidi Marie Bowman is a native of t he Shenandoah Valley situated under the Blue Ridge Mountains in Virginia. Upon graduatio n from Harrisonburg High School in 1997, she began her undergraduate studies at Blue Ridge Community College wher e she received an Associates degree in 2000. She wa s accepted into the horticulture program at West Virginia University (WVU) (Morgant own, WV) in 2000, and received her B.S. in horticulture in 2002. She then obt ained a Master of Agricultur e, Forestry and Consumer Sciences degree at WVU under the guidance of Dr. Sven Ve rlinden in 2004. In 2005, she enrolled in the interdisciplinary Doctor of Plant Medicine Program at the University of Florida under the supervision of Dr Robert McGovern. In 2007, she began a concurrent Master of Science program within the Department of Entomology and Nematology under the supervision of Dr. Marjor ie A. Hoy. She is currently a member of the Acarological Society of America, Syst ematic and Applied Acarology society, The Florida Entomological Society, and t he Entomological Society of America.