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1 INTERACTIONS BETWEEN FRANKLINIELLA FUSCA (THYSANOPTERA: THRIPIDAE), THRIPINEMA FUSCUM (TYLENCHIDA: ALLANTO NEMATIDAE), AND ENTOMOPARASITES ( TOMATO SPOTTED WILT VIRUS PANTOEA ANANATIS AND WOLBACHIA) By KELLY RENEE SIMS A DIS SERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010
2 2010 Kelly Renee Sims
3 To my f amily Ford Sims, Sandy Morgan, Laura Bonsky Olga Kapps, and especially James C. Dunford, Kia, Willie and Zigg y for their unconditional love and support while I pu rsued my entomological studies, making this milestone possible
4 ACKNOWLEDGMENTS I would firs t like to thank my advisors Joe Funderburk (University of Floridas North Florida Research and Education Center in Quincy, FL [UF NFREC]) and Drion Boucias (UF Department of Entomology and Nematology). Their advice, patience, guidance, and friendship have made this research possible. I would also like to thank my committee members Stuart Reitz (United States Department of Agriculture Agricultural Research S ervice Center for M edical, Agricultural, and V eterina ry Entomology in Tallahassee, FL [USDA -ARS-CMAVE] ), Ji mmy Becnel ( USDA -ARS CMAVE in Gainesville, FL) and Timur Momol ( UF NFREC in Quincy, FL ) for providing their time and expertise. Most of th is research would not have been possible without the ir help A very specia l thanks goes to the numerous i n dividuals who have provided knowledge equipment, supervision, and expertise on topics covered in this project: Byron Adams (Brigham Young University) Kostas Bourtzis (University of Ioannina) James Boyer (UFs Plant Science Research and Education Unit i n Citra, FL) Ellen D ickstein (UFs Plant Pathology) Karen and Kim Kelley (UFs Interdisciplinary Center for Biotechnology Research [ UF -ICBR]), Dean Paini (UF-NFREC), Dorith Rotenber g (Kansas State University) Savita Shanker (UF -ICBR), Mittrinjai Srivistava (UF -NFREC), Julie Stavisky (UF -NFREC), Michelle Stuckey (UF NFREC), Chris Tipping (UF NFREC) Carl Vining (UF -PSREU) Anne Whitfield (University of Wisconsin) and Donna Will i ams (UF -ICBR). I would like to thank numerous faculty, staff and students at the University of Floridas Department of Entomology and Nematology for making my research a rewarding and fulfilling experience: Carl Barfield Marc Branham Lyle Buss John Capinera John Denton Don Dickson Dan Fitspatrick Judy Gillmore Maria Gomez Debbie Hall Don Hall Amanda Hodges Nick Hostettler Pam Howell Marjorie Hoy Steve Lasley Verena Leitze Norm
5 Leppla Oscar Liburd Ale and Jim Maruniak Heather McAuslane Jane Medley James Nation Khu o ng Nguyen Linda Pedersen Pannipa Prompiboon Tamer Salem Nancy Sanders Mike Scharf Grover Smart Matt Tarver and Mary Kay Weigel. A very special thank you goes to those who contributed to my teaching experience at the University of Florida; they are: Doug Levey Elisa Livengood, Suzan Smith Carmella OSteen and Dale Witt. I would also like to thank those who opened their hearts and their homes to me: J udy Boucias Lincoln Brower and Miriam Funderburk. Most importantly, I would like to thank my family and friends for their support and patie nce: Bob and Maude Ashley; Marcia and Paul Blankenship; Laura, Michael, Mikayla, Taryn and Ryan Bonsky and Aaron Salazar; Brent Brooks ; Karen Buchanan and her family; Nic ole, Andrew and Brooklyn Butler; Glen Cannon and Jean Thomas ; Becky, Carl and Amanda Carter ; Frank and Mary Cassett; Karen and Alyssa Christner; Adie Davi es; Jim Kia, Willie, and Ziggy Dunford ; James F. Dunford; K im Dunford; K athy and Vance Eaddy; Imogene Floyd; Evelyn Green and Joe Hopkins; Marie Hershberger; Carol Kapps; Olga Kapps; Kat and Benn ett King; Lynne and Greg McCrae; Jennifer and Jason Meyer; Sandy and Mike Morgan; George, Dina and Caroline Nunn; Lisa Nunn, Pat Fry and Frankie DeAngelis; Gale and Jim Owens and their family Heather and Emmalee Larsen, Rachel, Jeff, Caitlyn and Lily Thomas and Jamie, Kelly and Erin Owens and Natalie Shaw; Andrea, Matt and Taryn Pawl; Kitty Phelps; John Ridg way; Betty Romano; Fran, Pete and Francine Rossi and Carol Field ; Carolyn Sabol and her family; Karen and Lee Schwind ; Ford Sims ; Teri Stuber; Glenn and Joan Taylor; April Terry; BJ Thornton; Margo and Roger Thrall; Laurie and Calvin Trenholm ; and Joanne Wilson
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 8 LIST OF FIGURES ............................................................................................................................ 10 ABSTRACT ........................................................................................................................................ 15 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW: RESEARCH BACKGROUND, OBJECTIVES, AND HYPOTHESES ....................................................................................... 17 Introduction and Literature Review ........................................................................................... 17 Thy sanoptera ........................................................................................................................ 17 Natural history of thrips ............................................................................................... 17 Biological control of thrips pests ................................................................................ 21 Frankliniella fusca (Hinds) (Thysanoptera: Thripidae) ............................................. 23 Entomoparasites Associated with Frankliniella fusca ...................................................... 26 Tomato spotted wilt virus (Bunyaviridae: Tospovirus) ............................................. 27 Pantoea ananatis (Serrano 1928) Mergaert et al. 1993 ............................................. 36 Wolbachia Hertig (Rickettsiales: Rickettsiaceae) ...................................................... 38 Entomophthorales spp. ................................................................................................ 42 Thripinema fuscum (Tylenchida: Allantonematidae) ................................................ 43 Goals, Objectives, Hypotheses, and Expected Outcomes ........................................................ 48 2 MOLECULAR IDENTIFICATION OF THE REPRODUCTIVE PARASITE WOLBACHIA IN HOST PARASITE POPU LATIONS OF FRANKLINIELLA FUSCA AND THRIPINEMA FUSCUM .................................................................................................. 58 Introduction ................................................................................................................................. 58 Materials and Methods ................................................................................................................ 59 Results .......................................................................................................................................... 65 Discussion .................................................................................................................................... 69 3 HISTOLOGICAL EXAMINATION OF FRANKLINIELLA FUSCA AND THRIPINEMA FUSCU M ............................................................................................................ 84 Introduction ................................................................................................................................. 84 Materials and Methods ................................................................................................................ 85 Results .......................................................................................................................................... 87 Discussion .................................................................................................................................... 95
7 4 THE IMACT OF A PARASITIC NEMATODE THRIPINEMA FUSCUM ON THE FEEDING BEHAVIOR AND VECTOR COMPETENCE OF FRANKLINIELLA FUS CA ....................................................................................................................................... 152 Introduction ............................................................................................................................... 152 Materials and Methods .............................................................................................................. 154 Results ........................................................................................................................................ 159 Discussion .................................................................................................................................. 161 5 THE MULTITROPHIC INTERACTIONS BETWEEN THE PARASITIC NEMATODE THRIPINEMA FUSCUM, PLANT PATHOGENS PANTOEA ANANATIS A ND TOMATO SPOTTED WILT VIRUS AND THE INSECT VECTOR FRANKLINIELLA FUSCA ....................................................................................................... 171 Introduction ............................................................................................................................... 171 Materials and Methods ( Pantoea anan atis )............................................................................. 174 Materials and Methods ( Tomato spotted wilt virus ) ............................................................... 179 Results ( Pantoea ananatis ) ....................................................................................................... 182 Results (TSWV) ........................................................................................................................ 185 Discussion .................................................................................................................................. 188 6 DATA SYNOPSIS AND FUTURE RESEARCH .................................................................. 205 APPENDIX A WOLBACHIA 16S RRNA, COI, AND MLST SEQUENCES ............................................... 209 B RECIPES FOR ELECTRON MICROSCOPY ....................................................................... 241 C RNA EXTRACTION, BACTERIAL IDENTIFICATION, AND MOLECULAR PROTOCOLS ............................................................................................................................ 242 LIST OF REFERENCES ................................................................................................................. 252 BIOGRAPHICAL SKETCH ........................................................................................................... 285
8 LIST OF TABLES Table page 2-1 Collection data including cohort population, locality, and date labeled for Wolbachia gene screen and 16S rRNA analysis .................................................................................... 73 2-2 16S, wsp and MLST loci a nd primer features .................................................................... 75 2-3 Wolbachia surface protein ( wsp ) loci and hypervariable region profi le (HVR1 4) for the six non -parasitized Frankliniella fusca samples selected for MLST. .......................... 76 2-4 The percentage of each Frankliniella fusca cohort population with a visible amplicon of each prime r by conventional PCR. ................................................................................... 76 2-5 Allelic profiles of the six nonparasitized Frankliniella fusca samples selected for MLST. ..................................................................................................................................... 77 2-6 The percentage of male and female Frankliniella fusca at adult eclosion after tetracycline (50 mg/ml) treatment as first instars. ................................................................ 77 3-1 Procedure for preparation of insect tissue for scanning (1 5) and transmission (16) electron microscopy. ............................................................................................................ 103 4-1 Mean longevity ( SE) of adult Frankliniella fusca .......................................................... 166 4-2 Mean total area of feeding ( SE) on leaf discs (mm2) fed on by Frankliniella fusca individuals for the initial 10 days of adulthood. ................................................................. 166 4-3 Proportion of viruliferous Frankliniella fusca cohorts trans mitting TSWV each day. .... 167 5-1 T he percentage of individual females containing bacteria cultivable on trypic soy broth agar plates from nonT. fuscum parasitized and T. fuscum parasitized Fran kliniella fusca laboratory and field populations. ........................................................ 194 5-2 The mean number of Pantoea ananatis colony forming unit counts standard deviation for early and late stage Frankliniella fusca plate d individuals, based on Thripinema fuscum parasitism status. ................................................................................. 194 5-3 Measurements of the zone of inhibition (in mm) for lysozyme standards (g/ml) used for determining the units of activity (E U/ml) in acid -derived T. fuscum parasitized Frankliniella fusca homogenates. ....................................................................................... 195 5-4 The average threshold cycle (CT) values standard deviation for TSWV and COI transcripts and relative quan tity of TSWV detected by quantitative PCR for viruliferous Frankliniella fusca cohorts. ............................................................................ 195
9 5-5 Efficiency of Tomato spotted wilt virus transmission by nonT. fuscum parasitized and T. fuscum parasitized Frankliniella fusca individuals. ............................................... 196 5-6 The percentage standard error of viruliferous Frankliniella fusca cohorts transmitting Tomato spotted wilt virus during each inoculation period. .......................... 196 5-7 The average threshold cycle (CT) values for TSWV and COI transcripts detected by quantitative PCR for individual viruliferous Frankliniella fusca .................................... 197
10 LIST OF FIGURES Figure page 1-1 The life cycle of Frankliniella fusca at 27C. ..................................................................... 51 1-2 Illustrations of Arachi s hypogaea and Tomato spotted wilt virus ....................................... 52 1-3 A collage of various fungi recovered from Frankliniella fusca laboratory and field popul ations in north central Florida ...................................................................................... 53 1-4 Differential interference contrast (DIC) microscope images of Entomophthorales sp. stages infecting Frankliniella fusca ..................................................................................... 54 1-5 Scanning electron microscope images of Entomophthorales sp. stages infecting Frankliniella fusca ................................................................................................................ 54 1-6 Entomophthorales sp. hyphal bodies in Frankliniella fusca ............................................... 55 1-7 The progression of an Entomophthorales sp. infection in the female Frankliniella fusca ....................................................................................................................................... 56 1-8 The life cycle of Thripinema fuscum in a female Frankliniella fusca host ........................ 57 2-1 An initial Wolbachia gene screen of 36 Frankliniella fusca and Thripinema fuscum populations .............................................................................................................................. 78 2-2 Neighbor joining phylogenetic tree based on 16S rRNA nucleotide alignment. ............... 80 2-3 Amino acid alignment of six wsp sequences from non -parasitized female Frankliniella fusca cohort populations. ................................................................................ 81 2-4 Conventional PCR detection of Wolbachia in Frankliniella fusca with 16S rRNA primers ( wspec ) after antibiotic therapy. ............................................................................. 82 2-5 Transmission e lectron micrographs of intracellular bacteria in the reproductive structures of non-parasitized and Thripinema fuscum parasitized Frankliniella fusca females. ................................................................................................................................... 83 3-1 The external dorsal view o f the Frankliniella fusca female head tagma with antennae, compound eyes, and ocelli. ................................................................................. 104 3-2 The mouthparts of a Frankliniella fusca female ................................................................ 105 3-3 The two wing morphs of Frankliniella fusca ..................................................................... 106 3-4 Scanning electron micrographs documenting the posterior opening of a Frankliniella fusca female. ......................................................................................................................... 107
11 3-5 A longitudinal thick section of a healthy Frankliniella fusca female. .............................. 108 3-6 The ovoid and tubular salivary glands of a Frankliniella fusca fe male ........................... 109 3-7 Transmission electron micrographs of Tomato spotted wilt virus in the ovoid salivary glands of a viruliferous Frankliniella fusca female ........................................................... 110 3-8 Digestive tract of the Frankliniella fusca female ............................................................... 111 3-9 Transmission electron micrographs showing midgut cells of the Frankliniella fusca female .................................................................................................................................... 112 310 Malpighian tubules of the Frankliniella fusca female. ...................................................... 113 311 Transmission electron micrographs showing the hindgut of Frankliniella fusca ........... 114 312 The genital region of a Frankliniella fusca female. ........................................................... 115 313 Longitudinal thick sections of the female Frankliniella fusca host documenting egg development. ......................................................................................................................... 116 314 Scanning electron micrographs of the abdominal cavity of a Frankliniella fusca female with egg. ................................................................................................................... 117 315 The developing oocytes of a fecund Frankliniella fusca female ...................................... 118 316 The progressive magnification of connections between a follicular cell and developing oocy te of a non-parasitized Frankliniella fusca female ................................. 118 317 The fat body of a Frankliniella fusca female. .................................................................... 119 318 The musculatu re of Frankliniella fusca .............................................................................. 119 319 Scanning electron micrographs documenting the external surface of the infective Thripinema fuscum female .................................................................................................. 120 320 Transmission electron micrographs showing the cross -section of an infective Thripinema fuscum female with lateral lines ...................................................................... 120 321 Differential interference contrast microscopic image (DIC) of an infective Thripinema fuscum female with anterior esophageal region. ............................................ 121 322 The cephalic region of the infective female Thripinema fuscum ...................................... 121 323 Scanning electron micrographs of the excretory pore of an infective Thripinema fuscum female. ...................................................................................................................... 122 324 Differential interference contrast microscopic images (DIC) of the parasitic Thripinema fuscum female.. ................................................................................................ 123
12 325 Scanning electron micrographs of the cuticular structure of the parasitic Thripinema fuscum female ....................................................................................................................... 124 326 Transmission electron micrographs documenting the cuticular structure of the parasitic Thripinema fuscum female.. ................................................................................. 125 327 Differential interference contrast microscopic images (DIC) of the Thripinema fuscum male .......................................................................................................................... 126 328 Scanning electron micrographs of the Thripinema fuscum male ...................................... 127 329 Differential interference contrast microscopic images (DIC) of the Thripinema fuscum juveniles. .................................................................................................................. 128 330 A collage of scanning electron micrographs documenting the various juvenile annulated cuticles of Thripinema fuscum .......................................................................... 129 331 A collage of transmission electron micrographs showing the various cuticular structures of juvenile Thripinema fuscum. .......................................................................... 130 332 Transmission electron micrographs showing the morphological structure of the juvenile Thripinema fuscum cuticle. ................................................................................... 131 333 Scanning electron micrographs of the anterior region of the juvenile Thripinema fuscum .................................................................................................................................. 132 334 Thripinema fuscum eggs ...................................................................................................... 133 335 Scanning ele ctron micrographs of the Thripinema fuscum eggs. ...................................... 134 336 Longitudinal thick sections documenting the in vivo life cycle of Thripinema fuscum in the female Frankliniella fusca host. ............................................................................... 135 337 Longitudinal thick section of a parasitized Frankliniella fusca female with juvenile nematodes throughout the host tagma. ................................................................................ 136 3-38 The life cycle of Thripinema fuscum ................................................................................. 137 339 A comparison of a non-parasitized and a Thripinema fuscum parasitized Frankliniella fusca female ................................................................................................... 138 340 A longitudinal thick section of a Thripinema fuscum parasitized Frankliniella fusca female .................................................................................................................................... 138 341 Scanning electron micrographs showing the internal abdominal cavit y of a Frankliniella fusca female parasitized by Thripinema fuscum ........................................ 139 342 Transmission electron micrographs documenting the parasitic Thripinema fuscum female in direct apposition to host m idgut cells. ................................................................ 140
13 343 Transmission electron micrographs showing the midgut cells of a Thripinema fuscum parasitized Frankliniella fusca female. ............................................................................... 141 344 Scanning electron micrographs documenting the accumulation of late -staged juvenile nematodes in the hindgut of Frankliniella fusca females .................................................. 142 345 Transmission electr on micrographs showing the hindgut of a Thripinema fuscum parasitized Frankliniella fusca female ................................................................................ 143 346 Scanning electron micrographs documenting a Thripinema fuscum juvenile inside the post erior region of a Frankliniella fusca female. ............................................................... 144 347 Transmission electron micrographs of the Malpighian tubule from the Thripinema fuscum parasitized Frankliniella fusca ............................................................................... 145 348 Gross dissection of the reproductive structures of a nonparasitized and Thripinema fuscum parasitized Frankliniella fusca female ................................................................... 146 349 Transmission electron micrographs showing a displacement of the female Frankliniella fusca developing oocytes as a result of parasitism by Thripinema fuscum .................................................................................................................................. 147 350 The progressive magnific ation of the connection between the follicular cells and developing oocyte in the Thripinema fuscum parasitized Frankliniella fusca female. 147 3-5 Transmission electron micrographs showing fat body of the Thripinema fuscum parasitized Frankliniella fusca female. ............................................................................... 148 352 Transmission electron micrographs showing an accumulation of lipid and glycogen in the juvenile Thripinema fuscum. ..................................................................................... 148 353 Transmission electron micrographs documenting the Thripinema fuscum parasitized Frankliniella fusca musculature .......................................................................................... 149 354 Scanning electron micrographs of the fractured Thripinema fuscum parasitized Frankliniella fusca documenting the partition between the host parasite interface ........ 150 355 Transmission ele ctron micrographs documenting the exocellular substance secreted by juvenile Thripinema fuscum juveniles ........................................................................... 151 4-1 Proportion of non -viruliferous and viruliferous Frankliniella fusca individual s surviving throughout adulthood. ......................................................................................... 168 4-2 Daily damaged area (mean SE) on leaf discs (in mm2) fed on by Frankliniella fusca individuals for the ini tial 10 days of adulthood ....................................................... 169 4-3 Mean cumulative frequency (MCF) of TSWV transmission to individual leaf discs over the lifetime of adult Frankliniella fusca. .................................................................... 170
14 5-1 Tr ansmission electron micrographs of the alimentary tract of a non -parasitized and Thripinema fuscum parasitized Frankliniella fusca female. ............................................. 198 5-2 Bacteria isolated from Frankliniella fusca in dividuals ...................................................... 199 5-3 A series of assays testing for antimicrobial activity from non Thripinema fuscum parasitized and T. fuscum parasitized Frankliniella fusca individuals against Pantoea ananati s. ................................................................................................................................ 200 5-4 The standard curve used to generate units of activity for acid -derived Thripinema fuscum parasitized Frankliniella fusca homogenates. ....................................................... 201 5-5 Reverse transcriptase PCR product of Frankliniella fusca cohort extractions using TSWV and COI primers. ..................................................................................................... 202 5-6 PCR product testing the suitability of using 28S as an internal control for Frankliniella fusca RNA extractions .................................................................................. 202 5-7 An amplification plot from a cohort qPCR reaction .......................................................... 203 5-8 The m ean cumulative frequency (MCF) of Tomato spotted wilt virus (TSWV) transmissions for non -Thripinema fuscum parasitized and T. fuscum parasitized Frankliniella fusca females for each inoculation access period (IAP). ............................ 203 5-9 A box plot of the number of Tomato spotted wilt virus (TSWV) copy numbers (data log transformed) according to the Thripinema fuscum parasitism status of viruliferous Frankliniella fusca females. ............................................................................ 204 510 A scatter plot of the correlation between Tomato spotted wilt virus (TSWV) copy number and ELISA optical density (OD405) values for Frankliniella fusca females. ...... 204
15 Abstract of Dis sertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INTERACTIONS BETWEEN FRANKLINIELLA FUSCA (THYSANOPTERA: THRIPIDAE), Thripinema fuscum (TYLENCHIDA: ALLANTONEMATIDAE), AND ENTOMOPARASITES (TOMATO SPOTTED WILT VIRUS PANTOEA ANANATIS AND WOLBACHIA ) By Kelly Renee Sims August 2010 Chair: Joseph Funderburk Major: Entomology and Nematology Frankliniella fusca (Hinds) (T hysanoptera: Thri pidae) i s an important vector of bacterial and viral plant pathogens in various cropping systems. The entomogenous nematode Thripinema fuscum Tipping & Nguyen (Tylenchida: Allantonematidae), a natural enemy of F. fusca is a key regulator of F. fusca popu lations and the diseases they vector. The obligate nematode renders host female thrips sterile and r educes secondary disease spread The data presented here suggest Thripinema parasites also reduce primary disease spread by modulating the density of entomop arasite s within their thrips host. The overall objectives of this research were to (1) id entify entomoparasite s associated with F. fusca using a combination of histological, molecular, and biological techniques and (2) determine if, and to what extent, the obligate parasite T. fuscum influences the biology of F. fusca and its association with entomoparasite s ( Wolbachia, Pantoea ananatis and Tomato spotted wilt virus ). Herein, I provide a detailed histological examination documenting the internal morphology of healthy F. fusca females, the life cycle of T. fuscum and identify cha n ges to thrips target tissues and cells resulting from T. fuscum parasitism. I also report that F. fusca and T. fuscum are infected by the reproductive parasite Wolbachia, and that parasitism by T. fuscum induces a switch in supergroup designation of the dominant Wolbachia
16 strain present in host thrips. I n addition, I document that parasitism by T. fuscum reduces the feeding r ates of F. fusca females by 65% and TSWV transmission by 50% Lastly, I demonstrate T. fusc um parasitism reduces both the incidence of F. fusca individuals harboring the plan t pathogens P. ananatis and TSWV a nd the pathogen titer s in infected individuals. Results from this project suggest that the response of a host to a parasite/path ogen is dependent upon its intera ction with other microorganisms From a broader perspective, the information generated by these studies may provide a better understanding of how parasites interface with entomogenous agents in their insect vectors Under standing how these interactions influence vector competence may one day provide targets for suppressing disease spread and provide a unique complement to existing disease management strategies for controlling thrips vector populations.
17 CHAPTER 1 INTRODUC TION AND LITERATURE REVIEW: RESEARCH BACKGROUND, OBJECTIVES, AND HYPOTHESES Introduction and Literature Review The following literature review pr ovides an overview of each organism involved in this multitrophic system, and addresses the most important aspe cts of each as they pertain to my research topic I have organized the literature review into three main sections: (1) Thysanoptera, (2) E ntomoparasite s of Frankliniella fusca and (3) Objectives, Hypotheses, Goals and Expected Outcomes There are numero us cross -referenc es throughout the dissertation which help to integrate the various topics as the y refer to Frankliniella fusca Thysanoptera Natural history of thrips The evolutionary history for Thysanoptera, or thrips (pl. and singl.), dates back 230 mi llion years with the recent world species dominated by taxa that evolved within the last 40 50 million years (Grimalidi et al., 2004). Thysanoptera presumably shared a common ancestor with Hemiptera (true bugs), Psocoptera (booklice), and Phthiraptera (su cking lice) based on homology of mouthparts, antennal sense cones, absence of ocelli in larvae, and reduction in Malpighian tubules ; these four orders currently comprise the hemipteroid insect lineage ( Ananthakrishnan, 1979; Mound, 1997; Gillot, 2005). Pr imitive thrips species fed on liquid from decaying tissues and fungal hyphae that were often associated with unstable microhabitats (Mound, 1997). The ability of a ncestral thrips to exploit ephemeral and optimal conditions facilitated the development of s everal r -selected biological characteristics including polyphagy, vagility, short generation time, tendency towards parthenogenesis, and a competitive breeding structure promoting aggregation and exploitation of localized optimal conditions (Mound, 1997) Over time, these traits predisposed the Thysanoptera to become opportunistic species successful in exploiting a
18 variety of habitats in tropical, subtropical, and temperate regions and many thrips species are now adapted to an invasive lifestyle (Ananthakrishnan, 1984; Mound, 1997; Morse and Hoddle, 2006). In recent years, human transport has played a significant role in the establishment of thrips populations in non-native habitats (Mound, 1983; Kirk and Terry, 2003). To date, thrips are cosmopolitan in distribution and numerous food, fiber, and ornamental crops serve as habitats for aggregating populations (Lew is, 1997a ; Mound, 1983, 2005). Thrips were first described by DeGeer in 1744 as Physapus but were later moved to the genus Thrips by Linnaeus in 1758 (Lewis, 1997b). In 1836, Thrips was raised to the order Thysanoptera by Haliday. The etymology of the word Thysanoptera comes from the Greek words thysanos, a fringe, and pteron, a wing. There are currently over 5,500 species of thrips descr ibed, and Mound (1996) speculates this number represents only 60% of total thrips species in existence. Thrips are divided into two suborders, the Tubulifera and Terebrantia. This division is based primarily on the shape of abdomen and ovipositor, althou gh wing characteristics and number of larval stages are also used to distinguish the suborders Tubulifera have the last abdominal segment modified into a tube like structure used for depositing eggs on vegetation. This group includes over 3,100 spec ies (known from one family, Phlaeothripidae) that feed on a wide range of resources including fungal spores or hyphae, mosses, tropical trees and shrubs, flowers, mites, lepidopteran eggs, scales, and whiteflies (Mound, 1997). The remaining seven Thysanoptera n families belong to the suborder Terebrantia. This suborder is distinguished from Tubuliferan thrips by the presence of a saw like ovipositor on the ventral side of the last abdominal segment that is used for inserting eggs into host plant tissue. The v ast majority of thrips within Terebrantia feed on leaves, flowers, pollen and grasses, and many are considered crop pests. The most economically important crop pests in the Terebrantia belong to
19 the family Thripidae ( Thrips and Frankliniella spp.) and include over 2,000 of the 5,500 described thri ps species (Mound, 1997, 2002). Thysanoptera are a remarkable group of insects with several defining biological characteristics. Thrips have an unusual development, often described as intermediate between holo ( complete) and hemi (incomplete) metabolous, in which there are two active larval stages and two to three quiescent pupal stages prior to adult emergence (Takahashi, 1921). Morphologically, thrips are the only insects that possess asymmetrical hypognathous mouthparts. The right mandible is resorbed during embryonic development and the single left mandible and two developed maxillae are reduced to an asymmetrical stylet used for punching and sucking cell contents (Chisholm and Lewis, 1984; Heming, 1978, 1 993; Moritz, 1997). Other defining characters include a protrusible arolium (or bladder) on the pretarsal end that can be everted by hemolymph pressure to allow thrips to walk vertica lly on surfaces (Heming, 1971). When present, wings are linear and frin ged with long marginal setae that function as slots to increase wing surface area and allow individuals to travel aerially over long distances (Lewis, 1997). Thrips are polymorphic in wing formation and can develop into macropterous (wings present), micro pterous or brachypterous (wings reduced to wing pads), or apterous (wingless) morphs (Mound, 2005). Wingless species, although not able to fly, are capable of aerial dispersion and some species have naturally dispersed over 1,000 miles (Mound, 1983). The mechanisms determining wing form are not well understood but are thought to be the result of both genetic and environmental factors such as photoperiod, crowding and food limitation (Hood, 1940; Koppa, 1970; Kamm, 1972; Zera and Denno, 1997; Mound, 2005). The small size (~1 5 mm) and thigmotactic behavior of thrips enables species to occupy diverse vegetative microhabitats and populations can be (but are not restricted to) anthophilous
20 (flower inhabiting), phyllophilous (leaf inhabiting), poephilous (gras sinhabiting), phlaeophilous (bark-inhabiting), or cecidicolous (gall -inhabiting) (Ananthakrishnan, 1984; Moritz, 1997; Sabelis and Van Rijn, 1997). Two thrips species are sub aquatic and reside in slime within the stalks and reeds of water plants (Lewis, 1973). The ability of thrips to occupy a wide variety of niches has resulted in the development of many diverse polymorphisms and behaviors (Moritz, 1997; Mound, 2005; Morse and Hoddle, 2006). Thrips can be phytophagous (tissue and pollen feeders), fung ivorous (mycophagous or sporophagous), predacious (on mites, aphid and whiteflies), omnivorous, cannibalistic, and ectoparasitic ( Darwin, 1876; Ananthakrishnan, 1984; Izzo et al., 2002; Terry, 1997, 2001) (Figure 1 1) Thrips behavior involves a broad range of visual ecological cues, host odors, and semiochemicals (Terry, 1997). Many thrips species display sexual dimorphism, with males possessing modifications in body form, cuticular structure, color, setae and genitalia ( e.g ., Mound et al., 1998; Mound, 2005; Tyagi et al. 2008). Thrips that do not display apparent sexual dimorphism compensate by posse ssing unique behavioral traits (Crespi, 1988, 1990). In one of the most extreme cases of dimorphism within the order, some gall -forming species exhibit te rritoriality wit h sophisticated social behavior including the development of soldiers with reduced reproductive potential that suggests the evolution of eusociality (Kirk, 1985; Crespi, 1992; Gillespie et al ., 2002; Mound, 2005). Sexual reproduction is the primary reproductive strategy in the haplodiploid Thysanoptera. However, many phytophagous thrips also exhibit arrhenotokous (unfertilized eggs develop into males), thelytokous (unfertilized eggs develop into females), or deuterotokous (unfertilized eggs develop into both genders) parthenogenesis (Moritz, 1997). In some thrips species, sex determi nation may be microbe associated ( Kumm and Moritz, 2008; See Chapter 2) T he number of offspring produced by a female varies upon dietary and environmental cond itions, but
21 most females are highly fecund and oviposit between 30 to 300 eggs over their lifetime (Lewis, 1997). A typical life cycle is completed in two to three weeks and the number of generations per year ranges from one to 15, the latter has species that are often multivoltine (Lewis, 1997). Most t hrips field populations are bisexual with sex ratios being primarily female-biased (Higgins and Myers, 1992; Tsuchida and Ohguchi, 1998; Hoddle, 2002; Reitz, 2002; VasiliuOromulu, 2002; Sims et al., 2005). However, some thrips species have populations that are unisexual (all female) or are either uni sexual or bixsexual (Ananthakrishnan, 1990) Biologica l control of thrips pests Thrips are highly mobile, opportunistic insects adapted to utilize intermittent resources such as those found in agroecosystems (Lewis, 1997; Mound, 1997; Morse and Hoddle, 2006). When a h abitat is found, thrips feeding and reproduction rates increase dramatically and populations can reach pest status in a short period of time (Kirk 1997). Because pest thrips occur throughout Thysanoptera, Mound (1997) speculated that the biological characteristics predisposing a species to becoming a pest are widespread within the group. Interestingly, < 1% of the described thrips species induce enough economic damage in various cropping systems to be considered pests (Morse and Hoddle, 2006). Nonetheless t his damage is estimated to cost growers billions of dollars in control cost s and productivity losses (Lewis, 1997; Ullman et al., 1997). Dam age to crops may be the result of direct feeding on plants as well as ovipositioning in or on plant tissue, or indirectly through the introduction of bacterial, fungal, and viral pathogens (Kirk, 1997). Visible feeding damage includes silvering or bronzin g of the leaves, scarring and deformation of fruit and flowers, rind blemish, premature flower loss, reduced seed production, soiling of leaves and fruit with fecal droplets, and a reduction of pollen and fruit (Ananthakrishnan, 1984; Childers and Achor, 1 995). Oviposition by females produces blotches or halos on the fruits of plant s resulting in aesthetic injury that can significantly affect the yield
22 and value of a particular crop. Although direct damage can be severe for polyphagous species, most thri ps are considered pests because of the indirect damage they cause to agroecosystems by introducing plant pathogens (Ullman et al ., 1997). Thysanopterans exhibit exponential population growth under optimal condi tions, and many biologists concluded density dependent factors such as natural enemies wer e not important for controlling thrips because they were considered unable to keep up with rap id population growth (Davidson and Andrewartha, 1948a b ; Andrewartha and Birch, 1954; Mound and Teulon, 1995; Butt an d Brownbridge, 1997; Loomans et al., 1997; Parella and Lewis, 1997; Parker and Skinner, 1997). However, extensive review by thrips ecologists confirmed that densitydependent parameters, particularly natural enemies, are important regulators of pest thrips populations (e.g ., Nicholson, 1958; Smith, 1961; Varley et al ., 1973; Stoltz and Stern, 1978; Nagai, 1990; Mound, 1997; Sabelis and Van Rij n,1997; Funderburk et al., 2000; Funderburk, 2002; Ramachandran et al., 2001; Hansen et al., 2003; Reitz et al., 20 03, 2006; Baez et al., 2004). Arthropod predators of Thysanoptera include Neuroptera, Diptera, Hymenoptera, Coleoptera, Dictyoptera, Orthoptera, He miptera Thysanoptera, and Acari (Sabelis and Van Rijn, 1997). The most common predators of Frankliniella s pp. are species of Orius (Hemipter a: Anthocoridae) and Amblyseius (Acari: Phytoseiidae) (Loomans et al., 1995; Riudavets, 1995). Controlling thrips populations with these natural enemies is only partially successful because day length and body size limits their performance and both are vu lnerable to insecticides (Butt and Brownbridge, 1997; Sabelis and Van Rijn, 1997). Entomogenous fungi within the Hyphomycetes ( Verticillium Metarhizium Paecilomyces and Hirsutella ) and Zygomycetes (Entomophthorales ) are important yet often overlooked thrips pathogens (Parker et al., 1996;
23 Butt and Brownbridge, 1997). Unlike arthropod biological control agents, fungi are not limited by day length, are active over a range environmental conditions and climates, do not need to be ingested to be pathogenic, and are easily mass produced (Parker et al., 1996; Butt and Brownbridg e, 1997). The biology of fungal entomopathogens and their potential to regulate thrips populations is reviewed in further detail below (see Entomo phthorales ). Some researchers have evaluated the potential of using entomopathogenic nematodes, particularly Heterorhabditidae and Steinernematidae (Rhabditida), as biological control agents because of their high level of virulence against soil -inhabiting stages of thrips (e.g., Chyzik et al., 1996; Ebssa et al., 2001; Buitenhuis and Shipp, 2005). However, high concentrations of infective juveniles are required to obtain high mortality, and although the nematodes are easily mass produced, the numerous inun dative spray applications required for thrips control are not economically feasible (Chyzik et al., 1996; Ebssa et al., 2001). Other parasitic nematodes, the entomogenous Tylenchida (= Allantonematidae ), are naturally occurring parasites of thrips. Unlike the entomopathogenic nematodes that kill their host in a short period of time, these parasites have negligible effects on thrips longevity or mortality (Sims et al., 2005). Thus, these parasites are self -sustaining and little effort is needed to maintain field populations Th e biology of allantonematid nematodes is reviewed in further detail below (see Thripinema fuscum ). Frankliniella fusca (Hinds ) (Thysanoptera: Thripidae) The first specimen of Frankliniella fusca Hinds was collected in Massachusetts a nd described as Euthrips fuscus by Hinds in 1902. In 1912, Karny placed E. fusca in Frankliniella. Watson (1922) was the first to i dentify thrips species in Florida peanut and included F. fusca in his research bulletin Currently F. fusca is distribute d throughout Canada, Mexico, and the eastern United States (Sakimura, 1963; Stannard, 1968; Chiasson, 1986; Johansen, 2002). Frankliniella fusca can be differentiated from other species in the genus by having short or
24 wanting postocul ar bristles the thir d antennal segment being yellowish and the fourth and fifth light grayish brown, the body generally dark brown to lighter brown, long, stout spines on the legs and a lack of comb on tergite VII (Watson, 1923). There are several adult taxonomic keys avai lable for distinguishing F. fusca from other Frankliniella species ( e.g ., Childers and Beshear, 1992; Oetting et al., 1993; Mound and Kibby, 1998; Moritz et al., 2001, 2004) ; however few larval keys exist because young instars possess few defining characte r states. Larval identification alternatives include rearing immature thrips to adulthood and molecular analyses (Brunner et al., 2002; Paini et al ., 2007). Numerous studies have been conducted on the biology of F. fusca (Watts, 1934; Newsom et al., 1953; Sakimura, 1963; Stannard, 1968; Lowry et al., 1992; Puche and Funderburk, 1992; Sims, 2003; Sims et al., 2005; Sims et al., 2009). Unlike other flower thrips species, F. fusca are relatively sessile and easy to manipulate for laboratory experiments. The postembryonic development of F. fusca includes a bean -shaped egg embedded within plant tissue, two larval instars, two non -feeding inactive pupal stages termed the pre -pupa and pupa, and an adult stage that develops either macropterous or brachypterous wi ng pads (Figure 1-1). Frankliniella fusca is unique in that it is only one of a few species within Thripidae that exhibit both macropterous (wings present ) and brachypterous (wings reduced to pads tha t range in size ) wing morphs (Mound, 1996). Stannard ( 1968) noted minor variations in structure and color between the two wing morphs of male and female F. fusca ; macropterous individuals are slightly larger and darker than their brachypterous counterparts. Frankliniella fusca are also sexually dimorphic and the larger, brown females can be easily differentiated from the smaller, yellow male adults (Figure 1-1).
25 The survival, development, longevity and reproduction of F. fusca on peanut at various temperatures (Lowry et al., 1992), as well as the intrinsic ra te of increase at different population densities, have been well documented (Lowry et al., 1992; Puche and Funderburk, 1992; Sims, 2003; Sims et al., 2005). The developmental time of F. fusca is typical for poikilothermic organisms; a developmental cycle (egg to egg) can be completed in 12 to 24 days under optimal (35C) and suboptimal temperatures (20C), respectively (Lowry et al., 1992, Sims, 2003; Sims et al., 2005). Egg development requires nearly half of the total developmental time with larval emer gence occurring seven days after ovipositioning between 2030C (Lowry et al., 1992). Overcrowded conditions reduce the survi vorship of larval thrips as a result of intraspecific competition for resources ( Paini et al., 2008). Adult females have a pre -vi tellogenic period of one day, after which they lay an average of two eggs per day (Heming, 1970; Lowry et al., 1992; Sims, 2003; Sims et al., 2005). The longest average female longevity reported for F. fusca is 13 days, which can be attributed to the addition of pollen to the female s diet (Sims et al ., 2005). Male and female F. fusca develop at the same rate, however females live on average four days longer (Sims, 2003). Frankliniella fusca generally maintains a 3:1 (female: male) sex ratio, however bec ause they exhibit arrhenotokous parthenogenesis, populations are able to survive in the absence of haploid males (Sims et al., 2005). Because of this attribute, genders are easily manipulated in laboratory populations with unmated and mated females always producing ma les and females, respectively. Frankliniella fusca is the most common thrips species found inhabiting and reproducing in groundnut and can comprise over 90% of the total thrips population in a legume field (Funderburk et al., 1998; Herbert et al., 2007). In Florida, populations of F. fusca are able to reproduce year round on groundnut and can produce as many as 15 generations per year
26 (Newsom et al., 1953; Lowry et al., 1992; Lewis, 1997). Larvae and adults inhabit the terminal buds and flowers, respectively, and the non-feeding pre -pupae and pupae tend to drop to the soil just below the plant. Larvae and adults feed in flowers and on the upper surface of plant leaves, causing a silvery scarring on the leaf surface and an upward curling of in jure d leaves (Newsom et al., 1953) Direct feeding injury to young groundnut by F. fusca does not result in significant yield loss unless other stressors such as post -emergence herbicide injury are present (Smith and Sams, 1977; Tappan and Gorbet, 1979; L ynch et al., 1984; Funderburk et al., 1998). Although F. fusca feeding damage can cause asthetic injury to plants, most crop losses are attributed t o their vectoring capabilities Frankliniella fusca adults colonize seedlings immediately after planting when the young plants are most susceptible to viral infection (Todd et al., 1996). Populations peak about 10 to 20 days after the planting date, and peak injury levels occur approximately 23 to 35 days after planting when peanuts start flowering (Tappan an d Gorbet, 1979; Todd et al., 1996 ). Thrips populations begin to decrease in May and eventually reach levels near extinction in mid -summer (Tappan and Gorbet, 1979; Todd et al., 1996 ; Funderburk et al ., 2002). Previous speculation for the population declines included physiological factors (e.g., diapauses) and natural enemies; the recent discovery of the sterilizing insect parasitic nematode Thripinema fuscum Tipping & Nguyen, is now known to be one of the so urces for the observed population decline ( Funderburk et al., 200 2). Entomoparasites A ssociated with Frankliniella fusca As with all insects, Thysanopterans have associations with a suite of prokaryotic, eukaryotic, and viral microorganisms. These symbionts can be temporary or permanent residents that establish a commensalistic, mutualistic, or parasitic relationship with their thrips host. This section covers the symbiose s of thrips entomoparasites and the interactions they have with their host. In this dissertation, the term entomoparasites refer s to the viral (TSWV), bacterial
27 (Pantoea ananatis and Wolbachia), fungal ( Entomophthorales spp.), and nematode ( Thripinema fuscum ) agents infecting F. fusca Tomato spotted wilt virus (Bunyaviridae: Tospovirus) The Bunyaviridae comprises the largest famil y (>350 spp.) of arthropod transmitted viruses and include s many important human pathogens such as Rift Valley fever ( Phlebovirus ), Crimean Congo hemorrhagic fever ( Nairovirus ), California encephalitis virus ( Hantavirus ), and La Crosse virus (Orthobunyavirus ) (Elliott, 199 7). Tospovirus is the sole plant infecting virus in the Bunyaviridae; the other genera ( Bunyavirus Phlebovirus Hantavirus Nariovirus, and Tenuivirus ) are vectored by hematophagous arthropods (mainly mosquitoes, ticks, and sand flies) o r rodents and infect humans and animals (Sherwood et al., 2001). There is a high degree of vector specificity for Tospoviruses ; all species are transmitted in a semi -persistent propogative manner exclusively by thrips in the Thripidae (Sakimura, 1963; Ull man et al., 1992; Nagata et al., 2002). Currently, the Tospovirus genus is composed of 19 species (Pappu et al., 2009). Species classification within the Tospovirus are primarily determined by the nucleocapsid (N) gene sequence although other factors suc h as host range, serological differences, and genome structure and organization have been used (Elliot, 1990; de Avila et al., 1990, 1993). Of the estimated 5,500 thrips species, 10 are known vectors of Tospoviruses (Pittman, 1927; Sakimura, 1963; Mound, 1996; Ullman et al., 1997; Whitfield et al., 2005). Most thrip s Tospovirus vectors are restricted to Thrips and Frankliniella genera within Thripinae and this vectoring ability i s thought to have evolve d independently in the two lineages (Mound, 1996). Tomato spotted wilt virus (TSWV) the type member of Tospovirus is economically the most important species within the genus. This pathogen is considered to be among the 10 most detrimental plant viruses worldwide and annual crop loss from TSWV has been estimated to exceed $1 billion (Goldbach and Peters, 1994; Prins and Goldbach, 1998). Tomato spotted wilt virus was first discovered in
28 1915 infecting tomatoes in Australia, found to be transmitted by thrips in 1927, and to be of viral origin in 1930 (Br ittlebank, 1919; Pittman, 1927; Samuel et al., 1930). Currently, there are seven substantiated thrips vectors of TSWV F. fusca (Sakimura, 1963), F. intonsa (Trybom) (Wijkamp et al ., 1995), F. occidentalis (Gardner et al., 1935), the dark form of F. schu ltzei (Trybom) (Sakimura, 1969), Thrips palmi Karny (Fujisawa et al., 1988), T. setosus Moulton (Fujisawa et al., 1988), T. tabaci Lindeman (Pittman, 1927), and F. bispinosa (Morgan) (Avila, 2006). Other reported thrips TSWV associations have been invalid ated by Mound (1996), who cited a misidentification of thrips spe cies by the authors. There are undoubtedly additional vectors of TSWV within the Thripidae a s well as new vectors that will evolve as th e Tospovirus -vector association change s over time T hrips tabaci for example, was at one time a primary vector but has since lost the ability to vector new isolates (Sakimura 1962, 1963; Mau et al., 1990; Wijkamp et al. 1995). These new isolates are formed when a host plant is coinoculated with different isolates that exchange genetic information through a reassortment of genome segments ( Qui et al., 1999). Tospoviruses are classified as single stranded, negative -sense tripartite RNA viruses (Class V of the Baltimore Classification of viruses). The spheri cal to pleomorphic virions measure 70 80 to 90120 nm in diameter and the outer shell of the virion consists of a host derived double layered membrane or envelope (5 nm thick) anchored with two viral glycoproteins (5 10 nm long) (Milne, 1970; Whitfield et al. 2005). The envelope encloses three non-covalently closed, circular ribonucleoproteins that are composed of the ssRNA segment, the nucleoprotein and multiple copies of the viral RNA -dependent RNA polymerase ( Fauquet et al., 2005; Whitfield et al ., 2005). The three linear single stranded RNA segments, S (2.9 kb), M (4.8 kb), and L (8.9 kb), code for four structural proteins and two nonstructural proteins from five open reading
29 frames. Collaborations between de Haan et al. (1990, 1991) and Kormelink e t al (1992) resulted in the fully sequenced genome of TSWV and provided a greater understanding of the viral organization, replication and function as herein described The ambis ense S segment codes for a 52.4 kDa nonstructural protein (NSs) in the vira l sense and a 29 kDa nucleocapsid protein (N) in the viral complementary sense (De Haan et al., 1990). The NSs functions as a silencing suppressor, and because this protein is not present in the mature virion, it is often used as a marker for viral replic ation in serological and molecular studies (de Haan et al., 1990; Kormelink et al., 1991; Kikkert et al., 1997; Takeda et al. 2002). The N protein is highly conserved among all TSWV isolates and is the primary gene used to for identification in molecular studies. T he ambisen se M segment codes for a 33.6 k Da nonstructural protein (NSm) in the viral sense and a 127.4 kDa precursor of the two glycoproteins (GP1 and GP2) in the viral complementary sense (Kormelink et al., 1992). The NSm protein is involved in cell to -cell movement through plasmodesmata and acts as an avirulence determinant (Kormelink et al., 1994; Storms et al., 1995, 1998; Margaria et al., 2007). The glycoproteins coordinate the TSWV entry process into thrips tissues and are required for transmissibility by thrips ( Bandla et al., 1998; Medeiros et al., 2000; Sin et al., 2005; Ullman et al., 2005). The negative -sense L segment codes for a 331.5 kDa RNA-dependent RNA polymerase (RdRp) (de Haan et al., 1991; Adkins et al., 1995). The TSWV p athway in the thrips vector is still unresolved, but the viral mode of infecti on has been shown to be system ic and time -dependent (Moritz et al., 2004; Whitfield et al., 2005). The acquisition access period (AAP) and the inoculation access period (IAP) ar e two important determinants for viral transmission by the thrips vector ( Wijkamp et al., 1995, 1996; van de Wetering et al ., 1996). Acquisition of TSWV occurs when first and early second instars ingest virus particles from infected plant tissue (van de W etering et al., 1996). The minimum AAP
30 reported for successful transmission is five minutes, although transmission rates are highest after an AAP of approximately 24 hours (Wijkamp et al., 1996). Young first instars are most susceptible to infection and the acquisition efficiency decreases with increased larval age (van de Wetering et al., 1996). When young larvae feed on infected plant tissue, the ingested virions migrate from esophagus to the midgut where glycoprotein one (GP1 ) located on the viral env elope bind to columna r epithelial cells through a 50 kDa cellular receptor located on the plasmalemma ( Bandla et al ., 1998; Medeiros et al., 2000). The virus fuses to the cell, possibly through pH -dependent receptor -mediated endocytosis, and releases its replicative contents into the cell ( Bandla et al., 1998; Whitfield et al., 2005). Viral mRNA is transcribed using the host machinery and the virion associated RdRps, assembled in the Golgi complex, and disseminated between cells via the exocytic pathway ( Kikkert et al., 1999; Whitfield et al., 2005). The virus spreads throughout the midgut and foregut, moving from the midgut epithelia to the surrounding visceral muscle cells (Kritzman et al., 2002). At this point, the route of infection from the midgut c ells to the salivary glands is speculative. The general consensus among thrips biologists is that virus movement is ontogenic -dependent, occurring in first instars when displacement of the salivary glands by the cibarial muscles causes a temporary fusion of the midgut to the visceral muscle cells surrounding the salivary glands ( Moritz et al., 2004). However, other viruses ( e.g ., Rhabdoviruses) are neurotropic in their insect hosts and the possibility that TSWV utilizes neurons and trachea to reach these glands cannot be ruled out (Ammar and Hogenhout, 2008; Ammar et al., 2009). This dissemination may occur at the midgut/muscle border because the gut epithelia are closely associated with the insect tracheal system (Ammar et al., 2009). It was reported th at the salivary organs formed a channel -like structure to the midgut and the virus utilized these ligaments as a means for accessing the salivary tissues, however in -depth
31 ultrastructural examination showed there is actually no mergence between the tubular salivary glands and the midgut (Ullman et al., 1989; Del Bene et al., 1999). Movement of TSWV through the hemolymph has also been ruled out through viral injection studies (Nagata and Peters, 2001). Receptors are widely distributed among different cell types and the bi nding of glycoprotein 2 (GP2) to 94 kDa thrips protein is likely involved in circulation of the virus within thrips tissues (Kikkert et al., 1998; Ammar et al., 2008). Studies conducted by Sin et al. (2005) have shown that both viral membr ane GP1 and GP2 are required for thrips transmissibility. The ovoid salivary glands are the primary site of replication for the virus and they must be heavily infected for successful transmission (Wijkamp et al., 1993; Nagata et al., 1999; Nagata and Pete rs, 2001). Transmission occurs when mature virus particles are released with saliva into a healthy plant cell during feeding by second stage larvae and adults ( Wijkamp et al., 1993). Thrips TSWV transmission rates are high even after short inoculation per iods which can have significant implications for dise ase spread in field conditions. Healthy adults do not become viruliferous after feeding on infected plant tissue even after long periods of exposur e (Sakimura 1963). Moritz (1997) suggested the inaccessibility of late larval and adult stages to acquire and transmit the virus is due to the extending and thickening of the peritrophic membrane with development, causing it to be less permeable to viruses. Sherwood et al. (2001) and van de Wetering et al. (1999) suggest differences in acquisition capabilities between larvae and adults reflect differences in feeding behavior. However, Ullman et al. (1992) found that both larval and adult thrips sufficiently ingest TSWV, and instead concluded that adult thri ps are unable to acquire the virus due to the presence of a midgut barrier. They observed that when larvae feed on infected plant tissue, virions enter the midgut epithelial ce lls by fusion at the apical mem brane of the midgut epithelial brush border (Ull man et al., 1995). However, when adults feed the
32 virions are degraded or altered in the midgut and/or epithelial cells so dissemination to other tissues cannot occur. Additional support for the barrier hypothesis comes from work conducted by Nagata et al (1997) who were able to develop tissue cell cultures supporting TSWV multiplication from the non -vectoring T. tabaci To conclude, the virus must penetrate through 7 internal barriers for successful acquisition and transmission to occur: (1) peritrophic envelope/laminae, (2) apical membrane of brush border in midgut lumen, (3) basement membrane of midgut columnar epithelial cells, (4) midgut basal lamina, (5) through the surrounding muscle cells, (6) basal lamina of salivary glands, and (7) plasma membra ne of salivary gland secretory cells ( Ullman et al., 1992; Ammar et al. 2009). Disrupting viral entry, movement, development, replication, or escape from any of these barriers can affect the vectoring capability of an individual (Nagata et al., 1999; Anan thakrishnan and Annadurai, 2007). The vector competence of thrips is dependent upon other intrinsic factors including the rate of viral multiplication in the insect, feeding behaviora l differences, gender genetic elements of both the virus and vector, ins ect immunity, and ontogeny ( Hardy et al., 1983; Nagata et al ., 2002; Arthurs and Heinz, 2003; Medeiros et al., 2004; Moritz et al., 2004; Cabrera La Rosa and Kennedy, 2007; Rotenberg et al., 2009; Sims et al., 2009). Tomato spotted wilt virus titer in hos t thrips has been shown to be a quantitative determinant of vector competence, and the existence of a dose -dependent infection threshold has been documented in other insect RNA virus associations such as mosquitoes and arboviruses (Hardy, 1983; Rotenberg e t al., 2009). Differences in f eeding behavior, particularly those between genders, often dictate the amount of virus secreted to infected plant cells. Sakuri et al. (1998) found that males have a higher transmission rate than females and suggested that because females feed intensively, they destroy
33 cells to the extent that virus replication is not supported in the plant cell. Males, on the other hand, puncture the cells and leave them suitable for virus replication and consequent infection of the plant ( van de Wetering et al., 1999). Differences in transmission efficiency between genders was further supported by work conducted by Rotenberg et al. (2009), who found males transmitted TSWV at a greater rate than females but harbored less TSWV N RNA. Host g enetic elements may also explain vector compe tence and has been well documented in other systems such as mosquito vectors and the pathogens they transmit (Gray and Banerjee 1999; Beerntsen et al ., 2000; Sim et al., 20 09). CabreraLa Rosa and Kennedy (2007) suggest vector competence to be under genetic control and inherited as a recessive trait, however sexually transmitted factors other than genes may be influencing thrips vectoring efficiency ( e.g ., Wolbachia). Medeiros et al. (2004) found that TSWV infec tion activates the insect innate immune system and the elicited defense responses are effective in r esisting infection. T he developmental stage in which the virus is in g ested is another critical step in determining viral competence. Extrinsic factors, su ch as density and environmental conditions, also affect whether an insect vector will acquire and transmit viruses (Hardy et al., 1993). For example, stable environmental conditions typically produce brachypterous thrips; these individuals probably do not transmit TSWV as efficiently as macropterous thrips because of their limited dispersal and contact capabilities (Wells et al ., 2002 a). Tospovirus epidemics can occur only when the thrips vector, tospovirus, and host plant coincide in a suitable environment (a.k.a. disease triangle) (Ullman, 1996). Tomato spotted wilt virus is currently known to infect over 900 monocotyledonous and dicotyledonous plants and both virus and host plants are distributed worldwide ( Sherwood et al., 2001; Culbreath et al., 2003; Campbell et al., 2009; Pappu et al., 2009). The large, overlapping host ranges of the virus
34 and vector increases the probability that a vector will contact an infected plant and makes this pathosystem extremely difficult to control (Mound, 1996; Pappu et al., 2009 ). Tomato spotted wilt virus was first reported on Florida groundnut in June of 1986, although it may have occurred there as early as 1974 (Kucharek et al., 1990). Frankliniella fusca is the key vect or of TSWV in Florida groundnut and causes extensive economic damage (Funderburk et al. 2002) Tomato spotted wilt virus cause systemic infection in their plant hosts and symptoms include circular light green and yellow ring spots on the foliage, yellowing of the leaves, tan spots or blotches, st reaking or mottling of the quadrifolates, deterioration of the root system, shrinking of the leaves, and an overall reduction in size of the plant (Figure 1-2) (Culbreath et al., 1993; Padgett et al., 1995). Tomato spotted wilt virus epidemics spread through groundnut fields in two main ways. Primary spread occurs when viruliferous thrips disperse to newly planted crops from surrounding vegetation and transmit the virus to healthy plants. The control of primary spread of TSWV by viruliferous thrips is considered to be a key component in disease management; most losses in groundnut fields result from an inability to control primary spread ( Camann et al., 1995; Puche et al., 1995; Gitaitis et al., 1998; Culbreath et al., 2003). Secondary spread occurs when larvae developing on infected plants within a crop acquire the virus and transmit it to other uninfected plants in the field after they have matured to adulthood. The control of TSWV secondary spread relies heavily on the application of broad-spectrum ins ecticides. However, insecticides alone do not prevent TSWV primary spread because adult thrips feed and transmit before insecticidal activity can kill the vector (Todd et al., 1996; Momol et al., 2004). A pplication of insecticides for thrips control also has been shown to have negative impacts on beneficial insects and can lead to thrips resistance (Newsom et al., 1953; Bielza et al., 2007, 2008). No single control measure has been effective in significantly reducing thrips transmitted
35 disease spread and many growers have implemented integrated control management systems that combine chemical, biological, and cultural practices (Sherwood et al., 2001; Culbreath et al., 2003). Cultivar selection, planting date, field location, plant population, row pattern, tillage, weed control, UV reflective mulch, and insecticides are all strategies used to minimize losses of crop plants ( Stavisky et al., 2002a; Culbreath et al., 2003; Reitz et al., 2003; Momol et al., 2004; Riley and Pappu, 2004). Additional control m easures, especially those utilizing natural enemies, are needed for reducing pest thrips in agroec osystems. Annual cropping systems fluctuate in their availability and suitability to insect pests (Kennedy and Storer, 2000). In winter months, F. fusca popu lations are highly brachypterous and commonly overwinter around harvested groundnut fields where volunteer groundnut and annual weed species take over ( Newsom et al., 1953; Chamberlin et al ., 1993). The ability of F. fusca to feed on a wide variety of hos t plants is a critical factor that promotes thrips dispersal and allows for bi -directional movement between cultivated (crop) and non-cultivated (weeds) host plants over time and space. Many alternate host plants serve as reservoirs for both TSWV and the F. fusca vector, allowing for virus acquisition and transmission between generations of thrips and crops ( Chamberlin et al ., 1992, 1993). Many publications have discussed the distribution and abundance of F. fusca in these cultivated and uncultivated host plants ( Chamberlin et al., 1992; Chellemi et al., 1994; Buntin and Beshear, 1995; Toapanta et al., 1996; Groves et al., 2001; Groves et al., 2002; Kahn et al., 2005; Paini et al., 2007; Northfield et al., 2008). Transmission of TSWV is limited in brachyp terous adults because of their inability to disperse long distances. Therefore, unless thrips diapause or emigrate from other regions, these winter plants and thrips are the primary means for TSWV to move across seasonal crop harvests. Dispersal from a h ost plant is dependent upon food, mates, oviposition sites, natural enemies,
36 and/or density. A major factor influencing dispersal from overwintering host plants is weather (Lewis, 1964; Puche and Funderburk, 1995). High temperatures promote dispersal and movement within a groundnut field whereas cool temperatures and/or rainfall induces stagnate populations (Harding, 1961). Increased photoperiod and temperatures associated with spring conditions encourages the development of winged individuals, and the highest dispersal rates frequently correspond with the highest level of macropterous individuals (Newsom et al., 1953; Hood, 1940; Koppa, 1970; Kamm, 1972; Chamberlin et al., 1992; Mound, 2005). This dispersal usually coincides with early stages of crop gr owth w hen the plants are most prone to damage (Northfield et al., 2008; Pappu et al., 2009). The pattern of dispersal into a groundnut field appears to be associated with the prevailing wind rather than the location of the overwintering crop (Garcia and B randenburg, 1995). However, one can discern the movement for viruliferous thrips by analyzing the spatial distribution of infected plants in the field (Puche et al., 1995). Vectors arriving from distant sources produce a random distribution of diseased p lants throughout the field whereas vectors from within the field tend to produce a clumped or aggregated pattern of diseased plants. Vectors coming from weed hosts bordering the field tend to generate a gradient of infected plants from the edge of the fie ld (Puche et al., 1995). Pantoea ananatis (Serrano 1928) Mergaert et al. 1993 Many phytophagous insects have facultative or obligate mutualistic associations with bacteria. For thrips, this relationship appears to be specific to the plant pathogenic Pantoea spp. (McKenzie et al., 1993; de Vries et al., 2001a,b, 2004, 2008; Wells et al., 2002b ; Gitaitis et al., 2003; C hanb usarakum and Ullman, 2008, 2009). Pantoea ananatis has been isolated from the guts of other insects such as plant hoppers, pyramids, an d flea hoppers (Takahashi et al., 1995; Watanabe et al., 1996; Bell et al. 2007). P antoea ananatis first described as Erwinia ananas (syn. E. ananatis, E. herbicola, E. uredovora), was discovered causing fruitlet brown rot of
37 pineapple in the Philippines Serrano (1928) placed this pathogen into the herbicola group of the Erwinia genus, a group proposed by Winslow et al (1920) for all plant associated, gram negative, non-spore forming, peritrichous, fermentative, rod-shaped bacteria. The species was moved to the genus Pantoea in 1989, and renamed P. ananatis and P. agglomerans (Beijerinck 1888) in 1997 (Gavini et al., 1989; Mergaert et al., 1993; Truper and DeClari, 1997). Ullman et al. (1989) first detected gram -negative bacteria in the gut of F. oc cidentalis and the bacteria were later isolated and characterized by de Vries et al (1995, 2001a). These microbe s reside in the hindgut and M alpighian tubules of all thrips life stages (Ullman et al., Dallai et al ., 1991, 1989; de Vries et al. 2001a). Tra nsmission of the gut bacteria occurs horizonta lly on the food source from feces or saliva or vertically through contamination of the egg shell (de Vries et al., 2001b). Currently, associations with Pantoea spp. gut bacteria are known for F. occidental is F. fusca and T. tabaci (Ullman et al., 1989; de Vries et al., 2001a b ; Wells et al., 2002; de Vries et al., 2008; Chanbusarakum and Ullman, 2008, 2009). Interestingly, F. occidentalis and F. fusca are rarely infected by any other gut bacteria (de Vri es et al., 2001a b ; Wells et al., 2002; Chanbusarakum and Ullman, 2009). These bacterial complexes are stable over time and space, suggesting the bacterial association is both symbiotic and widespread among Thrips and Frankliniella species (de Vries et al ., 2008; Chanbusarakum and Ullman, 2008, 2009). In 2002, isolates of P. ananatis recovered from laboratory and field collected F. fusca were shown to be pathogenic to onion (Wells et al., 2002). One year later, Gitaitis et al. (2003) reported that F. fusc a is an important vector of the plant pathogenic P. ananatis Similar to TSWV, P. ananatis is an emerging plant pathogen that infects both monocotyledonous and dicotyledonous plants and causes disease symptoms in economically important agricultural crops
38 (Moyer 1999; Rybicki and Pietersen, 1999; Couti nho and Venter, 2009). These bacteria have a quorum sensing system, a cell to cell communication by bacteria t hat determines local cell density and the biosynthesis of exopolysaccharides as a quorum -sensing signal molecule induces the onion rot disease (Morohoshi et al., 2007). This pathogen is broadly adapted to habitats and can function as an epiphyte, endophyte, pathogen, symbiont, and/or saprophyte (Coutinho and Venter, 2009). Pantoea ananatis functions as both a saprophyte inhabiting the gut microfauna of F. fusca and as a pathogen causing leaf blight, necrotic lesions, stalk rot, and bulb decay in onion (Wells et al., 2002; Walcott et al., 2002; Gitaitis et al., 2003 ). The severity of P. ananatis is highly dependent on environmental conditions ( e.g ., temperature, humidity, and precipitation) and causes 100% crop loss under fa vorable conditions (Gitaitis and Gay, 1997; Walcott et al., 2002). Once established in a field ( e.g ., by seed inoculums), P. an anatis likely spreads between crops from the movement of thrips vectors. For example, both Vidalia onion and groundnut are hosts for P. ananatis and F. fusca (Watson, 1922; Sether and DeAngelis, 1992; Gitaitis and Gay, 1997; Wells et al., 2002; Mullis et al., 2004). Current control measures for P. ananatis include developing resistant cultivars, using mulch and irrigation, and avoidance and eradication of initial inoculums (Gitaitis et al., 2004; Coutinho and Venter, 2009). Wolbachia Hertig (Rickettsiales : Rickettsiaceae) The intracellular bacterium Wolbachia w as first discovered in 1924 infecting the reproductive structures of Culex pipiens L. mosquitoes and are now one of the most abundant and widespread bacterial symbionts kn own (Hertig and Wolbach, 1924; Werren, 1997). In addition to insects, Wolbachia has been found in crustaceans, mites, spide rs, scorpions, filarial nematodes, and a plant nematode (McLaren et al., 1975; Breeuwer and Jacobs, 1996; Cordaux et al., 2001; Rowley et al., 2004; Baldo et al., 2007; Haegeman et al., 2009). Estimates suggest Wolbachia infect 20 to 66% of all arthropods species; however this estimate is higher when
39 testing for Wolbachia using modified PCR detection methods (Jeyaprakash and Hoy, 2000; Hilgenboecker et al., 2008). A variety of protocols have been developed for Wolbachia detection in arthropods; the most reliable and accurate protocols include histological examination, phenotypic identification, and multi locus sequence typing (MLST) of five Wolbachia specific ge nes (Hertig and Wolbach, 1924; Baldo et al., 2006) (see Chapter 2). Wolbachia belong to the alpha subdivision of the Proteobacteria, and are currently divided into nine clades or supergroups based on nucleotide sequence data (Wei sburg et al., 1989; Lo et al., 2007; Ross et al., 2009). All Wolbachia strains within the supergroups represent the single species W. pipientis ( Lo et al., 2007). These bacteria are found in cytoplasm of the host reproductive cells and are predominantly transmitted vertically from the mother to offspring (Wade and Stevens, 1985). By targeting their host reproductive system, Wolbachia employ a variety of strategies to enhance their transmission such as cytoplasmic incompatibility, feminization, parthenogenesis, and male killing ( Werren, 1997). These effects bias population sex ratios and may even cause speciation (Werren, 1997). In addition to manipulating host reproduction, Wolbachia has been shown to induce the upregulation of host immune genes (Brattig et al., 2004; Kambris et al., 2009). This upregulation, in turn, inhibits the establishment of other microorganisms such as nematodes, bacteria, and viral infection (Lowenberger et al., 1996; Kambris et al., 2009; Teixeria et al., 2008) (see Chapter 5). Although vertical tr ansmission from mother to offspring is the primary route for Wolbachia infection, there is ample evidence in the literature of horizontal transmission between taxa. This exchange may occur through intimate associations between hosts and their parasitoids (see Werren et al., 1995; Heath et al., 1999; Vavre et al., 1999), prey (see Johanowicz and Hoy 1996), host plants (see Sintupachee et al., 2006), or parasites (see Noda et al., 2001). Lateral
40 gene transfer is also widespread, and Wolbachia inserts have b een found in Drosophila, wasps, nematodes, and other bacterial symbionts (Werren et al. 1995; Dunning Hotopp et al., 2007; Darby et al., 2010) Ne arly the entire Wolbachia genome has been transferred to its Drosophila host (Dunning Hotopp et al., 2007). G ene transfer through the bacteriophage WO has also been demonstrated (Bordenstein and Wernegreen, 2004). Additionally, there is significant inter and intra specific variation in Wolbachia infection; i ndividuals within a population s can range from not be ing infected to being infected with multiple isolates (Boyle et al., 1993; Hilgenboecker et al., 2008; Kawasaki et al. 2 009). Individuals infected with multiple Wolbachia strains are thought to have risen by homologous recombination (Kondo et al., 2002; R euter and Keller, 2003; DunningHotopp et al., 2007). The ability of Wolbachia to undergo extensive gene recombination between strains suggests it may be used as a biological control against insect vector pest populations by serving as a vehicle for introducing anti viral genes into insect tissues that could interfere with pathogen replication or transmission ( Beard et al., 1993; Werren and Bartos, 2001). In most arthropods, these reproductive parasites are co nsidered facultative pathogens as cured individuals are physiologically unaltered (Werren, 1997; Duron et al., 2008). However, mutualistic associations with arthropods do exist. For example, Wolbachia are thought to function as nutritional mutualists by providing a fecundity benefit to dietary -stress ed Drosophila melanogaster Meigen (Brownlie et al., 2009). In fact, some of these mutualistic associations are obligatory. Antibiotic clearing of Wolbachia in Asobara tabida Nees (Hymenoptera: Braconidae) inhibits oogenesis and it is thought Wolbachia in teracts directly with the Sex lethal (Sxl) gene required for oogenesis ( Dedelne et al ., 2001; Starr and Cline, 2002). Recently, antibiotic clearing of Wolbachia in the collembolan Folsomia candida was shown to induce
41 sterility in all individuals despite t he production of eggs (Pike and Kingcomobe, 2009). Filarial nematodes also have an obligatory mutualistic association with Wolbachia, with antibiotic treatment resulting in negative e ffects to the worms reproduction and development (Hoerauf et al., 1 999; Taylor and Hoerauf, 1999). Complete genome sequencing of Brugia malayi Brug helped to delineate the mutualistic association between Wolbachia and its nematode host; Wolbachia provides nematodes with essential metabolites such as riboflavin, flavin adenin e dinucleotide, and heme, while the nematode supplies amino acids required for Wolbachia growth (Foster et al., 2005). Wolbachia are not obligatory symbionts for all filarial nematodes, as demonstrated by the inability to find the bacteria in some species such as Loa loa (Buttner et al., 2003). To date, Wolbachia has only been detected in one non-filarial nematode the banana root nematode Radopholus similis (Cobb) (Tylenchida: Pratylenchidae) and has yet to be detected in Tylenchid nematodes (Bordenstei n et al., 2003; Duron and Gavotte, 2007; Foster et al 2005; Haegeman et al ., 2009). Wolbachia has been reported in both arrhenotokous [ e.g., Echinothrips americanus Morgan, Gynaikothrips ficorum (Marchal), Suocerathrips linguis Mound and Marullo] and thel ytokous [ e.g., Franklinothrips vespiformis (Crawford DL), Hercinothrips femoralis (Reuter), Heliothrips hemorrhoidalis (Bouche), Parthenothrips dracaenae (Heeger)] thrips species and is hypothesized to be the causal agent for the observed parthenogenesis (Pintureau et al., 1999; Arakaki et al., 2001; Kumm and Moritz, 2008). High temperature or antibiotic treatment removed Wolbachia from thelytokous females and induced the production of sexually functional male s (Arakaki et al., 2001; Kumm and Moritz, 2008) Unfortunately, little is known regarding the effect of Wolbachia on the reproductive behavior of arrhenotokous populati ons of thrips (see Chapter 2).
42 Entomophthorales spp. Fungi isolated from, or proven pathogenic to, thr ips are in the Hyphomycetes or Z ygomycetes classes (Butt and Brownbridge, 1997). Despite the abundance of information on fungal pathogens of thrips (particularly F. occidentalis ) ( for a review see Butt and Brownbridge, 1997), there are few reports of fungi associated with F. fusca in fi eld conditions However, I have recovered a variety of parasitic and saprophytic fungal pathogens from F. fusca populations in north central Florida groundnut ( Fig ure 1-3). T he most common mycopathogen associated with F. fusca is Entomophthorales spp. (Z ygomycota: Entomophthoraceae) Entomophthorales spp. ar e common obligate pathogens of insects and many species have strict host specificity often restricted to a single species ( Alexopoulos et al., 1996). As the name implies (entomo=insect, phthor= destroyer), these pathogens are highly virulent and induce host mortality a few days after infection. These fungi can produce rapid mass epizootics in insect populations under conditions of high humidity (Bellini et al., 1992). For these reasons, fungal pathogens in this order are the focus of many biological control studies (Leathers et al., 1993). The life cycle of Entomophthorales involves two forms of conidia in the F. fusca host (Figure 1 4; Figure 1 5) Large primary conidia are forcibly ejected fr om the insect cadaver, producing a halo of protop lasm around the body. Smaller secondary conidia may develop from the primary conidia if a suitable substrate is unavailable for the primary conidia Th ese secondary conidia termed capilliconidia are elon gated cell s produced on a long, thin conidiophore that function as an attachment organ After the conidia attach to a suitable host, they germinate and a germ tube penetrates the insect cuticle. Once inside the insect, the myceli a fragment into hyphal bo dies that rapidly multiply in the insect (Figure 1 6). Within just a few days the hyphal bodie s proliferate throughout the host eventually breaking the cuticle and killing the insect (Alexopolous et al ., 1996) (Figure 1-7 ).
43 Thripinema fuscum (Tylenchida: Allantonematidae) Nematodes have an evolutionary history of ~750 million years, and the development of the protrusible stylet in ancestral forms laid the foundation for the evolution of the parasitic nematodes (Blaxter et al. 1998; Wang et al., 1 999; Sid diqi, 2001; Davis et al., 2004 ). Insect parasitism is thought to have arisen independently in four major groups of nematodes and it is speculated that the Tylenchida a rose from ancestors that had alternations of free living and parasitic generations on lower animals and plants (Poinar, 1975; Dorris et al., 1999; Blaxter et al., 1998; Stock ). The earli est fossil evidence of insect parasitism by a Tylenchid nematode was of a Howardula species (Tylenchida: Allantonematidae) parasitic in the body cavity of a phorid fly, recovered from Baltic amber dated at approximately 40 million years (Poinar, 2003). The first record of an insect -parasitic nematode association was in 1602, and it was not for three more centuries that these microorganisms were considered for controlling insect pests (Aldrovandus, 1602; Glaser, 1931). Currently, t here are 27 families of nematodes in eight orders that have associations with invertebrates that include the Tylenchida, Rhabditida, Spirurida, Strongylida, Oxyurida, Ascaridida, and Mermithida (Nguyen, 2010). All insect parasitic Tylenchida are placed in the superfamily Sphaerularioidea and the family Allantonematidae represents the basic type o f Sph aerularioidea (Remillet and Laumond, 1991). Allantonematid nematodes are specialize d, obligate parasites that attack their host within the most microhabitats of plant structures (Poinar 1975; Loomans et al., 1997). They are termed entomogenous nematodes because of their negligible effects on host longevity and mortality ( Harry Kaya, personal communication). The earliest record of a described entomogenous nematode occurring in thrips spp. was in 1895 when Uzel found a specimen infecting Thrips physopus L in Germany (Sharga, 1932). In 1910, Jones found a nematode in California infecti ng Heliothrips fasciatus Pergande (Russell,
44 1912). Another nematode was discovered in Russia infecting Stenothrips graminum Uzel in 1926 (Kolobova, 1926). Unfortunately, none of these species were named due to lack of details, descriptions, or figures (Sharga, 1932; Loomans et al., 1997). The first detailed study of the nematode Tylenchus aptini (Sharga) infecting Aptinothrips rufus (Gmelin) was reported by Sharga (1932). Lysaght (1936) later proposed the taxon should be revised to Anguil lina aptini T he species was afterwards transferred by Wachek (1955) to the genus Howardula. Nickle and Wood (1964) found a parasitic nematode infecting two species of blueberry thrips, F. vaccinii Morgan and Taeniothrips vaccinophilus Hood. Although their specimens w ere smaller than those discovered by Sharga, they determined the parasite to be Howardula aptini (Sharga). In 1972, Wilson and Cooley (1972) discovered H. aptini infecting F. occidentalis in Texas. Howardula aptini was again found infecting a new Megalurothrips sp. in South India in 1982 (Reddy et al., 1982), but this species differed slightly from that of Sharga (1932) and Nickle and Wood (1964). Siddiqi (2000) then changed the genus to Thripinema and separated the nematodes as Thripinema renirao, T. ap tini, and T. nicklewoodii respectively. A new species, T. khrustalevi was found parasitizing Thrips trehernei Prisner and T. physopus L. in Moscow (Chizhov et al., 1995). In 1997, a new nematode in the genus Thripinema was found parasitizing females of Thrips obscuratus (Crawford) in New Zealand (Teulon et al ., 1997). Also, Tipping et al (1998) discovered a nematode infecting an adult female F. fusca (Hinds) and named the new species T. fuscum Tipping and Nguyen. In 2000, Funderburk et al. (2002b) di scovered a subspecies of T. khrustolevi parasitizing F. australis (Morgan) in Chile. To date, there are five described Thripinema species that parasitize thi rteen species of thrips (Lim and Van Driesche, 2005). There are undoubtedly other undescribed species belonging to this little -studied genus.
45 Laboratory and field studies have addressed various biological aspects of the F. fusca and T. fuscum interaction (Funderburk et al., 2002; Sims, 2003; Sims et al., 2005; Funderburk and Sims, 2005; Sims et al., 2009). Thripinema fuscum parasitizes all stages of F. fusca with adult females being the most preferred host stage and males the least preferred (Sims et al., 2005). The intrinsic capacity of increase for T. fuscum when parasitizing adult females and ma les is 0.37 and 0.34, respectively (Sims et al., 2005). More than one Thripinema female is capable of parasitizing a single thrips host and the in vivo life cycle is approximately nine days (Sims et al., 2005). Parasitism does not affect survival or long evity of female F. fusca (Sims et al., 2005). Parasitism by Thripinema is initiated when a free -living infectious female or motherworm, penetrates a thrips host with her stylet through the coxal cavities or the soft, intersegmental membranes of the thor ax or abdomen (Tipping et al., 1998; Lim et al., 2001) After ingress, the motherworm undergoes a dramatic morphological transformation in the host hemocoel, in which she converts from a slender vermiform shape to an obese form comprised of a single swol len ovary (Tipping et al., 1998) (Figure 1-8 A-B). Significantly, development of the parasitic female is synchronized with the host, as demonstrated by differences in developmental time when parasitizing different stages of thrips; parasitic females enter ing early or late instars do not differentiate into the reproductive (ovarian) phenotype until the thrips begin to develop their adult r eproductive organs (Remillet and Laumond, 1991; Siddiqi, 2000; Sims et al., 2005). The stylet, esophagus, and alim entary system of the motherworm atrophies and the protective cuticle is shed and replaced with a thick, microvillar layer (Subbotin et al., 1993, 1994; Subbotin and Chizhov, 1996) Limited ultrastructural studies on females of Skarbilovinema laumondi (Tylenchid a: Iotonchiidae), a parasitic Tylenchid similar to Thripinema, revealed a well developed hypodermis containing an external or spongy layer of numerous interwoven
46 microvilli that are presumed to aid in the uptake nutrients required for egg production ( Sub botin et al., 1993). Studies on the integument structure of other insect parasitic Tylenchid females also reveal the presence of microvilli on the hypodermal surface, although the size, shape, and arrangement of the microvilli vary (Subbotin et al., 1994; Subbotin and Chizhov, 1996). In one instance, Howardula phyllotretae have ampullae formed from the invagination of the hypodermal membrane that presumably, like microvilli, increase surface area for absorption of nutrients ( Subbotin and Chizhov, 1996). Some Tylenchids also have an e xtracellular substance secreted by the hypodermis of parasitic females and juvenile nematodes that may aid in degrading host immune factors before these reach the absorptive microvilli (Subbotin et al., 1993, 1994). P arasitic females produce eggs within four to five days after ingress and typically have two to three eggs in their ovary at one time. These females are highly fecund; as many as 420 eggs have been reported in a single thrips host (Reddy et al., 1982; Loomans et a l., 1997). Eggs are produced continuously until death of either the host or the parasite, although the number of eggs produced decreases substantially during the later stages of parasitism (Mason and Heinz, 2002; Sims, 2003; Sims et al., 2005). The eggs hatch in the host hemocoel, and t he progeny nematodes feed on hemolymph in the thrips abdominal cavity and mature through three juvenile stages (Figure 1-8 CG) ( Tipping et al., 1998). Fully parasitized thrips contain all life stages of Thripinema and mor e than one Thripinema female is capable of parasitizing a host (Lim et al., 2001; Sims et al., 2005). The developed juveniles are believed to migrate from the hemocoel into the alimentary tract by using their stylet to breach tissue barriers, and, presumab ly, they exit via the anus or ovipositor as adults (Sharga, 1932; Lysaght, 1936; Reddy et al., 1982). Sharga (1932) reported seeing T. aptini juveniles in the abdominal hemocoel use their stylets to bore from the midgut or oviduct to the pyriform rectum w here they apparently
47 remained for a period of time befo re they exited through the anus with the insects frass. Reddy et al. (1982) observed H. aptini exiting through Megalurothrips sp. ovipositor. The partially free living females are small (0.25 t o 0.29 mm long) with a hypertrophied stylet, however m ales have a non -functi onal and nearly indistinguishable stylet. It is unknown how males migrate to the alimentary tra ct without a functional stylet, and one may speculate that m ales latch onto migrating fem ales or use a communal wormhole to exit Lacking a functional stylet, males are unable to parasitize a thrips host; supposedly, their only function in the free -living stage is to mate with females (Lysaght 1936; Nickle and Wood, 1964). Although never observed, it has been suggested that mating occurs during the free living stage (Lysaght, 1936; Nickle and Wood, 1964; Reddy et al., 1982). However, the risks of desiccation and failure to locate a female suggest that mating may occur inside the host. Aggregation s of male and female nematodes formed in the hindgut suggest that males may inseminate females just prior to their emergence This hypothesis is supported by observations that T. fuscum males emerge from the host later than females (Sims et al., 20 05) Mating within the host insect has been reported for Parasitylenchus an Allantonematid Tylenchid parasite of Drosophila recens (Perlman and Jaenike, 2001). Typically, Thripinema has a 19:1 (female: male) sex ratio, suggesting a tendency towards part henogenesis in the absence of males (Sims et al., 2005). The infective free living females, unlike the egg -producing females, have a cuticle similar to that of plant -parasitic nematodes. Under optimal conditions, free living female and male Thripinema su rvive an average of 86 and 61 hours, respectively (Mason and Heinz, 2002). Survival of male and female nematodes after emergence is short due to unstable moistur e and heat conditions (Mason and Heinz, 2002; Loomans et al ., 1997).
48 Thripinema spp. ha ve dram atic impacts on thrips populations in field conditions by reducing feeding and fecundity rates of females and subsequently decreasing TSWV spread The potential of this nematode to act as a biological control agent has been evaluated, and all reports suggest the potential of using Thripinema spp. as inoculative agents against pest thrips in both greenhouse and field conditions (Ar thurs and Heinz, 2002, 2003, 2005; Funderburk et al., 2002; Mason and Heinz, 2002; Lim and Van Driesche, 2004, 2005; Sims, 2003; Sims et al., 2005; 2009). In nature, the short -lived, free living male s and females are most commonly found in flower perianths where thrips aggregate to mate and feed on pollen, and which are hypothesized to be the primary site for transmission (Crespi 1993; Tipping et al., 1998; Sims et al., 2005). The timing underlying the spread of Thripinema in local thrips populations is not well understood. In groundnut, both the free -living nematode and the flowers are short lived mandating that the parasitiz ed thrips remain in close proximity with healthy conspecifics Goals, Objectives Hypotheses and Expected Outcomes The F. fusca / T. fuscum system is a unique and ideal model for examining the interface between a host insect and its obligate parasite Firs t, T fuscum has a direct life cycle that does not require an intermediate host; therefore, all physiological development occurs inside the thrips. Second, u nlike many other vertebrate nematodes that develop within host tissues, all Thrip i nema life stages exist in the hemocoel and can be readily collected. Third, Thripinema parasites depend upon their host for survival and transmission and therefore have negligible effects on thrips longevity or mortality (Sims et al ., 2005). Fourth, a generation of Thri pinema parasi tes can be completed in as few as nine days (Sims et al ., 2005). Finally F. fusca harbors numerous endo parasites and the multitrophic relationship between them and their host provides different approaches for determining how Thripinema may influence host vector competence. Importantly, the proof of concept established for the F. fusca/T. fuscum model may be extended
49 to other thrips pests such as viruliferous F. occidentalis parasitized by the entomogenous nematode T. nicklewoodi The overa ll goal of this research project was to determine if and to what extent, the obligate parasite T. fuscum influences the biology of F. fusca and its association with the entomopa rasite s Wolbachia, P. ananatis, and TSWV The specific objectives to reach th is goal are listed below In Chapter 2, I discuss utilizing a multigene approach using 16S rRNA, COI and MLST gene fragments to identify if one or more Wolbachia strains are associated with F. fusca and T. fuscum populations, and determine the influence of Wolbachia on F. fusca reproduction by comparing the development and reproductive biology (phenotype) of infected vs. antibioticallycured host individuals. In Chapter 3, I use histological techniques to document the (1) the internal morphology of non -par asitized F. fusca adult females ; (2) the life cycle of T fuscum and provide d a detailed in vivo account of this novel insect parasitic nematode including development, reproduction, and migration of the infectious female nematode and her progeny; and (3) c ompar ed and contrast ed the impact of T. fuscum on non -parasit iz ed and parasitized F. fusca females target tissues and cells In C hapter 4 I discuss the effects of gender, age, wing form, viral infection, and nematode parasitism on the feeding behavior an d TSWV transmission of F. fusc a. In Chapter 5, I report the the impact of T. fuscum on F. fusca to harbor the plant pathogens P. ananatis and TSWV using quantitative biological, molecular, and histological approaches. Chapter 6 is is a summary of the res earch findings and future di re ctions The central hypothesis is that parasitism by T. fuscum alters the biology and reduces the entomopathogen content of its F. fusca host The expected outcome of this project is to h ave a better understanding of how inse ct -para sitic nematodes interact with thrip s in vivo and s ubsequently, how parasites may suppress host vector competence. Deciphering t he mechanisms
50 underlying the in situ interactions between the parasite, the host thrips, and associated entomoparasites is not the goal of this dissertation (but see Chapter 6 for p otential mechanis ms). Results from this project may assist in identifying mechanisms that parasites/pathogens use to modulate the physiology of their respective insect vectors. For example elucidati ng the mechanisms responsible for shutting off egg production in parasitized thrips may provide novel avenues for regulating the intrinsic rate of increase of this pest insect. Likewise, understanding the mechanism(s) leading to reduced Tospovirus competency (acquisition and transmission) in parasitized thrips also may provide targets t o suppress the spread of disease From a broader perspective, the information generated by the se studies will enhance current understanding of host parasite dynamics and potentially provide a basis to develop this unique biological control agent to better regulat e this important insect vector.
51 Figure 1 1. The life cycle of Frankliniella fusca at 27C The number of days spe nt at each stage is in parentheses. (A) Egg embedded in plant tissue; (B C) Larval stages; (DG) Macropterous pupa and adults; (H K) Brachypterous pupa and adults.
52 Figure 1 -2 Illustrations of A rachis hypogaea and Tomato spotted wilt virus (A) Healthy A. hypogaea plant; (B) A. hy pogaea with TSWV symptoms; (CD) A. hypog aea leaflet showing TSWV symptoms. Scale: (C) 10 mm; (D) 1 mm.
53 Figure 1 -3. A collage of various fungi recovered from Frankliniella fusca laboratory and field populations in north central Florida.
54 Figure 1 -4. D ifferential interference contrast (DIC) microscope images of Entomopht h orales sp. stages infecting Frankliniella fusca (A B) Z ygospores; (C D) C onidi a ; (EF) Conidiophores Figure 1 5. Scanning electron mi croscope images of Entomophthorales sp. stages infecting Frankliniella fusca (A) F. fusca with a halo of primary conidia and a secondary saprophytic fungal infection; (B) P rimary conidia ; (CD) G erminating conidia ; (E F) C onidiophores
55 Figure 1 -6 Entomophthorales sp. hyphal bodies in F rankliniella fusca (A) A longitudinal thick section of a Frankliniella fusca female infected with an Entomophthorales sp.. Note the displacement of the thrips organs as a result of the abundance of hyphal bodies ; (B) Entomophthorales hyphal bodies with measurements (26.7 m 8.1 m) ; (C) T ransmission electron micrograph of the hyphal bodies inside F. fusca T he hyphal bodies are binucleate with a poorly defined wall and contain numerous lipid droplets.
56 Figure 1 -7 The progression of an Entomophthorales sp. infection in the female Frankliniella fusca (A) Healthy F. fusca female; (B) F. fusca female exhibiting symptoms of an Entomophthorales sp. infection (note the swollen abdomen); (C) Entomophthorales sp. spores penetrating through the cuticle of the F. fusca female ; (D) S ide profile of F. fusca female with Entomophthorales sp. breaking the insect cuticle. 1 mm 1 mm 1 mm 1 mm
57 Figure 1 -8. The life cycle of Thripinema fuscum in a female Frankliniella fusca host (A ) Adult F fusca female; (B) P rogressive enlargement of the parasitic female (right to left); (C) E ggs produced by the parasitic female ; (D F) F irst through third stage juveniles; (G) I nfectious free living females; (H) F ree living male. Ingress of (G) regenerates the cycle.
58 CHAPTER 2 MOLECULAR IDENTIFICATION OF THE REPRODUC TIVE PARASITE WOLBACHIA IN HOST -PARASITE POPULATIONS OF FRANKLINIELLA FUSCA AND THRIPINEMA FUSCUM Introduction The biology of Wolbachia is reviewed elsewhere (see Chapter 1). Currently the Wolbac hia genus contain s one species, W. pipientis with strain subdivisions representing eleven supergroups distributed throughout the Arthropoda and Nematoda phyla : arthropods (A B), filarial nematodes ( C-D), springtails (E), nematodes, arthropods, hexapods (F ), spiders (G), termites (H), mites (I), (J) and (K) (Lo et al., 2002; Baldo et al., 2006; Bordenstein et al., 2005; Ros et al ., 2009). Historically, many of these strains were placed in supergroups based on sequencing of the Wolbachia specific 16S rRNA ( wspec ) and surface protein ( w sp ) or cell division protein (ftsZ ) genes (Zhou et al., 1998). However, this technique has been scrutinized for not adequately typing and quantifying strain dive rsities within a host because of extensive intragenic recombinati on and strong diversifying selection at th e wsp gene ( Werren and Bartos, 2001; Jiggins et al., 2001; Jiggens, 2002; Reuter and Keller, 2003; Baldo et al., 2002, 2005 2006; Paraskevopoulos et al., 2006; Baldo and Werren, 2007). The recently developed multi locus sequence typing system (MLST) a process in which ~400500 internal fragments of five conserved housekeeping genes (gatB, coxA, hcpA, ftsZ, and fbpA ) are combined into a sequence type (ST) and the alleles at each locus are used as molecular markers t o genotype a strain, is a more rigorous technique for annotating Wolbachia strains (Maiden et al ., 1998; Urwin and Maiden, 2003; Paraskevopoulos et al., 2006; Baldo et al., 2006). Although not part of the MLST scheme, the Wolbachia surface protein ( wsp ) c an be used as an additional marker to type strains by examining shuffling of a relatively conserved set of amino acid motifs within four hypervariable regions (HVR1 HVR4) of the gene (Jolley et al., 2004;
59 Baldo et al. 2006). The complete MLST strain typin g system includes a web accessible central database that allows for extensive comparative analyses between Wolbachia strains (Jolley et al., 2004; Baldo et al., 2006). This molecular multigene approach in combination with biological phenotyping (see Chap ter 1) and histological examination (see Chapter 3), currently offers the most reliable method for identifying and classifying Wolbachia strain(s) as well as deciphering the impact of Wolbachia on host biology The previously observed female -biased sex rat io s of F. fusca and T. fuscum suggest Wolbachia may be dictating the reproductive biology of host and parasite populations (Sims et al., 2005). The objective s of this chapter were to (1) use a multigene approach to identify if one or m ore Wolbachia strain s are associated with F. fusca and T. fuscum populations and (2) determine the influence of Wolbachia on F. fusca reproduction by comparing t he development and reproductive biology (phenotype) of infected vs. antibiotically -cured host individuals Findings are supported by electron microscopic examination target ing the reproductive tissues of non -parasitized and parasitized F. fusca and T. fuscum Materials and Methods Sample collection and DNA extraction : To test for Wolbachia infection(s) in F. fusca an d T. fuscum populations, m ale and female thrips were collected from the flowers of groundnut (Arachis hypogaea and A. glabrata) between March 2007 and November 2009 at two locations: (1) The Plant and Science Research Education Unit of University of Florid as Institute of Food Agricultural Sciences facility at Citra in Marion County, FL (2924 N 8210 W), and (2) Fifield Hall of University of Florida's main campus at Gainesville in Alachua County FL (2938 N 8221 W). A synopsis of the collection data is listed in Table 2 -1. The excised flowers were placed into labeled Ziploc bags and transported on ice to the laboratory for processing. In the laboratory, flowers were destructively sampled and female F. fusca collected
60 with an aspirator attached to a vacuum pump. Aspirated thrips were placed in 1.5 ml Eppendorf microcentrifuge tubes with a 1 -cm peanut disc and maintained at 27C and a 14 hour light period for 24 h. To determine parasitism status of the thrips, individuals were transferred to a new tube, the old tube was rinsed with 200 l of water; and the rinsate was observed for free living nematodes at 50X magnification. Thrips were stored as non -parasitized or parasitized cohorts (n=20 unless otherwise stated ) in sterile 1.5 m l Eppendorf microc entrifuge tubes at 80C Free living female and male T. fuscum were collected from recovered parasitized females pooled, centrifuged, and sto red in cohorts of ~500 in 100 l of water at 80C. A synopsis of the F. fusca and T. fuscum collection data in cluding stage, gender, location, and date is listed below (Table 21) The number of cohorts used in each molecular analysis is listed accordingly in the results. DNA was extracted from the F. fusca and T. fuscum cohorts using the Epicentre MasterPure Ye ast DNA Purification Kit (Epicentre Biotechnologies, Madison, WI ) following the manufacturers protocol. Primer information polymerase chain reaction (PCR) and sequencing : A description of the genes and primer features are summarized in Table 2-2. All primer sequences and PCR conditions were kindly provided by K ostas Bourtzis ( University of Ioannina, Greece ). The primers for DNA amplification of the Wolbachia specific 16S rRNA gene (V6 -V8) and wsp (HRV1HRV4) were specifically designed to amplify varia ble and conserved regions within the genes, thus facilitating iden ti fication of Wolbachia infection(s) (Baldo et al., 2006; Petrosino et al., 2009). The five genes for MLST were selected based on the following conditions: (1) presence throughout the seque nced Rickettsiales, (2) a single copy in the Wolbachia genome, (3) a wide distribution in the Wolbachia genome, and (4) evidence of strong stabilizing selection within the Wolbachia genus (Jolley et al., 2004; Baldo et al., 2006). PCR amplification of all
61 genes was performed by using 1l of DNA, 2 l of 10X reaction buffer, 1.8 l of 25 mM MgCl2, 0.1 l of dNTPs, 0.5 l of forward primer (25 pmoles/l), 0.5 l of reverse primer, 0.1 l of Taq polymerase, and 14 l of sterile distilled water The thermocyc ler (GeneAmp PCR System 2700, Applied Biosystems Fost er City, CA) was programmed for an initial denaturing at 94C for 5 m, followed by 34 cycles of 30 s at 94C, 20 s at 55C, and 90 s at 72, one cycle of e xtension at 72C for 10 m, and a final hold at 4 C. PCR products were run on a 1.2% agarose gel and stained with ethidium bromide to confirm amplification of the expected product. PCR products were purified (QI Aquick PCR Purification Kit, QUIAGEN, Valencia, CA ) and sent to the University of Floridas Interdisciplinary Center for Biotechnological Research (UF -ICBR) for sequencing. Products were sequenced using a BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) and automated DNA sequencing (Applied Biosystems Model 3130 Genet ic analyzer). 16S rRNA : Electropherograms obtained from the sequenced 16S rRNA PCR products were visuall y inspected, and the sequences were assembled into contigs and edited using Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI) A nucleotide query was c onducted on the edited sequences using the BLASTn database ( http://www.ncbi.nlm.nih.gov/BLAST/ ) to confirm product identity to the Wolbachia 16S ribosomal RNA gene. The edited 16S rRNA sequences were comb ined with an additional 27 partial and complete 16S rRNA sequences that were selectively chosen and represent a subset of the Wolbachia supergroups A-K (obtained from Kostas Bourtzis) (Appendix A1 ). The partial 16S sequence for E scherichia coli served as the outgroup. All 16S sequences were aligned using ClustalX 2.0 (Thompson et al., 1997), trimmed and further edited in MacClade 4.0 as needed (Maddison and Maddison, 2000). P hylogenetic analyses were executed on the aligned sequences using the program P AUP 4.0b 4a ( Swofford,
62 2003). Phylogenetic trees were constructed using with distance analysis and neighbor joining, along with additional default settings in PAUP The robustness of the tree was tested using bootstrap and jackknife branch support values. A maximum of 1000 trees were searched for each data set followed by a 50% majority rule, strict consensus of the best fit trees. wsp typing : Electropherograms obtained from the s equenced wsp PCR products were edited as described above a nd the primer seq uences were marked I ndividual nucleotide sequences were trimmed using a reference template from the wsp gene of wMel, a supergroup A Wolbachia strain from Drosophila melanogaster located at the Wolbachia wsp typing protocol website ( http://pubmlst.org/wolbachia/wsp/ ) (Jolley et al., 2004) A single nucleotide sequence query was conducted on each of the edited nucleotide sequences using the default settings. The resu l ting query indicated either a direct or partial match of the wsp locus to others in the database. If an exact match was not found, a basic local alignment search tool (BLAST) was performed to identify the nearest allele and provided t he number of mismatches gaps, and alignment to the closes t allele In addition, the generated wsp amino acid (AA) sequence was divided into four consecutive hypervariable regions flanked by highly conserved regions: HVR1 (AA 52 to 84), HVR2 (AA 85 to 134), HVR3 (AA 135185), and HVR4 (AA 186 to 222) (Baldo et al., 2005) These regions were queried via BLAST to HVRs already in the database which provided a complete or partial match with similarities to the closest peptide(s) Alignments of the wsp AA profiles were generated using ClustalW2, a general purpose m ultiple sequence alignment program provided by the European Bioinformatics Institute ( http://www.ebi.ac.uk/ ), to identify similarities and/or differences in each hypervariable region. MLST : Non -parasitized F. fusca female populations that consistently provided a strong a mplification signal from a n initial screen of select genes were selected for the MLST (s ee
63 Figure 2-1). Electropherograms obtained from the sequenced gatB, coxA, hcpA, ftsZ, and fbpA PCR products were edited as described above S equences were trimmed by aligning each individual query sequence to a gene -specific polymorphic nucleotide map provided by the Wolbachia MLST database ( http://pubmlst.org/wolbachia/ ) (Jolley et al. 2004) A multiple lo cus query was conducted on the trimmed sequences of all five genes for each cohort population. Samples with identical nucleotide sequences at a given locus we re assigned the same allele number. For th e samples with unique nucleotide sequences ( i.e ., those not having an exact match to alleles in the database), the number of mismatches, gaps, and alignment to the closest allele was provided. The five MLST allele numbers for each cohort population were then combined and assigned an allele profile or sequenc e type (ST ). The MLST profiles obtained for each cohort population were then visually compared to the 16S rRNA and wsp profiles to determine potential factors ( e.g ., time, space, or parasitism) that may be driving Wolbachia nucleotide divergence in popula tions of F. fusca and T. fuscum Effects of Wolbachia on F. fusca reproduction : A series of l aboratory bioassays were conducted to determine the effect of antibiotic treatment on the sex ratio of F. fusca Thrips progeny from gene screen populations #13 and 14 (see Table 2 1) maintained in laboratory colonies were used for all the phenotype bioassays. For all treat ed groups, the antibiotic was offered by restricting F. fusca to the lid of a 1.5 -ml microcentrifuge tube with parafilm and inverting a microc entrifuge tube containing an antibiotic solution (antibiotic + 10% honey in double distilled water) over the lid (herein ref erred to as the therapy chamber ). A water solution (10% honey in double distilled water ) was administered to the control groups. F or each assay, an equal number of tubes without either solution w e re prepared to ensure F. fusca ingested the drinking water in the treatment therapy chambers as demonstrated by starvation of the confined
64 thrips in the empty tubes Unless otherwise stated, all laboratory bioassays were conducted at 25C and a 14 h light photoperiod. The first bioassay tested the efficacy of four antibiotics against Wolbachia in F. fusca Stock solutions of tetracycline, streptinomycin, and ampicillin (50 mg/ml; Sigma) and kanamycin ( 25mg/ml; Sigma) were stored at 20C prior to use. The stock antibiotics were diluted with a 20% honey solution to give a final concentration of 25 mg/ml for tetracycline, streptinomycin, and ampicillin, and 12.5 mg/ml for kanamycin. The antib iotic solutions were ad ministered orally to female thrips for five consecutive days. T he therapy chambers were refreshed daily with the appropriate solution (i.e ., antibiotic or water) After five days, females were removed from the enclosed lids of the therapy chamber and stored at -80C. The experiment was replicated three times. DNA was extracted as described above and PCR w as performed on DNA samples with Wolbachia specific 16S rRNA primers ( wspec ). The activity of each antibiotic against Wolbachia was determined visually by running the PCR product on a 1.2% agarose gel and staining with ethidium bromide. The second bioassay tested the effect of Wolbachia on gender determination of larval F. fusca The selected antibiotic was based on results from the prior experiment which provided the highest level of activity against Wolbachia. First instar F. fusca randomly collected from laboratory colonies (maintained as described in Chapter 2) were placed in cohorts (n=6) in the therapy chamber. After 48 h, larvae were removed from the lid of the therapy chamber and placed in a 1.5 -ml microcentrifuge tube with a peanut leaflet The thrips was placed in a new tube provisioned with a fresh peanut leaflet (1cm2) daily until adult eclosion. The gender of each thrips was recorded. The experiment was replicated three times.
65 The third bioassay tested if the arrhenotokous parthenogenesis displayed by F. fusca is the result of Wolbachia infection (s) Non -choice mating experiments involving virgin and mated female F. fusca with and without antibiotic were designed To collect virgin F. fusca late stage pupae were randomly collected from laboratory colonies and placed individually into a 1.5 -ml microcentrifuge tube provisioned with a 1 -cm2 peanut leaf disc. Eclos ed adults were collected and placed into a therapy chamber (with or without antibiotic) for 48 h. Adults were removed from the therapy chambers and the females were placed in a new tube containing only a leaf disc (virgin) or a leaf disc plus a nontreated or treated male (mated) for 24 h. The following day, females were individually placed into a new 1.5 -ml microcentrifuge tube provisioned with a leaf disc (1-cm2) and the males were discarded. The leaf discs were refreshed every 48 h for six days and th e old leafs were st ored for one week at 23C and 14 h light period. After a one week incubation period, l eaf discs were examined for emerging larvae for five days When present, the larvae were transferred to a new 1.5 ml tube and provisioned with a fresh leaf disc daily until adult emergence. The number of progeny and gender derived from each female was recorded. Histology : See Chapter 3 for the transmissi on electron microscopy protocol used for the histological examination of Wolbachia in individual no n-parasitized and T. fuscum parasitized F. fusca R esults 16S rRNA : A total of 44 DNA preparations were screened for infection(s) using Wolbachia specific 16S rRNA primers. Of these, 36 preparations representing samples from healthy F. fusca parasitized F. fusca and free -living T. fuscum populations were subjected to a gene screen. A moderate to strong amplification signal was detected in 30 of the 36 samples using the Wolbachia specific 16S rRNA primer s (Figure 2-1A) S equence data was obtained for 2 8 samples ( Ap pendix A2 ). The 28 e dited 16S rRNA sequences were subjected to a
66 phyl ogenetic analysis to determine relationships between the F. fusca and T. fuscum populations (Figure 2 2) The concatenated phylogenetic tree separated the samples into thr ee distinct subgroups (A1, B1, and B2). Subgroup A1 contained six free living T. fuscum five nonparasitized female, and five parasitized female F. fusca cohort populations (n=16) and clustered into Supergroup A The other two subgroups, B1 and B2, had high homology to Supergroup B. Subgroup B1 (n=10) was comprised of five non-parasitized female (n=5), two non -parasitized male (n=2), and three parasitized F. f usca female cohort populations. Subgroup B2 contained only 2 populations, one non -parasitized and one parasitized F. fusca female cohort. Free -living T. fuscum were not associated with subgroup B1 or B2. All healthy male popula tions clustered into subgroup A and parasitized male populations clustered into subgroup B. A total of 11 of the 15 coh ort populations associated with T. fuscum (either free living or parasitized) were in subgroup A. S even of the 13 healthy popula tions were clustered into subgroup B1. wsp typing : A total of 36 of the 44 samples were selected for a gene screen using wsp p rimers. The wsp pr imers amplified 26 of the samples as demonstrated by a moderate to strong band by PCR detection (Figure 2 -1B). S equence data w ere obtained for 25 samples ( Appendix A3). The edited wsp sequences of select nonparasitized F. fusca female cohorts (n=6) included all four hypervariable regions of the gene. Alignment of these sequences confirmed similarity of the gene acr oss five samples (#2, 6, 7, 8, 20) with distinct regions of nucleotide divergence in the HVR1 (Figure 2 -3 ). The wsp s equen ces from samples # 2 and # 6 were identical across the four conserved and hypervariable regions. Sequences # 7, # 8, and # 20 were identical across HVR2 4 and CR 34 but with distinct motifs in the HVR1 and CR2 regions Sequence # 11 had a high level of diverge nce from the other sequences in all hypervariable and conserved regions (motif in dark gray). Comparing similarities and differences in the wsp sequence data between the six
67 cohort populations mirror ed differences f ound for the 16S rRNA phylog eny with res pect to supergroup designation. The 16S rRNA phylogenetic tree group ed #2, 6, 7, 8 and 20 into subgroup B1 and #11 into subgroup B1. The wsp sequences for #2, 6, 7, 8, and 20 were highly homolo gous, whereas the wsp sequence for #11 ha d a high level of di vergenc e from the other sequences MLST : A gene screen was conducted on 36 of the 44 samples (Figure 2 1C G) There were 17 nonparasitized female F. fusca 10 parasitized F. fusca and nine free living T. fuscum in the analysis. Amplification from all s amples was most successful with t he coxA and ftsZ primers with a visible band detected by conventional PCR for 31 and 30 of the samples, respectively (Figure 2 -1C, F; Table 2 4) The gatB and ftbA primers successfully amplified Wolbachia in 19 and 12, res pectively, of the samples (Figure 2 1E, G; Table 2 4) Wolbachia was detected in only 8 of the samples using the hcpA primers (Figure 2 1D) Overall, the MLST primers successfully amplified product from 61, 62, and 38% of the nonparasitized F. fusca fem ales, parasitized F. fusca females, and free -living T. fuscum cohorts respectively ( Table 2 -4 ). Wolbachia was not detected by conventional PCR in two samples (non -parasitized F. fusca females (#39) and T. fuscum (#40)). Six non-parasitized F. fusca cohor t populations consistently provided a strong amplification signal in the initial gene screen as demonstrated by the presence of a band after detection by conventional PCR Partial and complete MLST sequ ence data was obtained for four and six of the popula tions respectively ( product was not detected in sample #11 using the hcpA primers and in sample #20 using the fpbA primers ) (Table 2-5 ; Appendix A4 ). Comparisons across samples indicated minor nucleotide divergences across loci, with sample #11 having the highest level of divergence. There was 100% homology to gatB in five of the six
68 samples and to coxA in three of the six samples All other alleles were unique. Two ST were identified f or the samples with complete MLST sequence data. Samples #7, 2, an d 6 share one ST and sample 8 differs in ST only by allele 15 for fbpA The variance at alleles between the four ST is at the same base nucleotide Effects of Wolbachia on F. fusca reproduction : A range of antibiotics were chosen that were expected to va ry in their activity against Wolbachia. Of the four a ntibiotics tested, ampicillin, streptomycin, and tetracycline (50 mg/ml) showed good activity against Wolbachia. Kanamycin (25 mg/ml) did not have any activity (Figure 2 -4). Visually, tetracycline ha d the greatest impact against Wolbachia as detected by conventional PCR and was the selected antibiotic for the phenotyping bioassays. First instar F. fusca were treated with tetracycline (50 mg/ml) for 48 h (n=174) and their gender recorded at adult eclos ion. A tot al of 29 trials were conducted and each trial included three control and three tetracycline -treated larval F. fusca Three control thrips and eight treated thrips died at t his concentration of antibiotic during the assay. The gender was record ed from 84 control thrips and 79 tetracycline treated thrips (Table 2 6). Larval thrips fed a liquid honey solution (= Wolbachia infected) emerged predominantly as females (66%), for a female to male ratio of 2 to 1. Larval thrips fed a liquid antibiotic solution (= Wolbachiacured) emerged predominatly as males (61%), f or a female to male ratio of 0.64 to 1. The last two bioassa ys examined the effects of removing Wolbachia by antibiotic treatment on offspring production of virgin and mated F. fusca females A total of five trials were conducted for each assay and each trial included three control and three tetracycline treated adult F. fusca females. The virgin control females (n=15) produced an average of 4.67 2.87 eggs (with a range of 0 12 total eg gs per female) and the virgin treated females (n=15)
69 produced an average of 4.07 2.7 eggs (with a range of 0 to 10 total eggs per female). All of the eggs in both treatments emerged as males after adult eclosion. The mated control females (n=15) produc ed an average of 4.47 2.56 eggs (with a range of 0 to 11 total eggs per female). The mated treatments (n=14) produced an average of 1.86 1.23 eggs (with a range of 0 to 4 total eggs per female). The female to male sex ratio of progeny from the mated control females was 2 to 1 (45 females and 22 males) and the progeny from mated treated females were 100% male (n=26). EM: Transmission electron micrographs of thin sections from non-parasitized and T. fuscum parasitized F. fusca females revealed the pres ence of numerous small (~1 m) bacteria in the yolk of the oocytes and the cytoplasm of follicle cells surrounding the oocyte (Figure 2 -5 ). These bacteria were not observed in any other F. fusca tissues. Discussion Use of the full MLST gene set for strai n typing indicates that non-parasitized F. fusca parasitized F. fusca and freeliving T. fuscum are all associated with one or more Wolbachia strains. Wolbachia has been detected in other arrhenotokous and thelytokous t hrips populations and in a plant parasitic nematode (see Chapter 1 ; Araki et al., 1999; Pintureau et al., 1999; Haegeman et al., 2009; Kumm and Moritz, 2009) To my knowledge, this is the first report of (1) multilocus sequence typing and supergroup designation of a Wolbachia strain(s) as sociated with a Thysanopteran and (2) detection of Wolbachia in an insect parasitic nematode. Horizontal transfer of Wolbachia has been documented between a host and parasite (see Chapter 1), but further analyses need to be conducted before concluding lat eral transmission between the two taxa occurred Interestingly, the F. fusca 16S rRNA sequence data was highly homologous to sequence data obtained from Bryobia sp. (Figure 2 2). These sap -feeding mites are commonly found on peanut and have been recovere d numerous times from peanut plants maintained in the
70 greenhouse for F. fusca colony rearing. Horizontal transfer of Wolbachia may also have occurred between Bryobia sp. and F. fusca Phylogenetic analysis of the 16S rRNA revealed two major groupings of W olbachia in the cohort populations. The presence of T. fuscum appears as t he dominant factor correlate d with the subgroup segregation in the cohort populations The majority of non-parasitized F. fusca clustered into Supergroup B (8/12) whereas the paras itized F. fusca and free living T. fuscum grouped into Supergroup A (11/16) These data suggest but do not conclude, parasitism may induce a switch in the dominant Wolbachia strain infecting F. fusca The cause of this switch is unknown. Initially, I s uspected the Wolbachia strain(s) associated with the T. fuscum nematode may be outcompeting (=destroying) the strain found in the non-parasitized F. fusca As reviewed in Chapter 1, Wolbachia is considered an obligatory mutualist in some insects and is re quired for successful oogenesis. This outcompetition, or killing of host Wolbachia, could potentially be the cause for the observed sterility in parasitized F. fusca However, antibiotically treated or Wolbachiacured femal es are still able to reproduce and there was not 100% homology in 16S sequence data between the T. fuscum and parasitized F. fusca population cohorts. An alternative hypothesis proposes the competitive intraspecific exclusion of Wolbachia strains within a parasitized F. fusca Wolbachia is predominantly found in the reproductive tissue s of infected individuals but can also be associated with somatic tissue (Min and Benzer 1997; Dobson et al., 1999; Cheng et al. 2000). H ealthy F. fusca have polymorphisms in the 16S rRNA electropherogr ams suggesting multiple Wolbachia strains within the examined population cohorts (data not shown) one associated with the reproductive system and one systemically infecting the s omatic tissues. As reviewed in Chapter s 1 and 2 T. fuscum causes a degene ration of the ovaries in parasitized F. fusca This degeneration may reduce the number of bacteria
71 associated with the reproductive tissues to non -detectable levels, and as a result, the Wolbachia specific primers were only able to detect the underlying s ystemic strain. However, thorough histological examination did not detect Wolbachia symbionts in tissues other than those associated with the reproductive system of F. fusca Bacterial associations with F. fusca individuals were restricted to the midgut and ovaries (see Chapter s 3 and 5 ). In the present study, Wolbachia influences the reproductive biology of the arrhenotokous F. fusca. Wolbachia did not alter the number of eggs or gender of offspring from virgin F. fusca but increased the fertility and production of female progeny in mated F. fusca. Additionally, the presence of Wolbachia in larval F. fusca increases the number of females upon adult eclosion As mentioned earlier, Wolbachia induces a variety of effects to host reproduction including c ytoplasmic incompatibility, parthenogenesis, feminization, and male killing (as reviewed in Werren, 1997). T o my knowledge, there is no information in the literature regarding the influence of Wolbachia on arrhentokous populations of haplodiploid insects Haplodiploid thylytokous populations of wasps revert to male production when antibiotically cured of Wolbachia (Legner, 1985; Stouthamer and Luck, 1991; Zchori et al., 1992). In my study, Wolbachiainfected females produced twice as many eggs as Wolbachia -cured mated females. These results suggest Wolbachia infection may induce male killing in the embryo of fecund F. fusca. The i nfluence of Wolbachia on the host parasite dynamics of F. fusca and T. fuscum are unknown. Parasitism rates under field conditio ns are time dependent and suggest extrinsic factors ( e.g ., temperature, humidity, rainfall, etc.) are critical for determining parasite success (data not shown) Parasitism rates in the laboratory are also time -dependent ( e.g ., laboratory parasitism rates from June to November increase from 25% to 85%) which suggests intrinsic
72 factor(s) (e.g ., physiological regulation by host or parasite) may also influence parasitism rates (data not shown) These physiological factors may be directly or indirectly relate d to Wolbachia infection. For example, Wolbach ia infection upregulates Aedes aegypti immune system and significantly inhibits the ability of Brugia filarial nematodes to develop inside mosquitoes (Kambris et al., 2009). Potentially, Wolbachia infection i n the thrips host upregulates immune factors that are active against T. fuscum Effects of these host immune factors on other en tomoparasites (e.g ., Pantoea ananatis and TSWV) in the F. fusca vector are discussed in Chapter 5. Clearly, more studies are n eeded to understand the interaction between Wolbachia and its F. fusca host and r esults from this chapter open avenues for new and unique management strategies to control thrips vector populations in agroec osystems (see Chapter 6).
73 Table 2 1. Collection data including cohort population, locality, and date labeled for Wolbachia gene screen and 16S rRNA analysis. Gene Screen # 16S Sample # Cohort population Collection locality Date 37 1 Non parasitized F. fusca females Unknown 2007 34 2 Non parasitized F. fusca females UF Campus 2008 40 3 Non parasitized F. fusca females UF Campus 2008 41 4 Parasitized F. fusca females UF Campus 2008 11 5 Freeliving T. fuscum UF Campus 2008 42 6 Non parasitized F. fusca females Citra (ornamental) Oct -08 30 7 Non para sitized F. fusca females Citra (ornamental) Oct -08 31 8 Non parasitized F. fusca females Citra (field) Oct -08 44 9 Parasitized F. fusca females Citra (field) Oct 08 10 10 Free living T. fuscum Citra (peanut field A population) 2008 1 11 Non parasitized F. fusca females UF Campus (first generation from colony) May -09 2 12 Non parasitized F. fusca females UF Campus (first generation from colony) May -09 3 13 Non parasitized F. fusca females UF Campus (first generation from colony) May -09 7 14 Non parasitized F. fusca males UF Campus (first generation from colony) May -09 4 15 Parasitized F. fusca females Citra (field) Aug 09 5 16 Parasitized F. fusca females Citra (field) Aug 09 6 17 Parasitized F. fusca females Citra (field) Aug 09 8 18 Free living T fuscum Citra (field) Aug 09 9 19 Freeliving T. fuscum Citra (field) Aug -09 13 20 Non parasitized F. fusca females* Citra (field) (first generation from colony) Nov -09 14 21 Non parasitized F. fusca females* Citra (field) (first generation from colony ) Nov -09 17 22 Parasitized F. fusca females* Citra (field) (first generation from colony) Nov 09 18 23 Non parasitized F. fusca males* Citra (field) (first generation from colony) Nov 09 19 24 Parasitized F. fusca males* Citra (field) (first generation from colony) Nov -09 21 25 Parasitized F. fusca males* Citra (field) (first generation from colony) Nov 09 23 26 Parasitized F. fusca pupae* Citra (field) (first generation from colony) Nov 09 24 27 Free living T. fuscum* Citra (field) (first generation from colony) Nov 09 25 28 Free living T. fuscum* Citra (field) (first generation from colony) Nov 09
74 Table 2 1. Continued 12 Non parasitized F. fusca females* Citra (field) (first generation from colony) Nov -09 15 Parasitized F. fusca females* C itra (field) (first generation from colony) Nov 09 16 Parasitized F. fusca females* Citra (field) (first generation from colony) Nov 09 20 Parasitized F. fusca males* Citra (field) (first generation from colony) Nov 09 22 Non parasitized F. fusca pupae* Citra (field) (first generation from colony) Nov -09 26 Free living T. fuscum* Citra (field) (first generation from colony) Nov 09 27 Free living T. fuscum* Citra (field) (first generation from colony) Nov 09 28 Non parasitized F. fusca fema les Citra (ornamental ) Oct -08 32 Non parasitized F. fusca females Citra (field ) Oct -08 33 Non parasitized F. fusca females Citra (field ) Oct -08 35 Freeliving T. fuscum UF Campus 2008 36 Non parasitized F. fusca females 38 Parasitized F. fusca females 2007 39 Free living T. fuscum 43 Non parasitized F. fusca females Citra (field) Oct -08
75 Table 2 -2 16S, wsp and MLST loci and primer features (taken from Baldo et al., 2006). Gene Product Primer Gene length (bp) Amplified nt range (bp) fragment size (bp) Designation Sequence (5 -3) wspec Wolbachia specific 16SrRNA WspecF YATACCTATTCGAAGGGATAG WspecR AGCTTCGAGTGAAACCAATTC wsp Wolbachia surface protein wsp_F1 GTCCAATARSTGATGARGAAAC 714 85688 546 wsp_R1 CYGCACCAAYAGYRCTRTAAA gatB aspartyl/glutamyl tRNA(Gln) amidotransferase, subunit B gatB_F1 GAKTTAAAYCGYGCAGGBGTT 1,425 421891 369 gatB_R1 TGGYAAYTCRGGYAAAGATGA coxA cytochrome c oxidase, subunit I coxA_F1 TTGGRGCRATYAACTTTATAG 1,551 491977 402 coxA_R1 CTAAAGACTTTKACRCCAGT hcpA conserved hypothetical protein hcpA_F1 GAAATARCAGTTGCTGCAAA 741 91605 444 hcpA_R1 GAAAGTYRAGCAAGYTCTG ftsZ cell division protein ftsZ_F1 ATYATGGARCATATAAARGATAG 1,197 274798 435 ftsZ_R1 TCRAGYAATGGATTRGATAT fbpA fructose bisphosphate aldolase fbpA_F1 GCTGCTCCRCTTGGYWTGAT 900 241749 429 fbpA_R1 CCRCCAGARAAAAYYACTATTC
76 Table 2 3. Wolbachia surface protein ( wsp ) loci and hypervariable region profile (HVR1-4 ) for the six non -parasitized F rankl iniella fusca samples selected for MLST. Locus Sample wsp HVR1 HVR2 HVR3 HVR4 11 372, 78 (91.91%) 1 (90.01%) 12, 71 (94.00%) 21, 109 (96.08%) 11 (84.62%) 20 424, 296, 162 (90.00%) 157, 17, 95, 80 (60.61%) 17 (100%) 88, 3 (98.04%) 2, 109 (96.43%) 7 424, 296, 162 (94.38%) 88, 65 (84.85%) 17 (100%) 88, 3 (98.04%) 2, 109 (96.43%) 8 424, 296, 162 (92.50%) 88, 65 (75.76%) 17 (100%) 88, 3 (98.04%) 2, 109 (96.43%) 2 424, 296, 162 (96.88%) 2, 77 (93.94%) 17 (100%) 88, 3 (98.04%) 2, 109 (96.43%) 6 424, 296, 162 (96.88%) 2, 77 (93.94%) 17 (100%) 88, 3 (98.04%) 2, 109 (96.43%) The number in parentheses represents the percent match to the alleles listed for each sample. Table 2 4. The percentage of each Frankliniella fusca cohort population wi th a visible amplicon of each primer by conventional PCR. Amplicon detected in sample Primer Non parasitized female F. fusca (17) Parasitized female F. fusca (10) Free living T. fuscum (9) 16S 88.24% (15) 90.00% (9) 66.67% (6) wsp 82.35% (14) 90.00% (9) 44.44% (4) coxA 82.35% (14) 90.00% (9) 88.88% (8) hcpA 29.41% (5) 20.00% (2) 11.00% (1) ftbA 52.94% (9) 30.00% (3) 0.00% (0) ftsZ 82.35% (14) 100% (10) 66.67% (6) gatB 58.82% (10) 70.00% (7) 22.22% (2) Total % 61.18% 62.00% 37.78% The number in parent heses represents the total number for each category.
77 Table 2 -5 Allelic profiles of the six non -parasitized Frankliniella fusca samples selected for MLST. Locus Sample gatB coxA hcpA ftsZ fbpA 1 43 (6) 32 (40) 6 (7) 15 (6) 20 39 14 (1) 6 (2) 7 (1) 7 39 14 6 (2) 7 (1) 9 ( 2) 8 39 14 6 (2) 7 (1) 15 (5) 2 39 14 6 (2) 7 (1) 9 (2) 6 39 14 ( 1) 6 (2) 7 (1) 9 (2) The number in parentheses represents the number of nucleotide differences to the closest allele. A dash ( ) designates sequence data was not obtained for th e listed gene. Table 2 6. The percentage of male and female Frankliniella fusca at adult eclosion after tetracycline (50 mg/ml) treatment as first instars. Treatment Female Male Control (84) 66% (56) 33% (28) Tetracyclin e treated (79) 39% (31) 61% (48) The number in parentheses represents the number for each treatment
78 Figure 2 -1 An initial Wolbachia gene screen of 36 Franklini ella fusca and Thripinema fuscum populations (A) 16S rRNA ; (B) wsp ;
79 Figure 2 -1 (continued) (C) coxA ; (D) hcpA (E) ftbA; (F) ftsZ ; and (G) gatB primers. A star (*) designates samples with a weak to no signal present.
80 Figure 2 2. Neighbor joining phylogenetic tree based on 16S rRNA nucleotide a lignment. Wolbachia strains are characterized by the names of their host species. Names in bold represent cohort populations in the dataset with the numbers in parentheses corresponding to those listed in Table 2 1. The supergroup designations are shown on the right. Bootstrap and jackknife values are depicted above and below the branches, respectively.
81 #2 EILPFYTKVDGITICTGKEKDSPLTRSFIAGGGAFGYKMDDIRVDVEGLYSQLAKD-TAVVNTSETNVADSLTAFSGLVNVYYDIAIEDMPITPYLGVGVGAAYISNPSKADAVKDQKG-FGFAYQAKAGVSYDVTPEIKLFAGARYFGSYGASFDKATKDD--NGIKNV----#6 EILPFYTKVDGITICTGKEKDSPLTRSFIAGGGAFGYKMDDIRVDVEGLYSQLAKD-TAVVNTSETNVADSLTAFSGLVNVYYDIAIEDMPITPYLGVGVGAAYISNPSKADAVKDQKG-FGFAYQAKAGVSYDVTPEIKLFAGARYFGSYGASFDKATKDD--NGIKNVVYSAI #7 EILPFYTKVDGITLCAGKVKDSPLTRSFIAGVGPFGYKMDDIRVDVEGLYSQLAKD-TAVVNTSETNVADSLTAFSGLVNVYYDIAIEDMPITPYLGVGVGAAYISNPSKADAVKDQKG-FGFAYQAKAGVSYDVTPEIKLFAGARYFGSYGASFDKATKDD--NGIKNV----#8 EILPFYTKVDGITLCAGKVNYSPLTRSFIAVVGPFGYKMDDIRVDVEGLYSQLAKD-TAVVNTSETNVADSLTAFSGLVNVYYDIAIEDMPITPYLGVGVGAAYISNPSKADAVKDQKG-FGFAYQAKAGVSYDVTPEIKLFAGARYFGSYGASFDKATKDD--NGIKNV----#20 EFLPFYTKVDGITLYEGKIDYSPLTTSFTALVVRIGYKMDDIRVDVEGLYSQLAKD-TAVVNTSETNVADSLTAFSGLVNVYYDIAIEDMPITPYLGVGVGAAYISNPSKADAVKDQKG-FGFAYQAKAGVSYDVTPEIKLFAGARYFGSYGASFDKATKDD--NGIKNV----#11 EFLPLFTKVDGITYKKDKIDYSPLKPSFIAVVGAFGYKMDDIRVDVEGVYSYLNKNDFKGVTFHPANTIADSVTAISGLVNVYYDIAIEDMPITPYIGVGVGAAYISTPLEP-AVNNQKNKFGFAGQVKAGVSYDVTPEVKLYAGARYFDSFGSNFDKSKEVDKVGGGKEIKVTKD *:**::******* .* ***. ** :*************:** *: ..*. ..:***:**:********************:**********.* :. **::**. **** *.***********:**:******.*:*:.***:.: .* *:: Figure 2 3. Amino acid alignment of six wsp sequences from non -parasitized female Frankliniella fusca cohort populations. The six samples used for the alignment were the same samples selected for the MLST. Amino acid motifs at each hypervariable region (HVR1 4) are grouped by color according to their similarity to other sequences. HV R1 HVR2 HVR3 HVR4 CR1 CR2 CR3 CR4
82 Figure 24. Conventional PCR detecti on of Wolbachia in Frankliniella fusca with 16S rRNA primers ( wspec ) after antibiotic therapy. (L) Marker Hyperladder II (5 l); (1) 50 F. fusca from laboratory colony (+ control); (2) Ampicillin (50 mg/ml); (3) 10 F. fusca from laboratory colony ( + control); (4) Kanamycin (25 mg/ml); (5) Strepomycin (50 mg/ml); (6) Tetracycline (50 mg/ml).
83 Figure 2 -5 Transmission electron micrographs of intracellular bacteria in the reproductive structures of non-parasitized and Thripinema fuscum parasi tized Frankliniella fusca females. (A ) Ovariole of a nonparasitized F. fusca parasitized individual with bacteria; (B) Magnification of the bacteria in the cytoplasm of the oocyte and surrounding follicle cell; (C) Ovariole of a T. fuscum parasitize d F. fusca female with bacteria; (D F) Progressive magnification of (C ).
84 CHAPTER 3 HISTOLOGICAL EXAMINATION OF FRANKLINIELLA FUSCA AND THRIPINEMA FUSCUM Introduction T here is very limited information available in the literature on the internal anatomy and morphology of F. fusca despite this thrips importance as a vector of Tomato spotted wilt virus (TSWV, Bunyaviridae: Tospovirus) and Pantoea ananatis (Eubacteriales: Enterobacteriaceae) (Sakimura, 1969; Heming, 1970a,b; Wells et al., 2002; Gitaitis et al., 2003). Most histological studies conducted on Thysanoptera have been restricted to the F. occidentalis vector ( Dallai et al ., 1991, 1996, 1997; Del Bene et al ., 1991; Hunter and Ullman, 1989, 1994; Kumm, 2002; Ullman et al., 1989). However, thrips m orphologies vary greatly between species and may deline ate persistent vectors from semi -persistent vectors and non -vectors (Ullman et al., 1989). A better understanding of F. fusca vector biology is essential for developing strategies to manage this crop pest In addition, there have been few studies pictorially document ing the in vivo life cycle of Allantonematidae nematodes and the pathological changes these parasites i nduce to host tissues (Schmidt and Platzer, 1980; Tomalak et al., 1984, 1988). The potential exists for T. fuscum to be commercialized as a natural enemy of F. fusca but the in situ relationship between the parasite and host thrips has not been investigated. Therefore, t he objective of this chapter was to conduct a histological examinati on and (1) document the internal morphology of healthy F. fusca females, particularly select tissues and cells affecting vector competence and reproduc tion; (2) examin e the life cycle of T. fuscum including in vivo location and development of the parasitic female and juveniles, reproduction, and migration of late stage juveniles out of the thrips host ; and (3) identify changes to host thrips target tissues and cells resulting from T fuscum invasion and replication. Information gathered from these studies may provide insight into how the
85 parasitic T fuscum alters host morphology and how such alterations influence F. fusca vector competence, and as a result, offer new strategies for controlling pest insect vector s. Materials and Methods Specimen collection: Female F. fusca were collected from peanut flowers ( Arachis hypogaea L.) in Marion Co., Florida at the Institute of Food and Agricultural Science Pl ant Research and Education Unit (2924 N 8210 W ). Specimens were selected from established non -parasit ized and T. fuscum parasitized F. fusca laboratory colonies maintained as previously described (Sims et al., 2005). Light microscopy : Non -parasitized and parasitized F. fusca females were dissected in a droplet of double distilled water on a glass slide u sing minuten pins. Dissected specimens were photographed with the Auto -Montage Pro System, a program which uses a series of captured images focused at different heights to form a high quality focused composite (Auto -Montage Pro 5.02.0096; Syncroscopy, Fre derick, MD, USA ). Free living T. fuscum were collected by rinsing 1.5 -ml microcentrifuge tubes that previously held parasitized F. fusca adult females with 200 l of H epes -buffered saline (pH 6.8). The buffer from each tube was collected and pooled into a single tube, centrifuged at 5,000g for 3 minutes, and exchanged with warm (~37C) 2.5% gluteraldehyde buffered in 0.1M sodium cacodylate containing CaCl2 (1 mg/ml). The recipes used for histology ( i.e., fixative, buffers, post -fixatives, etc.) are liste d elsewhere (Appendix B). The in vivo stages of T. fuscum were collected by dissecting fully parasitized F. fusca females in a 10 l droplet of H epes buffered saline T he droplet of nematodes was transferred into a 1.5 m l microcentrifuge tube containing warm fixative for 10 minutes. N ematodes were then placed individually in a droplet of glycerin, covered with a cover slip and photographed under a differential interference contrast microscope with a RT Spot Diagnostics Imaging System (Spot Imaging Solut ions, Sterling Heights, MI)
86 Scanning electron m icroscopy : Adult F. fusca females were immobilized on ice and adhered to double -sided tape attached to a microscope slide. A 10 l droplet of warm fixative was immediately placed o ver the specimen T he sub merged insect was pierced with a finely pulled capillary tube through the cuticle of each body segment to allow for the exchange of fluids After five min utes the specimen was transferred to a 1.5 -ml microcentrifuge tube with warm, fresh fixative for two hours Stages of T. fuscum were collected as described above and fixed for two hours at room temperature F ixed s pecimens were transferred to a specimen holder that sandwich es microorganisms between nucleopore filters (0.4 m; Nucleopore Corp., Pleasant on, CA ) to reduce elimination of samples during the multiple fluid transition s required for processing (Kurtzman et al., 1974). Using a syringe, s amples were washed post -fixed, and dehydrated ( Table 3-1 ). Following the final dehydration in acetone the syringe was removed and the specimens were dried using the critical point method (BalTek 030 CPD, Cheshire, UK) The sp ecimen holder was then disassembled and the nucleopore filters holding either F. fusca females or T. fuscum were attached to SEM stubs w ith double -sided adhesive. To process p arasitized F. fusca females for SEM, individuals were fracture d o n a copper stub adhesive by lightly dragging a razorblade over the surface of the insect and prying open the cuticle using fine forceps and minuten pin s. All stubs were coated with gold for 130 s in a sputter -coater (Denton Vacuum Desk II (Au/Pd), Moorestown, NJ, USA) and examined in a Scanning Electron Microscope (Hitachi FE -S4000, Hitachi High Technologies America, Illinois, USA ) at 10 kV Measuremen ts from digital images were taken with the Quartz PCI Image Management System (Vancouver, Canada). It is important to note that shrinkage resulting from fixation typically results in a 30% size reduction of specimens and the reader should see Tipping et a l. (1998) for exact measurements of T. fuscum stages
87 Transmission electron m icroscopy : Frankliniella fusca females and T. fuscum individuals were prepared for transmission electron microscopy following the protocol described in Table 3 -1. When needed, so dium bis (2-ethylhexyl) su lfosuccinate (Aerosol O.T. 100% ) was used to break the surface tension and help submerge thrips in fixative All samples were embedded in a 1 % agarose block prior to post -fixation. Dehydrated samples were infiltrated with Epon -A raldite resin (cat no. 13940, EMS; Hatfield, PA, USA ) and Z60 40 embedding primer (cat no. 5044010, EMS) was added to help the resin adhere to insect cuticle. Samples were placed in plastic molds and polymerized in a 65C oven Resin blocks were sectio ned using a Reichert Jung Ultracut E Microtome. Thick sections (0.5 m) were stained with 1 % toluidine blue in 1% borax for 15 s covered with Permount and a cover slip and viewed under a compound microscope. Images were collecte d with the RT Spot Diagn ostics Imaging System Ultrat hin sections (7090 nm) were taken with a diamond knife (Diatome; Hatfield, PA, USA ), and collected on Formvar Carbon Coated Copper Grids ( 200 mesh; EMS, Hatfield, PA, USA ). Sections were post -stained in 0.5% uranyl acetate f or 10 minutes followed by Reynold s lead citrate for 5 minutes and viewed at 75 kV with a Hitachi H 600 electron microscope Results Healthy female F. fusca: Results herein are concordant with the few histological studies available in the literature that document the internal morphology of F. fusca and F. occidentalis (Heming, 1970; Ullman et al., 1989; De Bene et al., 1991; Dallai et al., 1991, 1996; Hunter and Ullman, 1992, 1994; Kumm, 2002). Externally, t he head holds the antennae, compound eyes, ocell i, and piercing sucking mouthparts in a hypognathous position (Figure 3 -1 Figure 3 -2). Internally, the h ead contains the brain, ganglia, and nerves. The thorax, the second body segment, encloses the ovoid salivary glands and the anterior foregut. In ad dition, muscles predominate in the thoracic tagma and serve as po ints of attachment for the external wing and leg
88 appendages. Frankliniella fusca have macropterous and brachypterous wing form s ( Figure 3 -3 ). The abdome n, the largest of the three body segm ents, is comprised of 13 segments that encl ose the alimentary tract and reproductive structures A saw -like ovipositor is present on the ninth segment (Figure 3 -4). Abdominal f at body and muscles underlie the female thrips cuticle. A thick section of a healthy F. fusca female taken at 400X exhibits the location and fine detail of these structures (Figure 3 -5). Herein, the internal anatomy of F. fusca will be br iefly described according to those systems and/or tissues most affected by Thripinema fuscum p arasitism (i.e., invasion, replication, and emergence) : the dige stive system, the reproductive system fat body and muscle. The digestive system of F. fusca consists of the salivary glands, foregut, midgut, hindgut, and Malpighian tubules These organs fl ank the surrounding muscle tissue in the thoraci c and abdominal tagma The F fusca salivary glands are comprised of two morphologically distinct type s: short, ovoid glands that lie proximally to the esophagus in the thoracic region and long, tubular glan ds that extend from the ovoid glands to the anterior portion of the midgut (Figure 3 6A-B). The salivary glands produce and release saliva used for digesting food The glands are endodermal in origin and easily recognizable under the electron microscope by the presence of cells containing spheroidal secretory granules and a thin dense cuticle -lined lumen (Del Bene et al. 1999) ( Figure 3 6C D). Importantly, the ovoid salivary glands are the principal site for TSWV replication (Figure 3 -7). The foregut ( stomodauem ) sits i mmediately posterior to the salivary glands in the thoracic tagma and is the primary organ for ingestion. The foregut develops from invaginating ectodermal tissue and consists of a thin layer of epithelial cells lined by cuticular intima ( Figure 3 -8A-B) The midgut, located posterior to the foregut, is the principal site for secretion of enzymes and digestion and absorption of food particles (Figure 3 -
89 8A,C D) The F. fusca midgut is convoluted and forms three loops designated as Mg1, Mg2, and Mg3 (Figure 3 8D) The midgut is composed of a single -layered epithelium resting on a continuous basement membrane (Figure 3 9A -C). Circular and longitudinal muscles lie just beyond the basement membrane and serve to contract t he midgut The micro villi of the midgut cells are distinct in each midgut region, and are surrounded either by a myelinlike membrane (Mg1 2) or glycocalyx that form a brush border (Mg3) (Figure 3 9D) The midgut is endodermal in origin, and because it lacks a cuticular inti ma, thrips have an extracellular membrane termed the perimicrovillar membrane (PM) that supposedly functions to compartmentalize digestion and/or aid in the absor ption of essential amino acids Frankliniella fusca have four Malpighian tubules at the base of the midgut which open into the digestive tract at the pyloric region where the midgut meets the ileum of the hindgut (Figure 3 10A). The two anterior tubules appear to be adhered for a short distance to the midgut and the two posterior tubules are connected to the ovarioles via small ligaments. The Malpighian tubules function together with the hindgut as excretory organs to remove waste materials from circulation within the hemolymph and to also reabsorb useful substances. Intracellular granules, sphe res, and vacuoles are common ly found in the Malpighian tubules and serve as collection sites for nitrogenous waste and other excretory products ( e.g., uric acid as well as other orga nic and inorganic material) ( Figure 3 10B -C). The hindgut (proctodaeum) c onstitutes the posterior part of the alimentary canal (Figure 3 8A) This rather simple tube extends from the pyloric valve to the anus and is supported posteriorly by suspensory muscles ext ending from the abdominal wall. The hindgut is derived from ectodermal tissue and lined by a thin layer of cuticular intima ( Figure 3 11A ). This cuticle functions to protect the underlying epithelial cells and is shed with each molt. The formation of primary urine in the Malpighian tubules is accompanied by a loss of useful substances and the hindgut
90 acts to selectively reabsorb those substances from the lumen back into the hemolymph. The hindgut also secretes additional waste components into the urine. Both t he midgut lumen and the hindgut are associated with numer ous bacteria (Figure 3-1 1B -D, Figure 5 1A -B). The female reproductive system of F. fusca consists of the ovaries, calyx, two lateral oviducts, a median oviduct, a spermatheca, accessory glands, and a vagina or genital chamber. Females have one pair of pan oistic ovaries consisting of two ovaries with four ovarioles each located underneath the alimentary tract. Each ovariole is divided into five zones: (1) the terminal filament; (2) the germarium; and (3-5 ) the vitellarium zones IIIa -c The ovariole s merg e posteriorly at the calyx and unify to a median oviduct which forms the vagina. The spermatheca is located just anterior to the vagina and serves as a storage organ for the sperm (Figure 3 12). The accessory gland is situated against the ovipositor and produces substances that attach eggs to the substrate during oviposition (Figure 3 12). The ovipositor is formed from a modification of the eighth and ninth abdominal segments, and females insert eggs into plant tissue with this structure (Figure 3 -4) D uring oogenesis, the e ggs fill nearly the entire abdominal cavity (Figure 3 13). Eggs are full of lipid droplets and proteins (yolk) that are transported from the fat body to the ovaries by the hemolymph (Figure 3 14). The developing oocytes are of diffe rent developmental stages and appear healthy with prominent and well defined nuclei in the follicle cells (Figure 3 15). The follicle cells are tightly linked to surrounding the oocytes by ladder like extensions (Figure 3 16) The f at body of F. fusca lo cated as thin lobes immediately under the female thrips abdominal cuticle, is the major tissue for metabolism and nutrient storage (Figure 3 -5 ). In this tissue, a dipocytes store energy reserves as lipid droplets composed of triglycerides. There is also a n abundance of glycogen in these cells. The fat body is the principal site for the synthesis of
91 vitellogenin, the egg yolk precursor protein In addition, the fat body synthesi zes a ntimicrobial peptides hemocyte proteins, metabolites, storage proteins, lipophorins, and hormones The fat bodies are heterogenou s, appearing as smooth and regular masses with globules of fat droplets and glycogen deposits (Figure 3 17). Frankliniella fusca possess hundreds of individual muscles (Figure 3 18) A typical inse ct muscle is composed of either bundles of or loosely aggregated muscle fibers made of myofibrils, each which contain actin, myosin, and contraction proteins in repeating sarcomere units (Figure 318B -C). Sarcoplasmic reticulum run s longitudinally on the surface of the muscle fibers and store calcium ions used in the contraction process (Figure 318C) The muscles also contain many large mitochondria (sarcosomes) and intracellular tracheoles to provide energy and oxygen for their use (Figure 3 18B -C). T hripinema fuscum : Infective female s (n=10 ). The average body length is 199 m 17.80 m (range of 175.3 m 221.0 m) ; the a verage body width is 7.97 m 1.02 m (range of 6.65 m 9.8 m) The female body is straight to slightly curved when heat -r elaxed (Figure 3 19A ). The cuticle is a nnulated with transverse striations averaging 0.55 m and bearing lateral fields (=cuticular ridges) that extend the length of the body ( Figure 3 19B E, Figure 3 20). E ach lateral field is 1.56 m 0.26 m wide and composed of two thick lateral lines measuring 0.25 m 0.03 m. F emales have a distinct pr otrusible hollow stomatosylet composed of a stylet shaft and cone ( etymology is tyl=knobbed; enchos= spear) (Figure 3 21). T he mouth is moderately sclerotized and consists of a stoma (=pore) (0.20 m 0.65 m) surrounded by four submedian lobes (Figure 3 22) Females have an excretory pore opening on the ventral side of the anterior region ( Figure 3 23). The uterus is well -developed with a single prodelphic o vary.
92 Mature parasitic female (n= 7 ). The average body length is 169.27 m 47.31 m (range of 143.5 m 219.8 m); the average body width is 60.95 m 17.52 m (range of 36.82 m 91.48 m) The female body is oval -shaped, often with part of uteru s protruding from the vulva ( Figure 3 24) A l ateral field and excretory pore was not observed. Females lack a stylet and esophagus and their b ody wall is modified with a hypodermal layer (Figure 3 25). U pon closer examination the hypodermis is covere d with microvilli and punctations (Figure 3 26). The o vary is long and convoluted and the uteru s usually with one to two eggs Free -living male (n= 1 ). The body length is 160.85 m; the body width is 8.2 m. Body is curved dorsally when heat relaxed ( Fig ure 3 27). The male cuticle is similar to the infective female and a lateral field is present The s tylet is indistinguishable (Figure 3 27). The posterior region possesses the following reproductive structures (Figure 3 28) : caudal alae (=bursa) large with crenate margins (Figure 3 28BD), paired spicules and a thin well -sclerotized gubernaculum (Figure 3 28EF ). Juveniles (n=1 3). The average body length is 124. 31 m 35. 43 (range of 82.83 m 161.95 m); the average body width is 9.88 m 1.81 m (range of 7.47 m 13.02 m). Different stages of juveniles are found in F. fusca ranging from small and wide for early -staged juveniles (J1 J2) to long and slender for late -staged juveniles (J2 J3) (Figure 3 29). The j uveniles shed four cuticles (Fi gure 3 -x) and the c uticle develops structure with each molt (Figure 3 30). Upon closer examination, the juvenile cuticle contain s modifications that are likely for nutrient assimilation (Figure 3 31, Figure 3 32). The lip region and stylet develop in late-staged juveniles ( Figure 3 33). Reproductive structures are not obvious at this stage Eggs (n= 11). The average egg length is 34. 03 6. 03 (range of 23.05 46.02); the average egg width is 16.64 3.97 (range of 11.52 17.37). Eggs are oval -shaped (Figure 3 34)
93 and with bumps (Figure 3 35). The embryo is often observed through a transparent chorion (Figure 3 35). Life cycle: A series of parasitized thrips sampled at intervals shows the generalized in vivo life cycle of a T. fuscum female ( Figure 336). Immediately upon parasitization, the infectious female begins the dramatic transformation to the obese phenotype (Figure 3 -36A) During this time (day one to day three post -parasitization) a mass deterioration of F. fusca body structure is apparen t The cuticle of T. fiuscum degenerates and is replaced by a microvilliated layer interspersed with nodules and cuticular pits (Figure 3 25, Figure 3 26). The parasitic female is directly apposed to midgut cells where she likely sequesters nutrients for egg production ( Figure 336A ). The swollen female produces eggs within four days post -parasitization and eggs are continuously produced until death of either the host or parasite (Figure 3 36B ). The eggs hatch into corpulent juveniles that morph to the characteristic vermiform juvenile shape Fourth -stage juveniles emerge from the posterior end of male and female T. fuscum nine days post parasitization thus completing the life cycle. Typically, the female motherworm, eggs, and immature nematodes were found in the anterior abdominal regions of the thrips, whereas the fully mature juveniles were localized in a sac-like hindgut region ( Figure 3 -36C). Occasionally the juveniles migrate to the thoracic and head tagmata ( Figure 3 37). D iagram s of the complete T. fuscum life cycle can be seen in Figure 1-7 and Figure 3 38. Histopathology : Externally, there are no detectable symptoms or morphological changes induced by parasitism even though the abdominal cavity of a T. fuscum parasitized female is filled w ith nematodes (Figure 3 39) However, l ight microscopy of thi ck sections demonstrated the significant impact of T fuscum on the female thrips (Figure 3 40) Parasitization induces a displacement and invasion of the alimentary tract, an atrophy of the ovaries and fat body, an
94 accumulation of numerous electron -dense vesicles in hemocoel, and a degradation of muscle tissue. Scanning e lectron microscopy of fractured parasitized F. fusca thrips revealed numerous juvenile nematodes orient ed longitudinally in t he abdominal hemocoel (Figure 3 41A -D). The densely -packed nematodes pressed against the midgut forming indentions on the basal side of the basement membrane (Figure 3 41E -F). Transmission electron microscopy of thin sections from parasitized thrips revealed the parasitic T. fuscum female was always located proximal to the midgut with her microvilliated layer in direct contact with the basement membrane (Figure 3 42). The midgut cells appeared similar in size, shape, and organelle content to the cells o f nonparasitized F. fusca (Figure 3 43). The midgut of parasitized F. fusca shows the presence of what appears to be a peritrophic matrix in the lumen (Figure 3 43D-F ). T his matrix was not visible in any of the healthy thrips however, suggest ing this e xocellular secretion ma y instead be a sloughing of microvilli (Nation, personal communication) Late -staged juvenile nematodes move from the hemocoel and penetrate into the hindgut lumen where they form a mass aggregation (Figure 3 44A-C, F ). Upon closer examination, this aggregat e appears to be where mature males inseminate females prior to their exodus from the host (Figure 3 44D -E Figure 3 46). Transmission electron microscopy of thin sections from parasitized F. fusca female shows the hi ndgut lumen full of late -staged juvenile nematodes (Figure 3 45). Examination of the alimentary tracts of the parasitized thrips revealed the absence of the microbiota associated with healthy thrips (Figure 3 45D -E, Figure 5 3). Lastly, the Malpighian tu bules are full of secretory vesicles and uric acid crystals that probably serve as collection sites for T. fuscum excretory waste product filtered from the host hemolymph (Figure 3 47)
95 Gross dissection of parasitized thrips revealed the ovaries of parasit ized T. fuscum were half the size of those in non-parasitized thrips (Figure 3 48). The reduced ovarioles were significantly displaced as a result of the numerous juvenile nematodes in the hemocoel (Figure 3 40). As a result, the zones of the oocytes wer e compressed against either juvenile nematodes or other host tissues (Figure 3 49). Transmission electron micrographs show a degradation of t he oocytes with apoptotic bodies and nuclei of different sizes in the surrounding follicular epithelium (Figure 3 50). In addition to the reproductive tissues, apoptotic bodies w ere present in fat deposits of parasitized F. fusca females The atrophied fat bod ies in parasitized thrips lacked lipid droplets but contained numerous dense vesicles filled with granular material (Figure 3 51A-B). There was a depletion of glycogen in many of the fat bodies as demonstrated by the space void of glycogen and organelles (Figure 3 51C D). This depletion in glycogen coincided with an increased glycogen content inside juvenile ne matodes (Figure 3 52). Parasitized F. fusca also had storages of glycogen in the abdominal muscle tissue (Figure 3 53A -C). Numerous l esions and ruptured mitochondria were also visible in the muscle tissue ( Figure 3 53D-F) In many sections, progeny nema todes present in the hemocoel were partitioned away from insect tissues by an exocellular membrane that formed Thripinema-containing vacuoles void of insect cells (Figure 3 54, Figure 355). Discussion The internal morphology of non parasitized F. fusca is similar to other thrips species (Ullman et al., 1989; De Bene et al., 1991; Dallai et al., 1991, 1996; Hunter and Ullman, 1992, 1994) In the following paragraph, I only discuss F. fusca morphological characters that differ from those previously describe d in the literature for Frankliniella sp. First, Dallai et al. (1991) reported that the anterior Malpighian tubules of F. occidentalis lie free in the hemocoel but that
96 the posterior tubules are briefly adhered to the hindgut wall before their separation into the hemocoel. This is not the case for F. fusca The two anterior tubules appear to be adhered for a short distance to the midgut and the two posterior tubules are connected to the ovarioles via small ligaments. Second, Terebrantian thrips within t he Frankliniella genus have a single accessory gland that is evident as a large apical bulb (Heming, 1970; Dallai et al., 1996; Kumm, 2002). The apical bulb of F. fusca is connected to the pyloric region via small ligaments. This is the first record of s uch an attachment in Frankliniella sp. and it can be assumed that either other species of Frankliniella do not have these ligaments or the fragile ligaments were broken during dissection. Examination using light and electron microscopy provided insight into the life cycle of T. fuscum The T. fuscum life cycle is typical to that of other Allantonematidae nematodes ( Sharga, 1932; Lysaght, 1936, 1937; Nickle, 1963; Nickle and Wood, 1964; Ashraf and Berryman, 1970; Wilson and Cooley, 1972; Thong and Webster, 1975; Reddy et al., 1982; Tomalak et al., 1988; Chizhov et al., 1995; Green and Parrella, 1995; Teulon et al., 1997; Funderburk et al., 2002b ; Poinar et al., 2004; Zeng et al., 2007). The free living T. fuscum emerge from a parasitized male or female F. f usca host in the moist flower per ia nths of peanut where thrips aggregate to feed on pollen. The free -living juveniles can survive up to 48 h outside of a host under optimal conditions (data not shown). The infectious females remain in the flowers and, upon contact with a new host, enter the thrips through the intersegmental membranes of the abdomen or the coxal cavities of the leg. After the infectious female enters a thrips host, she undergoes a dramatic phenotypic transformation that is likely triggere d by exogenous stimulation by host factors (Croll, 1970; Sukhdeo and Sukhdeo, 2004). The infectious T. fuscum female sheds her cuticle and transforms her epicuticle into a microvilliated layer with pits The ultrastructure of
97 the integuments of Tylenchid parasitic female nematodes has been well documented (Riding, 1970; Poinar, 1972; Cliff and Baldwin, 1985; Subbotin et al., 1993, 1994, 1996). The female body surface contains a microvilliated layer with numerous ampullae and vacuoles on the outer hypoder mal membrane. In Skarbilovinema laumondi (Tylenchida: Iotonchiidae), the microvilli of the hyperdermis are densely packed and form a spongy layer. The hypodermal processes (microvilli, ampullae, and vacuoles) are absorptive organs and the spongy layer aids in primary digestion (Subbotin et al. 1993). In my study, the parasitic T. fuscum female nestles against the host midgut and appears to directly absorb nutrients from the host midgut cells. This assimilation of nutrients provide s the energy requir ed for the substantial numbers of T. fuscum eggs produced. Reddy et al. (1982) reported up to 420 Howardula aptini eggs in a female Megalurothrips sp. host. Eggs laid by the parasitic T. fuscum female are released into the host hemocoel and hatch after t wo to three days. The eggs and juveniles are partitioned from the host hemolymph by a secreted layer or surface coat. This surface coat is an external, extra cuticular labile layer that protects against host immunogens (Blaxter et al., 1992). Similar to the parasitic female, the juveniles likely sequester nutrients for development through their cuticle. In early -staged juveniles, there are cuticular pits on the surface which may aid in nutrient assimilation. Late -staged juveniles develop a definite c uticular layer and a functional stylet. Tomalak et al. (1988) reported juvenile Sulphuretylenchus spp. stylet feeding caused mass destruction to host beetle organs but Neoparasitylenchus Allantonema and Contortylenchus sp. caused only minor tissue damag e to host beetles. I did not observe direct damage to host tissues by stylet feeding of juvenile nematodes in my study and hypothesize the stylet of juvenile T. fuscum functions to penetrate the host alimentary tract. Juvenile T. fuscum were never observ ed in the foregut or midgut lumen of parasitized individuals. Based on this observation, I suggest
98 late -staged juvenile nematodes (J3s) enter the alimentary tract through a pocket located at the pyloric valve where the proximal region of the Malpighian tubules meet the posterior midgut and anterior hindgut. Invasion of the nematodes into this junction has been documented by Serrao et al (2008) for nematode parasites of Hypocryphalus mangiferae (Coleoptera: Curculionidae). Male T. fuscum who lack a funct ional stylet, would also be able to enter through this region. After the late -staged male juveniles enter the alimentary tract, they migrate to the hindgut and aggregate to inseminate females prior to their exodus from the posterior region of the host. F rom an evolutionary perspective, it is preferential that the female nematodes mate inside the host rather than outside in the harsh environment where they are prone to desiccation and isolation Often, more than one parasitic T. fuscum female parasitize s a thrips host which reduces consanguinity in the nematode populations Parasitism by T. fuscum cause s substantial pathological changes to female F. fusca host tissues and organs There was a significant displacement of host tissues that increased with nem atode density T he tissues most significantly affected by T. fuscum parasitism were the fat body and reproductive tract First, fat bodies were reduced and replaced with large dense vacuoles. In addition, there was an obvious depletion of host glycogen i n the fat bodies that was simultaneous with an observed glycogen increase in juvenile nematodes. Fat body is likely metabolized by the nematodes for energy, resulting in a significant reduction in this tissue Parasite -induced depletion of host fat body tissue is a common outcome of parasitism ( see Bailey and Gordon, 1973; Condon and Gordon, 1977; Schmidt and Platzer, 1980; Tomalak et al., 1984, 1990). The transfer of glycogen from F. fusca to T. fuscum suggests the parasite utilizes host glycogen as a c arbohydrate/glucose source for body energy. Cheng and Snyder (1962) reported that cells
99 recently involved in glycogen digestion have aggregates of glycogen granules that form amorphous masses in the cytoplasm of host snail cells. As trematode parasitism progresses, host cell glycogen is depleted and an increase of glycogen within the parasites is observed. Decreased levels of glycogen or glycogenesis in fat body has also been reported for Schistocerca gregaria infected with Mermis nigrescens (Gordon and Webster, 1971), larval black flies Prosimulium miuxtum P. fuscum and Simulium venustum infected with Neomesomermis flumenalis (Condon and Gordon, 1977), mosquito Culex pipiens infected with Romanomermis culicivorax (Schmidt and Platzer, 1980), and Lymant ria dispar infected with the microsporidium Vairimorpha sp (Hoch et al., 2002). The dense vacuoles replacing contents of the fat body may be excretory T. fuscum waste product Cheng and Snyder (1962) reported similar large clumps of granular ma terial in snails infected with trematodes which without doubt represent cercarial excreta The second and most pronounced change induced by Thripinema i n its F. fusca host is a reduction in size and shape of the reproductive organs (Lysaght, 1937; Green and Parel la, 1995; Loomans et al., 1997; Arthurs and Heinz, 2003). Thripinema fuscum operating as a parasitic castrator, causes a rapid sterilization of parasitized thrips (Sims et al., 2005). The time frame to induce sterility depends upon the stage is parasi tized; adult female F. fusca parasitized as larvae do not produce any eggs whereas females parasitized as adults stop laying eggs within 2 3 days (Sims et al., 2005). The physiological mechanisms driving sterility are not well understood in this genus (bu t see Chapter 6) Lysaght (1937) suggested that Thripinema rendered female thrips sterile by either depriving thrips of protein required for normal development or by secreting a toxin that damages the reproductive organs. Hocking (1967) proposed that juv eniles stopped oogenesis by directly feeding on the reproductive organs or associated tissue, a term otherwise
100 known as mechanical castration. Green and Parrella (1995) speculated that massive numbers of juvenile nematodes in the abdomen stimulate stretch receptors signaling the ovaries to halt oogenesis as if maximum egg capacity had been attained. Sims et al. (2005) concluded that the parasitic T. fuscum female is responsible for stopping oogenesis because sterility is induced before the production of j uveniles in the host hemocoel. Numerous families within the Nematoda have been reported to induce partial or complete sterility in their insect host [e.g., Sphaerulariidae and Musca autumnalis (Treece and Miller, 1968), Scolytus ventralis (Ashraf and Berr yman, 1970a,b), Vespa simillima (Sayama et al., 2007); Allantonematidae and Scolytus spp. (Oldham, 1930), Dendroctonus pseudotsugae (Thong and Webster, 1975), Hypothenemus hampei (Castillo et al., 2002); Neotylenchidae and Sirex noctilio (Bedding, 1972)], but few mechanisms for the observed sterility have been provided. Since pathogens and their host share the same resources, theory suggests that a parasitized host is forced to convert fecundity resources toward sustaining infection (longevity) or that the parasite consumes host resources that would otherwise be used for host reproduction (Beckage, 1985; Hurd, 1990; Bonds, 2006). Perlman and Jaenike (2003) cross infected Drosophila spp. with different allopatric and sympatric Howardula spp. and concluded t hat sterility is host determined because an individual host response to infection (measured by degree of sterility) was the same regardless of the infecting nematode species. Roseler and Roseler (1973), who determined that the yolk proteins of parasitized queen Bombus terrestris are of a lower concentration than those of the nonparasitized conspecifics, suggested that the nematode causes injury to the yolkproducing corpora allata gland. Tenebrio molitor L. beetles parasitized by the rat tapeworm exhibit reduced fertility and fecundity and an increased lifespan (Hurd et al., 2001). The metacestodes produce a small effector peptide(s) that stimulates the tr anscription of vitellogenin mRNA but suppresses
101 translation resulting in decreased levels of vitell ogenin in parasitized Tenebrio females (Webb and Hurd, 1999; Warr et al., 2006). In addition, components transiently present in the hemolymph retard the uptake of yolk proteins by the ovarian follicles (Major et al., 1997). Studies have suggested the uni dentified effector(s) disrupt hormonal activity (Webb and Hurd, 1995). In addition to inhibiting vitellogenin synthesis, the tapeworms also induce resorption of the developing eggs that results from the induction of an apoptotic program in the fat body ce lls (Warr et al., 2005, 2006). Similarly, I found that parasitized F. fusca females had a significant reduction in fat body and apoptosis in the follicle cells surrounding the ovarioles. Based on these observations, I suggest T. fuscum nematodes deplete fat body (as described above) which reduces the synthesis of vitellogenin necessary for oocyte development and maintenance (see Chapter 6) Alternatively, the endosymbiont Wolbachia localized in host ovarioles may be dictating the reproductive biology of parasitized F. fusca (see Chapter 2). The mechanism T. fuscum utilizes to escap e detection by F. fusca probabl y involves deceiving the non -self recognition of the host immune system. Vetter et al. (1978) and others (Smith et al., 1981; Smith et al., 1983; Edwards et al., 1990; Politz and Philipp, 1992; Rockey et al., 1 983; Fattah et al., 1986; Badley et al., 1987) reported nematodes can evade host immune factors by sloughing off their cuticle and any surface -bound antibodies. Mastore and Bri vio (2008) added both insect parasitic Stein ernema feltiae nematodes and their isolated cuticles to cultures of Galleria mellonella larval hemocytes and discovered that the parasite cuticle is essential for immunoevasion. Through a series of e laborate assays, they show that S. feltia e cuticular lipids bind host proteins and create a coat or disguise mechanism against host hemocyte recognition The cuticular surface of T. fuscum is dynamic and responsive to the host as demonstrated by the transformation of the infecti ve to the parasitic female. There were few
102 hemocytes observed in the hemolymph and phagocytosis, encapsulation, nodule formation, or melanization of nematodes in the host hemocoel was not observed Scanning electron micrographs did show host factors atta ched to the cuticle of all T. fuscum stages In many instances, the juvenile T. fuscum nematodes were partitioned from the host hemolymph by an exocellular membrane T h ese sequestration mechanism s p otentially protect the nematodes from the up-regulated t hrips cellular and humoral defense systems How ever, further studies using cytochemical stains and/or cryo-sectioning and antibody or lectin probes are needed to determine the biochemical properties of the T. fuscum surface layer and its function in immune evasion (see Chapter 6). In summary, parasitism by T. fuscum induces numerous morphological and behavioral alterations (e.g., sterility, a reduction in feeding and vector competence) to its F. fusca host (see Sims et al., 2005 and Chapters 4 and 5) The ability of the naturally -occurring T. fuscum to induce these alterations without significantly impacting longevity or mortality make this nematode an important candidate for use in thrips integrated management strategies.
103 Table 3 1. Procedure for prepar ation of insect tissue for scanning (15) and transm ission (16) electron microscopy. Step Stage Chemical Time 1 Fixation 2.5% gluteraldehyde 2 hrs @ RT or overnight @ 4C 2 Buffer wash 0.1M Cacodylate buffer 3X @ 15 min. each 3 Post fixation 1% osmium tetroxide 2 hrs @ RT or overnight @ 4C 4 Water wash ddH 2 0 3X @ 15 min. each 5 Dehydration 10% e thanol 10 min 30% e thanol 10 min 50% e thanol 10 min 70% e thanol 10 min or overnight @ 4C 80% e thanol 10 min 90% e thanol 10 min 95% e thanol 10 min 100% e thanol 15 min 100% e thanol 15 min 100% a cetone 15 min 100% a cetone 15 min 6 Infiltration 25% resin/75% absolute acetone 4 hrs or overnight @ RT 50% resin/50% absolute acetone 4 hrs @ RT 75% resin/25% absolute acetone 4 hrs @ RT 100% resin 4 hrs or overnight @ RT 100% resin 6 hrs @ RT
104 Figure 3 1. The external dorsal view of the Frankliniella fusca female head tagma with antennae, compound eyes, and ocelli.
105 Figure 3 -2 The m outhparts of a Frankliniella fusca fem ale. (A) Ventral view showing the mouth cone (arrow); (B) A scanning electron micrograph of the tip of the mouth cone showing the numerous sensory pegs and stylet; (C) Maxillary s tylet with labral pad and food canal
106 Figure 3 -3 The two wing mor phs of Frankliniella fusca (A C) Scanning electron micrograph s showing the wings of a macropterous female. The wings are fr inged, with fine long setae; (D F) Scanning electron micrographs showing the wing pads of a brachypterous female; (G) Macropterous male (left) and female (right) F. fusca ; (H) Brachypterous male (left) and female (right) F. fusca
107 Figure 3 4. Scanning electron micrographs documenting the posterior opening of a Frankliniella fusca female. (A E) Various views of the females genital opening and saw -like ovipositor (arrows) used to cut slits into plant tissue and deposit eggs; (F) The retracted ovipositor inside the abdomen of a F. fusca female (arrow). .
108 Fig ure 3-5. A longitudinal thick section of a healthy Frankliniella fusca female. The female has an abundance of muscle in the thorax, a convoluted alimentary tract, numerous lobes of fat body, and a robust reproductive system with ovarioles containing lipid -filled eggs and an ovipositor .
109 Figure 3 6. The ovoid and tubular salivary glands of a Frankliniella fusca female. (A) A gross dissection documenting the ovoid salivary glands (osg) proximal to the esophagus in the thoracic region and tubular salivary glands (tsg) running parall el to the foregut; (B) A longi tudinal thick section showin g the ovoid salivary gland; (C D) Transmission electron micrographs of the tubular salivary gland (tsg) with microvilli (mv) and numerous secretory vesicles (sv). tr=trachea, bm=basement membrane, cu=cuticle.
110 Figure 3 7. Transmission electron micrographs of Tomato spotted wilt virus in the ovoid salivary glands of a viruliferous Frankliniella fusca female. (A B, D E) Progressive enlargement of TSWV virions released from the salivary gland cell cytoplasm into the lumen; (C,F) Clusters of TSWV virions in the ovoid salivary gland cell of an infected female. Arrows point to a group of enveloped virions in the cytoplasm.
111 Figure 3 -8 Digestive tract of the Frankliniella fusca female. (A) A longitudinal thick section showing the foregut, midgut, and hindgut; (B) A l ongitudinal section through the thorax showing the cuticle lined foregut; (C) A longitudinal section through the a bdomen showing the midgut coiled around the surrounding reproductive organs; (D ) A l ong itudinal thick section through the abdomen showing the convoluted midgut and midgut lumen.
112 Figure 3 9. Transmission electron micrographs showing midgut cells of the Frankliniella fusca female. (A) Midgut epithelium with well -organized brush border ( mv) extending into the gut lumen (lu) and regenerative cell Note the well defined nucleus (n) and trachea (tr) apposing the basement membrane; (B) Midgut epithelial cell with invaginations (in) of the basement membrane (bm) and microvilli (mv) on the a pica l face forming a brush border; (C) Typical epithelial cell of a F. fusca midgut. Insects secrete secretory vesicles (sv) from the midgut cells to the lumen to help digest meals. Cells undertaking constitutive secretion are often characterized by sui table amounts of golgi bodies (g) These golgi complexes consist of parallel cistern and numerous small vesicles ; (D) Transverse section through the microvilli of a F. fusca midgut with mitochondria (mt) and glycogen (gly) deposits
113 Figure 3 10. Ma lpighian tubules of the Frankliniella fusca female. (A) F our Malpighian tu bules extend from the digestive tract at the pyloric region between the posterior midgut and anterior hindgut. The two anterior tubules are adhered for a short distance to the midgut and the two posterior tubules are connected to the ovarioles via small ligaments; (B) A transmission electron micrograph of a transverse section through a F. fusca Malpighian tubule. Microvilli (mv) form the brush border lining the lumen (lu) and t he basal surface of the cells rest on a basement membrane (bm). The Malpighian tubules a re surrounded by hemolymph (he) ; (C) U ric acid crystal (ua) within the cytoplasm of a Malpighian tubule. These crystalline spheres are formed from nitrogenous waste (inorganic salts, uric acid, urates, proteinaceous material, etc.) and are eventually released into the tubule lumen. Invaginations of the plasma membrane (in) allow for elasticity and an increase in surface area for exchanging ions a nd molecules. mt=mitochondria.
114 Figure 3 11. Transmission electron micrographs showing the hindgut of Frankliniella fusca (A) The general rectal wall consists of a thin epithelial layer (ep) lined with cuticle (cu). Note the glycogen granules (gly) in the rectal epithe lium ; (B) The h indgut with vacuoles (v) muscle fibers (mf), and glycogen aggregates in the rectal epithelium. Note the cuticular intima (arrows) lining the hindgut lumen (lu) ; (C) The h indgut wedged between a developing oocyte (ooy) and a rectal pad (rp). T he hindgut lumen (lu) is filled with numerous bacteria (b) (see Chapter 5); (D) The rectal pad (rp) contains numerous pleomorphic mitochondria (mt) muscle fibers (mf), and nuclei (N) along the cuticle -l ined border.
115 Figure 3 12. The genital r egion of a Frankliniella fusca female. The apical bulb of the accessory gland is connected to the pyloric region via small ligaments. The spermatheca is a dhered closely to the vagina and ovipositor.
116 Figure 3 13. Longitudinal thick sections of the fe male Frankliniella fusca host documenting egg development. (A) A pre -vitellogenic female and a (B) fecund female The reproductive structures are robust, with the ovarioles extending to the first abdominal segment and numerous eggs in the ovaries. Note how the well -developed ovaries flank the defined alimentary tract in healthy females.
117 Figure 3 14. Scanning electron micrographs of the abdominal cavity of a Frankliniella fusca female with egg. (A B) A deve loping egg (arrows); (C) Interior struc ture of the developing egg (arrow); (D) Y olk protein (arrow) inside the egg.
118 Figure 315. The developing oocytes of a fecund Frankliniella fusca female. (A C) The progressive magnification of the follicle cells and developing oocytes. The follicle cells are distinct with prominent and welldefined nuclei (n). Figure 3 16. The progressive magnification of connection s between a follicular cell and developing oocyte of a non-parasitized Frankliniella fusca female (A) Developing oocyte; (B) Nucleus in the follicle cells and organelles within the cytoplasm of the oocyte; (C D) The zipper like or ladder like connections between the two layers are strong and distinct.
119 Figure 3 17. The fat body of a Frankliniella fusca female. The fat body is composed of large lipid droplets ( asterics ) and small dense glycogen granules (arrowheads). Figure 3 18. The musculature of Frankliniella fusca (A) A t hick section show ing the various muscles in the thoracic cavity of F. fusca ; (B) A longitudin al section of the thoracic muscle. The I band (I) contains thin actin filaments that extend from the Z line (Z) to the thick myosin filaments (A, A band). The arrow represents the lighter zone where the thick myosin filaments are in the absence of the ac tin filaments. Trachea (tr) typically indent the cell membrane of the muscle cells, thus minimizing the distance for oxygen diffusion. nf=nerve fibers; mt=mitochondria; (C) Thoracic muscles of F. fusca An abundant supply of mitochondria (mt) is neede d to fuel the muscle cells for flight. The circular fiber bundles (mf) are enclosed by the sarcolemma (sl) and the dark lines within the circular fibers are likely sarcoplasmic reticulum (sr). Z=z lines.
120 Figure 3 19. Scanning electron micrographs documenting the external surface of the infective Thripinema fuscum female (A) Ventral view of a T. fuscum female; (B E) Side profile of a T. fuscum female with lateral lines extending anterior to posterior ends; (F) Anterior view of an infective T. f uscum female. Note the well defined cuticular structure in all micrographs. Figure 3 20. Transmission electron micrographs showing the cross -section of an infective Thripinema fuscum female with lateral lines (arrows). (A B) Progressive magnificati on of the lateral lines of a developing nematode.
121 Figure 3 21. Differential interference contrast microscopic image (DIC) of an infective Thripinema fuscum female with anterior esophageal region. Figure 3 22. The cephalic region of the infective female Thripinema fuscum (A B) Enface view showing square mouth plate and stylet lumen ( black arrows); (C D) Lateral view show ing the labial region with lips ( white arrows). Note the well -defined cuticular structure in the micrographs.
122 Figure 3 23. Scanning electron micrographs of the excretory pore of an infective Thripinema fuscum female. (A) The excretory pore is located on the ventral side beneath the esophageal region; (B) A higher magnification of the excretory pore and surrounding pore c uticle.
123 Figure 3 24. D ifferential interference contrast microscopic images (D IC) of the parasitic Thripinema fuscum female (A, D) Dorsal view of a fecund T. fuscum female; (B) A fecund T. fuscum female with two eggs; (C) Two parasitic T. fuscum females; (E) A fecund T. fuscum female with egg emerging from the uterus; (F) A montage image of the parasitic T. fuscum female
124 Figure 3 25. Scanning electron micrographs of the cuticular structure of the parasitic Thripinema fuscum female. (A) Dorsal view of a parasitic T. fuscum female with slightly everted uterus; (B H) Progressive magnification of the cuticular structure of the T. fuscum female documen ting the pit like structures (F G) and knoblike projections (E H).
125 Figure 3 26. Tr ansmission electron micrographs documenting the cuticular structure of the parasitic Thripinema fuscum female. (A) Cuticle of the parasitic female with microvilli (arrow); (B D) Various micrographs showing the microvilliated cuticle; (E F) Knob -like projections extending from the cuticle of the parasitic T. fuscum .
126 Figure 3 27. Differential interference contrast microscopic images (DIC) of the Thripinema fuscum male. (A D) Arrows point to the copulatory structures of the bursa (arrow) and guber naculums (arrowhead ), a sclerotized structure used for guiding the spicule. Males are easily differentiated from females by their curved body structure.
127 Figure 3 28. Scanning electron micrographs of the Thripinema fuscum male. (A) Dorsal view of th e free -living male; (BC) Bursa or caudal alae used for grasping a female; (DF ) Progressive magnification showing the posterior region of a male with bursa, gubernaculum, and spicules
128 Figure 3 29. Differential interference contrast microscopic i mages (DIC) of the Thripinema fuscum juveniles. The juveniles develop from the short a nd wide phenotype to the (AB) long and slender phenotype (C F).
129 Figure 3 30. A collage of scanning electron micrographs documenting the various juvenile annulated c uticles of Thripinema fuscum
130 Figure 3 31. A collage of transmission electron micrographs showing the various cuticular structures of juvenile Thripinema fuscum
131 Figure 3 32. Transmission electron micrographs showing the morphological structure of the juvenile Thripinema fuscum cuticle. (A) The cuticle of a juvenile T. fuscum nematode is separated from its ho st by an exocellular substance; (B D) The cuticular structures of different aged T. fuscum juveniles. The cuticular pits (cp) likely ar e used to assimilate nutrients through the hemolymph, similar to the microvilli of the parasitic females. ecl=extra cortical layer; icl=inner cortical layer; hyp=hypodermis; cp=cuticular pits; sl=striated layer; mf=muscle fibers.
132 Figure 3 33. Scannin g electron micrographs of the anterior region of the juvenile Thripinema fuscum (A F ) Various orientations of the anterior T. fuscum region with stylet lumen and lips visible ; (G H) Enface view of T. fuscum ; (I J) Stylet protr uding from stylet lumen
133 Figure 3 34. Thripinema fuscum eggs (A) Differential interference contrast microscopic ima ges (DIC) of eggs; (B) Transmission electron micrograph of curled embryo with chorion inside the abdomen of a Frankliniella fusca host.
134 Figure 3 35. Scanning electron micrographs of the Thripinema fuscum eggs. (A) Dorsal view of egg; (B -C) Progressive magnification of the chorion structure of (A); (D F, G I) Egg with progressive magnification of the chorion; (J K) Egg with protuberances; (M O) Various orientations of nematode embryos within the egg chorion.
135 Figure 3 36. Longitudinal thick sections documenting the in vivo life cycle of Thripinema fuscum in the female Frankliniella fusca host. (A) Day three post -parasitization with parasi tic T. fuscum female lodged against midgut; (B) Day five post -parasitization with T. fuscum eggs and early -staged juveniles in the host hemocoel ; (C) Day nine post parasitization with all stages of T. fuscum present in the host hemocoel. Note the nema tode aggregate form ing a peristoferous vesicle in the abdomen.
136 Figure 3 37. Longitudinal thick section of a parasitized Frankliniella fusca female with juvenile nematodes throughout the host tagma. (A) A juvenile located in the head (arrows) and (B) thoracic tagma. The majority of the juvenile nematodes reside in the abdomen.
137 Figure 3 38. The life cycle of Thripinema fuscum The eggs hatch and develop through three juvenile stages (J1J3) before emerging as fourth stage males (J4 and infectious females (J4 The infectious female parasitizes a Frankliniella fusca female in peanut and develops into a parasitic female (P
138 Figure 3 39. A comparison of a non-parasitized and a Thripinema fuscum parasitized Frankliniella fusca female. (A) Dorsal view of the non -parasitized (top) and parasitized (bottom) female; (B) Gross dissection of a non-parasitized F. fusca female with eggs; (C) Gross dissection of a parasitized F. fusca female with three parasitic T. fuscum fema les and progeny. Figure 3 40. A longitudinal thick section of a Thripinema fuscum parasitized Frankliniella fusca female. Parasitism results in a displacement of the alimentary tract, depletion of fat od y, atrophy of ovari es, and the accumulation of dense vesicles. Numerous nematodes fill the body cavity.
139 Figure 3 41. Scanning electron micrographs showing the internal abdominal cavity of a Frankliniella fusca female parasitized by Thripinema fuscum (A D ) Juvenile T. fuscum are aggregated lon gitudinally in the host hemocoel; (E F) This aggregation compresses the midgut (arrow) forming depressions in the alimentary tract.
140 Figure 3 42. Transmission electron micrographs documenting the parasitic Thripinema fuscum female in direct appositi on to host midgut cells. (A) Parasitic female (P against basement m embrane (bm) of midgut cell; (B D) Magnification of parasitic female against midgut cells with microvilli (arrows) touching basement membrane ; (E H) Parasitic female with ovar y (ov) closely adhered to the host midgut cells n =nucleus.
141 Figure 3 43. Transmission electron micrographs showing the midgut cells of a Thripinema fuscum parasitized Frank liniella fusca female. (A) Juvenile nematodes (arrows) located at the basem ent membrane of the host midgut (mg) with vacuoles (v) ; (B) M idgut epithelial cells with nucleus (n) and microvilli (mv) on apical surface forming the gut lumen; (C) M idgut epithelial cell with well organized brush border (mv) extending into the gut lu men insert shows autophagic body ; (D) M idgut lumen with perimicrovillar membrane (pm) and microvilli ; (E) Progressive magnification of D; (F) Longitudinal and transverse section through the microvilli with microfilament bundles (long arrow) and gly cocalyx (arrowhead).
142 Figure 3 44. Scanning electron micrographs documenting the accumu lation of late -staged juvenile nematodes in the hindgut of Frankliniella fusca females (A C) Progressive magnification of juvenile orgy i n the hindgut; (D E) Pr o gressive magnification of male with caudal alae (=bursa) potentially inseminating a female in the hindgut; (F) Gross dissection of posterior abdomen of a F. fusca female with aggregation of nematodes forming an orgy (arrow) in the hindgut.
143 Figure 3 45. Transmission electron micrographs showing the hindgut of a Thripinema fuscum parasitized Frankliniella fusca female. (A B) Late -staged juveniles (arrows) accumulate within the hindgut lumen which is characterized by the cuticular intimal lining (cu); (C) The hindgut lumen of a parasitized F. fusca female is free of bacteria but with excretory product; (D) Progressive magnification of the excretory product in (C). n=nucleus.
144 Figure 3 46. Scanning electron micrographs documenting a Thripinema f uscum juvenile in side the posterior region of a Frankliniella fusca female. (A C) Various images of the juvenile (arrows).
145 Figure 3 47. Transmis sion electron micrographs of the Malpighian tubule from the Thripinema fuscum parasitized Frankliniella fu sca (A) The tubule (mt) with secretory vesicles (sv) is displaced against host tissues as a result of the numerous juveniles in host hemocoel ; (B) Parasitized F. fusca have an a ccumulation of secretory vesicles (sv) and uric acid (ua) crys tals in the cell cytoplasm; (C D) Progressive magnification of the waste product in the tubule
146 Figure 3 48. Gross dissection of the reproductive structures of a non-parasitized and Thripinema fuscum parasitized Frankliniella fusca female. (A) The non -parasit ized F. fusca female has a robust reproductive system with fully mature eggs in the ovary (egg); (B) The parasitized F. fusca female has a reduced reproductive system with an undeveloped ovary (ov). Note the ovary of (B) parasitized F. fusca is half the size of (A) nonparasitized F. fusca a p=apical bulb, sp=spermatheca.
147 Figure 349. Transmission electron micrographs showing a displacement of the female Frankliniella fusca developing oocytes as a result of parasitism by Thripinema fuscum (A F) Juvenile nematodes in the hemocoel (arrows) push against oocyte and alter their shape and function Sterility is the outcome of infection. Figure 350. The progressive magnification of the connection betwe en the follicular cells and developing oocyte i n the Thripinema fuscum parasitized Frankliniella fusca female. (A) Nucleus in the follicle cell surrounding an oocyte; (B) The zipper -like or ladder -like connections between the oocyte and follicle cell are torn; (C) Apoptosis in the follicle cell o f an oocyte characterized by a migration of heterochromatin to the edge of the nucleus and a poorly-defined nucleus.
148 Figure 3 51. Transmission e lectron micrographs showing f at body of the Thripinema fuscum parasitized Frankliniella fusca female. (A B) Electron dense vesicles are abundant in the fat bodies underlying the thrips cuticle ; (C D) There is a n obvious depletion of glycogen (arrows) in the fat bodies of parasitized F. fusca. Figure 3 52. Transmission electron micrographs showing an accumu lation of lipid and glycogen in the juvenile Thripinema fuscum (A D) Glycogen (gly) and lipid (l) deposits in juvenile Thripinema fuscum nematodes are likely seque stered from the host fat body.
149 Figure 3 -53 Transmission electron micrographs documenti ng the Thripinema fuscum parasitized Frankliniella fusca musculature (A B) Progressive magnification of the muscle fibers (mf) and mitochondria (mt) with the presence of numerous (C) glycogen granules; (D ) Ruptured mitochondria in the muscle fibers of parasitized F. fusca despite otherwise properly fixed tissue; (E F) Progressive magnification of the ruptured mitochondria.
150 Figure 3 54. Scanning electron micrographs of the fractured Thripinema fuscum parasitized Franklini e lla fusca documenting th e partition between the host -p arasite interface. (AD) Eggs part itioned from host tissues by an exocellular substance (arrows); (E I) Juveniles are partitioned from the host tissues by the same exocellular substance (arrows).
151 Figure 3 55. Transmis sion electron micrographs documenting the exocellular substance secreted by juvenile Thripinema fuscum juveniles (A E) The exocellular substance forms a sheath which protects the nematode from host tissues (arrows); (D) T he secreted substance likely pr otects against host immune factor s.
152 CHAPTER 4 THE IMACT OF A PARAS ITIC NEMATODE THRIPINEMA FUSCUM ON THE FEEDING BEHAVIOR AND VECTOR COMPETENCE OF FRANKLINIELLA FUSCA Introduction The tobacco thrips, Frankliniella fusca (Hinds) (Thysanoptera: Thripidae), is a polyphagous insect pest that feeds on agriculturally important plants in North America (Lewis, 1997). It is one of at least seven thrips species capable of transmitting Tomato spotted wilt virus (TSWV, Bunyaviridae: Tospovirus) in crops such as groundnut tobacco, tomato, pepper, as well as in numerous ornamentals, grasses, and weeds (Parrella et al., 2003; Whitfield et al., 2005). Frankliniella fusca populations contain both brachypterous and macropterous adults with the proportion of each wing for m changing seasonally (Chamberlin et al., 1992). Ecologically, wing form plays a role in thrips dispersal (Chamberlin et al., 1992; Groves et al., 2003), yet no studies have investigated if wing form has a direct effect on TSWV replication and transmissio n. Transmission of T ospoviruses by viruliferous adult thrips is the only significant form of inoculation during natural epidemics and the transmission rate is dictated primarily by the feeding behaviors exhibited by the respective vector (Culbreath et al ., 2003; Ananthakrishnan and Annadurai, 2007). TSWV is transmitted in a propagative manner by thrips (Ullman et al., 1993; Wijkamp et al., 1993). Acquisition of TSWV occurs when first and early second instars feed on infective plant tissue, and transmission occurs via late instar and adult thrips that have acquired the virus as larvae (van de Wetering et al., 1996; Wijkamp et al., 1996b). Larvae and adults feed by piercing plant cells and sucking out the cell fluids, which produces a silvery scarri ng on the plant tissue (Hunter and Ullman, 1989). It has been difficult to resolve a consistent pattern in the pathological effects of tospovirus infection on thrips as there have be en reports of negative (Stumpf and Kennedy, 2005), neutral (Wijkamp et al., 1996a), and positive (Maris et al., 2004; Stumpf and Kennedy, 2007) effects on thrips. Recent work has shown
153 genetic variation of both thrips populations and Tospovirus isolates, as well as the quality of infected host plant tissue, are important factors in pathogenicity ( Stumpf and Kennedy, 2005; Stumpf and Kennedy, 2007). Because vector transmission varies based on both the quality and quantity of feeding of thrips (van de Wetering et al., 1998; van de Wetering et al., 1999), I hypothesize manipulation of fe eding behaviors may provide an avenue for decreasing the spread of Tospoviruses in agroecosystems. The insect parasitic nematode Thripinema fuscum Tipping and Nguyen (Tylenchida: Allantonematidae) renders female F. fusca sterile without any negative effects on their survival (Sims et al., 2005). Parasitism significantly reduces the longevity of male F. fusca and the effects of T. fuscum on male reproduction are unknown (Sims et al., 2005). Parasitism by T. fuscum is initiated when a free -living female ent ers the host through the intersegmental membranes of the coxal and/or abdominal cavities (Tipping et al., 1998). Once inside the host, the parasitic female produces eggs that hatch and develop through three juvenile stages before boring into the alimentar y tract and emerging via the anus as the fourth -stage adult (Sharga, 1932). All stages of F. fusca are capable of being parasitized, with young adult females the most preferred (67%) and males least preferred (25%) in laboratory experiments (Sims et al., 2005). Parasitism of adult F. fusca has been reported to exceed 80% in field conditions (Funderburk et al., 2002). Parasitism of F. fusca larvae on groundnut averaged 49% and 28% in laboratory and field experiments, respectively (Sims et al., 2005). Th e lower rate of larval parasitism in field conditions is most likely due to differences in microhabitat between the larvae and parasitized females; larvae remain within the terminal buds of groundnut and parasitized adults typically aggregate in the flower s where they feed on pollen and where free living T. fuscum are able to easily contact new hosts.
154 Recent observations suggest that parasitism by Thripinema spp. may suppress TSWV transmission by reducing host feeding rates (Sims et al., 2005). Related research has shown that the feeding by Frankliniella occidentalis (Pergande) is suppressed significantly by the nematode Thripinema nicklewoodi Siddiqi; however, the effects of parasitism on vector competence are unclear (Arthurs and Heinz, 2003; Lim and Van Driesche, 2004). Arthurs and Heinz (2003) reported that TSWV infection of F. occidentalis did not affect susceptibility to T. nicklewoodi but fewer parasitized thrips became active transmitters and their per capita frequency of disease transmission was r educed by 50%. Alternatively, Lim and Van Driesche (2004) reported that parasitized F. occidentalis did not subsequently acquire Impatiens necrotic spot virus as readily as their non-parasitized counterparts but that rates of transmission remained the same between the two viruliferous groups. The interaction between F. fusca, T. fuscum groundnut and TSWV serves as an ideal multi -trophic system to examine the impact(s) of a chronic disease on vector competence. The first objective of this chapter was to e xamine the effects of gender, wing form, virus infection, and nematode parasitism on the feeding behavior of F. fusca The second objective was to determine what effects gender, age, and nematode parasitism have on TSWV transmis sion. From these experimen ts, I provide a framework for better understanding how the highly host specific T. fuscum parasite interfaces with plant viruses/insect vector associations. Materials and Methods Maintenance of F. fusca TSWV inoculum, and T. fuscum : Colonies of healthy a nd parasitized F. fusca were maintained as described by Sims et al. (2005). Leaves of groundnut (Arachis hypogaea L.) showing TSWV symptoms were collected in Alachua Co., Florida, and confirmed to be TSWV positive by double antibody sandwich enzyme -linked immunosorbent
155 assay (DAS -ELISA), a technique used to determine the presence or absence of TSWV by detecting viral structural (nucleocapsid) proteins (SRA 39300; Agdia Inc., Elkhart, IN). TSWV was maintained in groundnut by F. fusca transmis sion. For viru s acquisition, first instars were placed on infected tissue and allowed to feed for 24 hours. Larvae were then transferred to 15 -cm polypropylene containers with a 5 -cm diameter ventilation hole covered with fine mesh. The polypropylene containers were stored in a sealed plastic crisper lined with moist paper towel and maintained at 27 10h dark photoperiod. Fresh tetrafoliate leaves were deposited into containers every day until adult emergence. Transmission of the virus to Florunner groundnut was achieved by allowing viruliferous adults to feed for 72 h on 3 to 6 wk old healthy plants enclosed in cages (12.7 -cm diameter cl ear plastic cylinders with 6.35cm diameter screen holes). Host plants were held at 25 30 C in a greenhouse. After an incubation period of approximately 10 20 days following the end of the inoculation access period, symptomatic plants were confirmed to be TSWV positive using DAS ELISA. Effect of gender, wing form, virus infection, and nematode parasitism on survivorship, feeding behavior and TSWV transmission: Approximately 200 females wer e confined for 48 hours in four cages containing TSWV infected groundnut for oviposition. Newly eclosed first instars were allowed to feed on TSWV infected leaves for a 48 hour acquisition period to generate viruliferous thrips. After this 48 hour period, half of the larvae from each cage were transferred in groups (n = 20) to 1.5 -ml microcentrifuge tubes containing two T. fuscum -parasitized adult female F. fusca excreting nematodes and a 1 -cm diameter groundnut leaf disc. Larvae were held with parasitized females for 72 h to achieve optimal levels of parasitism. The remaining half of the larvae were transferred to tubes with two healthy adult females and a 1 -cm diameter groundnut leaf disc for 72 h to serve as non -parasitized
156 controls. Corresponding co ntrol thrips ( i.e., uninfected by TSWV) were reared in the same manner, except for being given healthy groundnut leaves rather than TSWV infected groundnut leaves. Individual larvae were then placed (after the 48 h virus acquisition period and 72 h parasit ization access period) in tubes with a fresh groundnut leaf disc until adult emergence. After thrips emerged as adults, they were each transferred individually (n = 213) to a new tube that was provisioned with a fresh groundnut leaf disc (1 cm2) with the top (adaxial) surf ace placed upward every 24 h until d eath. Immediately after each 24 h feeding period, the upper surfaces of the discs were photographed for feeding injury (Auto -Montage Pro, Syncroscopy) and analyzed (SigmaScanPro 4.02, Jandel Corporation 1995) using a modified color defined protocol of Kerguelen and Hoddle (1999) that detected the amount of silvered areas caused by thrips feeding. Background noise was accounted for by subtracting the mean pixel count of unfed control leaf discs from the pixel count of leaf discs with feeding injury. Pixel counts were converted to area of feeding (in mm2). After measurements were taken, leaf discs were placed on sterile water in 24 -well plates for 5 days at 25 C to amplify the virus titer. Leaf discs we re then stored at 70 C until analysis by DAS ELISA. Leaf discs were recorded as positive if their ELISA (optical density) value was greater than the determined threshold value for the plate (mean of control leaf disc readings + 3[SD]). At death, thrips w ere dissected and the gender, wing form, and presence of T. fuscum were recorded. Dissected thrips were then stored individually in tubes with 150 l of PBS -PVP buffer at 80 C until further analysis could be conducted using antigen coated plate (ACP) -Ind irect ELISA to confirm presence of viral replication in adults. The monoclonal antibody probes (Ascites cell lines 1C1A7 lot# A16714 and ascites cell line 6B1C1 lot# A16779; Agdia Inc.
157 Elkhart, IN) used in these assays detect a non-structural protein enco ded by the small RNA segment of TSWV, thus differentiating thrips in which the virus is actively replicating and are capable vectors from those that have merely ingested the virus by feeding on infected plant tissue (Bandla et al., 1994). Statistical analyses: To determine survivorship rates, thrips were classified according to gender and T. fuscum parasitism, giving four treatment groups for non -viruliferous thrips (non parasitized females, parasitized females, non -parasitized males, parasitized males) (n = 144). However, because only two parasitized, viruliferous males, which died within one day, were obtained, only three treatment groups were available for viruliferous thrips (non -parasitized females, parasitized females, non -parasitized males) (n = 67) This classification enabled us to make similar analyses for viruliferous and non-viruliferous thrips. Survival distribution curves according to gender, virus infection and nematode parasitism were generated using Kaplan Meier estimates (Proc LIFETEST, S AS 2004), and the Cox Proportional hazards model (Proc TPHREG, SAS 2004) was used to determine how survival rates of adults differed among the four treatment groups of non-viruliferous thrips (non parasitized females, parasitized females, non -parasitized m ales, parasitized males) and among the three treatment groups of viruliferous thrips (non -parasitized females, parasitized females, non -parasitized males). Comparisons were made to examine the impact of parasitism status and gender on host longevity. For the feeding and transmission analyses, a subset of 10 individuals per treatment (5 of each wing form) that survived 10 d or more were randomly selected (RANDBETWEEN function, Excel 2000). It was not possible to determine the parasitism and viral status of thrips until after all experiments had been conducted, therefore I was unable to have an equal number
158 of thrips for all categories. The effects of independent variables on the amount of feeding were assessed using a mixed model repeated measures ANOVA wit h data normalized via a log (y+1) transformation before analysis (Proc MIXED, SAS 2004). The daily amount of feeding for each individual thrips was the repeated measure. Because of the potential serial correlations in feeding within each thrips, I used a first order autoregressive covariance structure in the models. Due to the lack of viruliferous males parasitized by T. fuscum initial analyses were run separately for females and males to determine the effects of wing form, virus infection and parasitis m on feeding injury. These initial models showed that wing form and virus infection did not affect feeding by males and females and were therefore taken out of the analyses. Further analyses (slice option within Proc MIXED, SAS 2004) were conducted to de termine the effects of gender and nematode parasitism, and their interaction on feeding over time. To observe transmission over time, m ean cumulative distribution plots were generated for each treatment to (Proc RELIABILITY, SAS 2004). To determine if ove rall rates of transmission varied ( i.e ., number of days with transmission/total days of adulthood), comparisons of transmission frequencies among the three groups of viruliferous thrips (nonparasitized females, parasitized females, non -parasitized males) were conducted using a generalized linear mixed model ANOVA (Proc GENMOD, SAS 2006). In this case, transmission was a binary response variable because each observed leaf disc was either infected or not infected. The generalized linear mixed model account ed for the response variable being expressed as the proportion of leaf discs infected relative to the number of leaf discs observed for each individual (Madden et al., 2002). Pairwise comparisons among the treatment groups were made using the LSMeans opti on.
159 To determine if the likelihood of transmission changed over the course of a thrips lifetime, and if this was related to thei r gender or parasitism status, I analyzed the gap times from one transmission event to the subsequent one for the three types of viruliferous thrips (non parasitized females, parasitized females, non -parasitized males) [(Johnston and So, 2003; Nelson, 2003) Proc TPHREG, SAS 2004]. The robust sandwich estimate for the covariance was used to account for potential correlations in ga p times within individuals. To determine if the amount of feeding each day affected the likelihood of a thrips t ransmitting TSWV on that day, I analyzed if the probability of transmission increased with the amount of feeding for each of the three virulifer ous thrip s states. For these analyses, I used a logistic analysis, with a repeated effect being the 10 daily observations for each thrips (Proc GLIMMIX, SAS 2006). Amount of feeding was log transformed before analysis. Analyses were conducted separately for the three groups of viruliferous thrips because of differences in the amount of feeding among the groups (see results below). Results Survivorship: The longevity of nonviruliferous thrips was first compared to determine if T. fuscum parasitism affec ted the longevity of females and males (Figure 4-1A). Comparisons of the longevity of parasitized and non -parasitized thrips of each gender showed that females did not differ in their longevity ( =0.08, P = 0.78), but parasitism led to a significant reduction in longevity among males ( = 16.77, P < 0.0001) (Table 41). The proportional hazard ratios were similar between each comparison except for parasitized males; there was a 78% chance that a parasitized male would die before a non -parasitized male. The survival distribution graphs showed no difference in longevity between non-viruliferous and viruliferous F. fusca cohorts, however, the onset of mortality occurred earlier for viruliferous t hrips than for non -viruliferous
160 cohorts (Figure 41). Longevity did not differ between viruliferous non-parasitized ( = 0.55, P = 0.46) and parasitized ( = 0.67, P = 0.41) female thrips (Figure 41B, Table 1). Feeding behavior: The feeding rates for female F. fusca were much higher than for males (F1,60 = 119.34, P < 0.0001) (Figure 42). There was a significant in teraction between thrips gender and parasitism status (F1,60 = 16.24, P = 0.0002) on the feeding behavior of F. fusca adults indicating that the effect of parasitism on feeding differed between females and males. Parasitized females fed significantly less than non -parasitized females (F1,38 = 54.82, P < 0.0001), however there was no significant difference in the feeding rates of parasitized and nonparasitized males (F1,22 = 1.25, P = 0.28). There were no significant differences in the mean feeding rates within genders between brachypterous and macropterous individuals or within genders between viruliferous and non viruliferous individuals (Table 4-2). Because the 3 -way interaction between gender, parasitism and time (F9,540 = 0.84, P = 0.58) was not signi ficant and the resulting model with two-way interactions had a lower Akaikes Information Criterion, the 3 -way interaction was deleted from the analysis. The lack of an interaction suggested that differences in feeding between parasitized and non parasiti zed thrips within each gender were consistent over time. There was not a significant gender by time interaction (F9,540 = 1.33, P = 0.22), indicating that females and males showed similar dayto -day variation in feeding (Figure 4-2). Transmission rates: There were significant differences in transmission rates of TSWV among the three different groups of viruliferous thrips ( = 46.49, P < 0.0001). Non-parasitized females transmitted more than non -parasitized males ( 1 = 19.54, P < 0.001). Parasitism significantly reduced virus transmission by females ( 1 = 41.48, P < 0.0001). There were no significant differences in tran smission rates of parasitized females and non -parasitized males ( 1
161 = 1.52, P = 0.22). There were significant differences in the gap times between transmissions among the three groups of viruliferous thrips ( 13.78, P = 0.001), with viruliferous nonp arasitized females having a 67% and 74% chance of transmitting before a viruliferous parasitized female and a viruliferous non -parasitized male, respectively. There were also significant differences in the time during the thrips life that transmission occ urred ( = 41.34, P < 0.0001). The mean cumulative distribution plots showed for all treatments that the frequency of transmissions decreased with age; older thrips did not transmit as efficiently as younger thrips (Figure 43, Table 43). The hazard ratio for the overall comparison was ~0.90, so the likelihood of a thrips transmitting TSWV decreased about 10% for each additional day of adulthood. This decline in transmission with age was consistent across categories of thrips ( = 0.76, P = 0.68), reinforci ng the idea that parasitism reduced transmission throughout adulthood. Nonparasitized females transmitted at a greater rate than both parasitized females and non -parasitized males, and these differences were consistent throughout the thrips lifetime (Fig ure 4-3). The likelihood of transmission increasing with the amount of feeding per day differed according to parasitism status of the thrips. For non -parasitized females and males, the likelihood of transmission did not increase with their amount of feedi ng each day. The regression slopes were not significantly greater than 0 for non parasitized females (t 89 = 1.31, P = 0.10) or for non -parasitized males (t 89 = 0.94, P = 0.18). In contrast, the likelihood of parasitized females transmitting on a partic ular day increased with their amount of feeding (t 80 = 1.87, P = 0.03). Discussion The comprehensive nature of this research allowed for comparisons in feeding and transmission rates to be made for both male and female thrips and provided insight into how T.
162 fuscum may be modulating the physiology of its obligate F. fusca host. Feeding rates of female F. fusca were reduced nearly 65% by T. fuscum parasitism, a conclusion that was consistent with results from previous studies on T. nicklewoodi parasitizing F. occidentalis (Lim and Van Driesche, 2004; Arthurs and Heinz, 2003). Arthurs and Heinz (2003) proposed that parasitized female thrips fed less because developing T. nickle woodi juveniles distended the abdomen and triggered stretch receptors of F. occid entalis However, feeding rates of parasitized females were reduced immediately upon adult emergence and juveniles were not observed in the host hemocoel until much later. Furthermore, parasitism by T. fuscum did not appear to affect the feeding rates of male thrips in our study. Although male F. fusca produced fewer T. fuscum they are significantly smaller than females, and likely, fewer T. fuscum would distend their abdomen in an amount proportionate to parasitized females (Sims et al., 2005; Reitz et al., 2006). Because male thrips feeding behavior was not affected by T. fuscum parasitism, it is also unlikely that T. fuscum is disrupting host neurological systems or that stomach lesions resulting from the continual migration of T. fuscum progeny into and out of the alimentary tract reduce the hosts ability to ingest food. A much more likely hypothesis is that F. fusca may be diverting nutrients from egg production towards sustaining Thripinema development (Hurd, 1990). Previous studies show that adult diet affects egg production of thrips and more energy ( i.e ., food intake) is likely needed for thrips oogenesis than is needed for sustaining T. fuscum infection (Teulon and Penman, 1991). Despite the fact that T. fuscum reduced the amount of feeding by female F. fusca parasitism di d not decrease their longevity. Still T. fuscum parasitism had pathological effects on males, as parasitized males had lower survivorship than non parasitized males despite feeding at similar levels. Lim et al. (2001) suggested that because healthy male thrips feed less than
163 females, their limited nutrient reserves are depleted quicker when parasitized by Thripinema thus reducing their longevity. Tomato spotted wilt virus infection does not appear to have a significant effe ct on F. fusca longevity, which supports the findings of Wijkamp et al. (1996a) and Arthurs and Heinz (2003) for F. occidentalis These results suggest that viruliferous adult F. fusca have a delayed onset of mortality, and this phenomenon may be an adapt ive strategy of TSWV to enhance transmission by its host. The efficiency with which thrips transmit TSWV decreased with age. van de Wetering et al. (1999) suggested that TSWV transmission is a function of food ingestion; a higher consumption rate is associated with more viral particles being egested into plant tissue. Because feeding rates were consistent over the course of the thrips lifetime but transm ission declined in our study, I hypothesize that viral titer may be reduced in older thrips because of senescence of salivary glands and/or degradation of viral particles. In this study, non-parasitized females transmitted TSWV more efficiently than non parasitized males. Sakurai et al. (1998), van de Wetering et al. (1998), and van de Wetering et al. (19 99) reported that male F. occidentalis transmit with a higher efficiency than females because of differences in feeding behavior. Females tend to feed more frequently and for longer intervals, which irreversibly destroys cell contents and can prevent vira l replication within the target plant. In contrast, males feed with a higher frequency of shallow probing and induce only minor cell damage so the cells are better able to support viral infection. The difference in transmission eff iciencies between gende rs in this study and those mentioned may be due to differences in thrips species and host plants used, as both Sakurai et al. (1998) and van de Wetering et al. (1998, 1999) tested the effects of TSWV transmission by F. occidentalis on Datura stramonium (L)
164 Thripinema fuscum parasitism reduced TSWV transmission of adult female F. fusca by approximately 50%. Non -parasitized females fed more and had much higher transmission rates than parasitized females. This difference in transmission remained relatively constant throughout adulthood, which emphasizes the permanent impact of parasitism on thrips vector competence. Prior virus transmission data (Sakimura, 1963) has shown transmission of TSWV by viruliferous F. fusca adults to be sporadic. Transmissions for both non-parasitized and parasitized females in our study were sporadic and the gap times between transmissions were greater for parasitized females. I initially suspected vector capabilities would be reduced because T. fuscum lowers host feeding, and by doing this, reduces the viral titers delivered into plant tissue. This trend was observed when comparing the feeding and transmission rates for males and females females fed more and transmitted at a higher rate than males. However, non -parasitized males fed less than parasitized females but transmitted TSWV at similar rates, suggesting other mechanisms may be operating to reduce transmiss ion by parasitized females. Results from the logistic analysis may indicate that parasitism not only reduces feedi ng (which indirectly affects transmission), but also that parasitism has some type of direct effect on virus replication. It may be that T. fuscum is sequestering important nutrients from the host that are required for successful development, and by doing so, alters the physiology of salivary gland cells and their ability to replicate virus. Alternatively, it has been shown that activation of the immune system of F. occidentalis by TSWV infection induces the upregulation of antimicrobial and other immune system -related proteins (Medeiros et al., 2004). It is possible that both parasitism and TSWV infection may be inducing a synergistic immune response in the thrips that is detrimental to TSWV development.
165 Allantonematid nematodes induce various behavioral and physiological changes in their insect hosts (Hurd, 1993). By sterilizing female F. fusca, T. fuscum aids in reducing secondary spread of TSWV in field conditions (Sims et al., 2005). By reducing feeding, T. fuscum also aids in reducing primary sprea d of TSWV. Experiments in Chapter 5 were designed to determine whether T. fuscum reduces primary spread by inducing a direct effect on the titer of plant pathogens in the F. fusca vector. Understanding how these alterations influence vector competence ma y one day provide targets for suppressing disease spread.
166 Table 4 1. Mean l ongevity ( SE) of adult Frankliniella fusca Parasitism s tatus Viruliferous1 Longevity (days) Females Males Not parasitized No 15.15 0.74 (52) 13.17 1.06 (30) Yes 15. 21 0.72 (39) 15.4 1.4 (10) Parasitized No 14.74 1.03 (46) 8.25 0.72 (16) Yes 17.72 1.51 (18) 1.0 0 (2) 1Thrips were categorized as viruliferous if they tested positive for the NSs protein of TSWV by ACP Indirect ELISA. N umber s in parentheses represent the total number s of thrips per cohort. Table 4 2. Mean t otal area of feeding ( SE) on leaf discs (mm2) fed on by Frankliniella fusca individuals for the initial 10 days of adulthood Total feeding over 10 days (mm 2 ) Parasitism sta tus Viruliferous 1 Wing form Females Males 2 Not parasitized No Brachypterous 3.60 0.76 (5) 0.72 0.13 (5) Macropterous 4.37 0.80 (5) 0.35 0.10 (5) Yes Brachypterous 3.07 0.38 (5) 0.30 0.05 (5) Macropterous 3.80 0.68 (5) 0.51 0.13 (4 ) Parasitized No Brachypterous 1.32 0.21 (5) 0.31 0.08 (3) Macropterous 1.15 0.16 (5) 0.34 0.13 (2) Yes Brachypterous 1.33 0.25 (5) Macropterous 1.24 0.21 (5) Sample sizes are given between parentheses (total n = 64) 1Thrips were categorized as viruliferous if they tested positive for the NSs protein of TSWV by ACP Indirect ELISA. 2No parasitized viruliferous males survived beyond 1 day.
167 Table 4 3. Proportion of viruliferous Frankliniella fusca cohorts transmitting TSWV each day Day Non parasitized females Parasitized females Non parasitized males 1 0.5 (10) 0 (10) 0.22 (9) 2 0.7 (10) 0.3 (10) 0.56 (9) 3 0.8 (10) 0.4 (10) 0.44 (9) 4 0.9 (10) 0.3 (10) 0.33 (9) 5 0.7 (10) 0.3 (10) 0.33 (9) 6 0.6 (10) 0.1 (10) 0.33 (9) 7 0. 5 (10) 0.4 (10) 0.11 (9) 8 0.8 (10) 0.4 (10) 0.67 (9) 9 0.8 (10) 0.5 (10) 0.33 (9) 10 0.7 (10) 0.3 (10) 0.78 (9) 11 0.86 (8) 0.67 (9) 0.33 (6) 12 0.5 (8) 0.11 (9) 0.17 (6) 13 0.5 (6) 0.25 (8) 0.33 (6) 14 0.33 (6) 0.29 (7) 0.33 (6) 15 0.67 (6) 0.14 (7) 0.17 (6) 16 0.5 (4) 0.17 (6) 0.17 (6) 17 0 (4) 0.17 (6) 0.17 (6) 18 0 (3) 0 (5) 0.25 (4) 19 0.33 (3) 0 (4) 0 (2) 20 0.33 (3) 0 (4) 0 (2) 21 0 (2) 0 (4) 0 (1) 22 0.5 (2) 0 (3) 23 0 (2) 0 (3) 24 0 (1) 0 (1) 25 0 (1) 0 (1) 26 0 (1) 0 (1) 27 0 (1) N umber s in parentheses represents the number of thrips per cohort alive on that day
168 Figure 4-1. Proportion of (A) non -viruliferous (n = 144) and (B) viruliferous (n = 69) Frankliniella fusca individuals surviving throughout adulthood.
169 Figure 4 -2 Daily damaged area (mean SE) on leaf discs (in mm2) fed on by Frankliniella fusca individuals for the initial 10 days of adulthood (n = 64).
170 Figure 4 3. Mean cumulative frequency (MCF) of TSWV transmission to individual leaf discs over the lifetime of adult Frankliniella fusca. The flattening of all three curves indicates that the frequency of transmission decrease s with thrips age.
171 CHAPTER 5 THE MULTITROPHIC INT ERACTIONS BETWEEN TH E PARASITIC NEMATODE THRIPINEMA FUSCUM, PLANT PATHOGENS PANTOEA ANANATIS AND TOMATO SPOTTED WILT VIRUS AND THE INSECT VEC TOR FRANKLINIELLA FUSCA Introduction Frankliniella fusca is an important vector of viral and bacterial plant pathogens (see Chapter 1). The obligate parasite T. fuscum significantly reduces the vectoring capacity of F. fusca to transmit TSWV (see Chapter 4 ). Most, but not all, of the reduction in TSWV transmission to groundnut could be explained by a decrease in feeding rates of parasitized F. fusca There were no significant differences in transmission rates of parasitized females and non parasitized males, but males fed significantly less than parasitized F. fusca females. In addition to altering host feeding behavior, parasitism by T. fuscum potentially could induce a host response detri mental to plant pathogens in the insect vector. Information on the impact of intra and extra cellular parasites/pathogens on the competence of insect vectors to transmit disease agents is scarce. One system that has demonstrated an impact on vector compet ence i nvolves microsporidian intracellular parasites of malaria vectors ( Aedes and Anopheles spp.) (Becnel, 1993). Microspori dian infection increases mortality, reduces longevity, and decreases the reproductive capacity of mosquito vectors (Undeen and Alger, 1975; Haq et al., 1981; Kelly et al., 1981). In addition to reducing population densities, infection also interferes with the plasmodial development in both Anopheles and Aedes vectors. Mosquitoes infected with microsporidia are less susceptible to P lasmodium infection (Bano, 1958; Hulls, 1971; Bargielowski and Koella, 2009). Bano (1958) found plasmodial development was inhibited as reflected by a decrease in both oocyst size and in number of ookinetes infecting the midgut. He speculated microsporid ia may (1) sequester nutrients required for proper development of Plasmodium (2) alter midgut cells and preventing
172 penetration by Plasmodium oocysts, or (3) cause the Malpighian tubules to releases toxic substances that interfere with Plasmodium developme nt. Additionally, Hulls (1971) observed degenerated oocysts in the midgut lumen of Anopheles infected with microsporidia, a significant decrease in sporozo ites in the salivary glands, and a reduction in sporozoite viability. Recent work suggests microspor idia infection primes the host immune system, thus impeding the development of Plasmodium parasites. For example, Bargielowski and Koella (2009) injected Sephadex beads into microsporidia infected Anopheles and found an increased melanization response a nd reduced number of Plasmodium oocysts In addition to the limited research on microsporidia, both the gut microflora and endosymbionts of insects have been reported to impact vector competence. Ingestion of a bloodmeal has been shown to stimulate bacte rial replication in the midgut of mosquitoes ( De Maio et al., 1996). Pumpuni et al. (1993) orally challenged adult Anopheles with a gametocyte culture spiked with bacterial preparations. Examination of these mosquitoes revealed a reduction in the number oocysts associated with the midgut tissue. The observed reduction in oocyst density was positively correlated to bacterial concentration. The authors speculated that bacteria may be non specifically binding to the parasite or midgut epithelium, thus preventing entry of Plasmodium ookinetes into midgut cells. Additionally Dong et al. (2009) demonstrated bacteria within Anopheles midgut inhibit infection of Plasmodium ; removal gut bacteria through antibiotic treatment resulted in a near doubling of the oocysts in the midgut. Dong et al. (2009) fed mosquitoes a Plasmodium infected blood meal containing either live or heat killed bacteria and found fewer oocysts developed in the midguts. Interestingly, injection of bacteria into the hemocoel prior to chall enge with Plasmodium infected blood significantly reduced the number of oocysts in the midgut. These results suggested an indirect effect of the bacteria on plasmodial
173 development. Utilizing a microarray -based genome -wide gene expression strategy, Dong et al (2009) like other investigators (Dimopoulos et al., 1996, 1997; Barillas -Mury et al., 1996; Hernandez -Martinez et al., 2002; Hillyer et al. 2003; Aguilar et al. 2005 ) found bacterial challenge to Anopheles upregulates the innate defense resulting in the production of immune genes ( e.g ., cercopins, defensins, gambicin, serine proteases, and pattern recognition receptors). These antimicrobial components disrupt the gut microflora that serve as a defensive barrier against Plasmodium development. The in hibition of parasite development by gut bacteria has been reported in other insect vectors including triatomines, tsetse flies, and sand flies (Schlein et al., 1985; Welburn and Maudlin, 1999; Azambuja et al., 2004). In addition to manipulating reproduction (see Chapter 2), endosymbiotic bacteria such as Wolbachia protect insects from viral infection Hedges et al. (2008) compared survival rates of Drosophila melanogaster infected with a range of pathogenic RNA viruses in the presence or absence of Wolba chia and discovered a de lay in virus accumulation and mortality in Wolbachiainfected flies. Teixeira et al. (2008) reported a laboratory D. melanogaster strain treated with tetracycline had higher viral titers of the same RNA viruses The authors specul ate Wolbachiainduced host resistance to RNA viruses may be caused by either a cell autonomous or systemic effect. Autonomously, Wolbachia could compete for resources in the cell cytoplasm, reduce cell meta bolism, and/or actively interfere with vir al repl ication in the cell. Systemically, Wolbachia may pre activate the host immune system allowing for a faster response upon viral infection. Recently, Wolbachia endosymbiont s have been shown to modulate infection with Dengue, Chikungunya, Plasmodium and Bru gia nematodes in the Aedes vector ( Kambris et al., 2009; Moreira et al., 2009; Mousson et al., 2010). Wolbachia infection likely activates the innate immune system and renders the host more resistant to pat hogen invasion and replication. The
174 tradeoff for constant immune upregulation is a shorten ed lifespan (Libert et al., 2007 ). The life shortening phenotype also reduces disease transmission by reducing the number of older ( i.e ., those that have surpassed the pathogens extrinsic incubation period) individuals within a vector population (Cook et al., 2008). To my knowledge, the only work examining the impact of a parasite on competence of insect vectors in a plant system has been conducted on Thripinema spp. (see Chapter 1). Research findings concurred t hat the T hripinema induced reductions in host feeding is a significant factor for the decrease in Tospovirus transmission rates (Arthurs and Heinz, 2003; Lim et al., 2004; Sims et al., 2008; see Chapter 4). The goal of this chapter was to observe if paras itism by T. fuscum impacts the titer of the plant pathogenic TSWV and P. ananatis in the F. fusca vector. Specifically, the first objective was to survey the cultivable bacterial fauna associated with field and laboratory F. fusca populations, and investi gate the impact of T. fuscum parasitism on the capacity of F. fusca to harbor the plant bacterial pathogen P. ananatis The second objective was to quantify and compare TSWV titers and TSWV transmission rates between non -parasitized and parasitized adult female F. fusca Potential mechanisms underlying the associations between T. fuscum and F. fusca and how these mechanisms may influence pathogen titer in th e insect vector, are discussed. Materials and Methods (Pantoea ananatis ) Survey of bacteria associ ated with F. fusca : Female F. fusca were randomly selected from laboratory colonies (n=64, maintained as described in Chapter 2) and PSREU field populations (n=50, collected as described in Chapter 2). Thrips were rinsed by submerging individuals in successive washes of 0.6% hypochlorite solution, 70% ethanol, and sterile 0.85% saline for 15 s each. After adults were washed, they were dissected in 10 l of sterile saline to determine parasitism status (as described in Chapter 2). Dissected individual th rips were placed
175 in a sterile 1.5 -ml microcentrifuge tube with 90 l of saline and sonicated at a setting of 10% for 10 s with a Fisher Sonic Dismembrator microprobe (Model 300, Fisher Scientific, Pittsburgh, PA). An additional 100 l of saline were added to the tubes, the tubes were vortexed for 10 sec, and 100 l of the sample were spread onto a tryptic soy broth agar plate (TSBA; 15 g TSB + 7.5 g agar up to 500 ml of water). The remaining 100 l of sample were stored at 4C. Plates were incubated at 2 5C, and the bacterial phenotypes and number of colonies were recorded after 24 h. Individual clones were isolated and propagated by transferring single colonies of selected phenotypes to individual plates. Gram staining was performed on the two predomina nt phenotypes. The isolated bacteria were sent to the University of Floridas Bacterial Identification and Fatty Acid Analysis Laboratory (Gainesville, FL) for MIDI analysis, a system that uses gas chromatography to identify and compare fatty acid profile s against a bacterial species library. Statistical analyses were conducted using a logistic regression (Proc GENMOD, SAS 2006) that compared the ratio of thrips harboring bacteria between the different categories of thrips surveyed. Thrips category was de fined as the class variable. The genmod procedure allows for unbalanced design and comparison of ratios without data transformation (Neter et al., 1990). Means were compared using the least -square statement of SAS. Coinfection assay with P. ananatis and T. fuscum : Adult F. fusca were collected fr om Marion (2924 N 8210 W) and Alachua (2938 N 8221 W) County, FL, and maintained in laboratory colonies as previously described (see Chapter 2). The P. ananatis stock preparation was obtained by inocula ting one single colony of P. ananatis to 5 ml of LB broth and vortexing at 150 rpm overnight at 25C. After 12 hours, the concentration of bacterial solution reached
176 approximately 5.0 x 109 cells/ml. Groundnut leaf discs ( 1-cm2) were cleaned by successiv e washes of 0.6% hypochlori te solution 70% ethanol, and sterile 0.85% saline for 15 s each Discs were dipped into a concentrated P. ananatis stock solution and air dried. A single one day -old female was selected randomly from the colony and placed in a 1.5 -ml microcentrifuge tube that contained a leaf disc inoculated with P. ananatis After a 48 h exposure to bacterial coated leaf discs, females were transferred either to a 1.5 -ml microcentrifuge tube with leaf disc (controls) or with leaf discs contai ning two parasitized F. fusca adult females that were excreting nematodes. Thrips remained in these tubes for an additional 48 h before individuals were removed, washed as described above, air dried, and individually transferred to a 1.5 -ml microcentrifug e tube with a fresh cleaned leaf disc. The following day, thrips were transferred to clean tubes. The prior tubes were rinsed with 200 l of water and the rinsate was examined for free living T. fuscum to identify the two parasitized F. fusca that served as an inoculums source for these assays. Seven and 12 d after the initial exposure to the P. ananatis treated leaf discs, individuals were dissected in 25 l of saline to determine parasitism status, sonicated, and 2 l of homogenate spot plated in ten -fo ld serial dilutions. Resulting colony forming units (CFUs) were determined after 24 h of incubation at 25C. Attempts were made to obtain 50 thrips for each treatment per replicate experiment. The experiment was replicated three times. For the statistic al analyses, colony forming units were long transformed prior to analysis and the capability procedure (Proc CAPABILITY; SAS 2006) was used to test for normal distribution of the transformed data. The frequency procedure (Proc FREQ, SAS 2006) was used to generate a goodness of fit test and frequency table to determine if the presence of P. ananatis is associated with parasitism status (Proc FREQ, SAS 2006). Multiple comparison tests among treatment frequencies were made using Tukeys style multiple compar ison of proportions (Zar,
177 1999). A mixed model anova (Proc MIXED; SAS 2006) was performed on logtransformed data to test for differences in bacterial CFU counts between parasitism treatments over time. Comparisons between treatment means were made using the LS means option. Assays for antimicrobial activity: Individual F. fusca females collected from field populations (see Chapter 2) were dissected in Hepes -buffered saline to determine parasitization status and placed in a 1.5 -ml microcentrifuge tube wi th 50 l buffer. The two treatments, non parasitized and parasitized F. fusca females, each had two tubes of 50 thrips each. Thrips were homogenized with a plastic pestle on ice, mixed with an additional 50 l buffer, and centrifuged at 2,000 x g for 3 minutes at 4C to pellet debris. The supernatant (100 l) was transferred to a 0.45m filtered microcentrifuge tube and centrifuged at 10,000 x g for 5 m. To obtain the acidderived homogenate, 50 non-parasitized and parasitized F. fusca females were dis sected and placed in a 2.0 -ml conical screw cap microcentrifuge tube (Fisher Scientific, Pittsburgh, PA, USA) with 0.5 ml of 4% acetic acid. Five 2.0 -mm Zirconia beads (BioSpec products, Inc., Bartlesville, OK, USA) were added to each of the tubes and samp les were homogenized for 20 s using a multi tube homogenizer (Thermo Savant Fast Prep Homogenizer, Savant, Markham, Ontario). The tubes were individually inspected under a dissecting microscope to ensure thorough homogenization of thrips. Samples were bo iled for two minutes to precipitate large molecular weight proteins and centrifuged at 10,000 x g for 10 m to remove heat denatured proteins. The resulting supernatant was frozen at 70C overnight and freeze -dried the following day. A tube containing 0. 5 ml of 4% acetic acid served as a negative control. After freeze drying, 40 l of sterile 0.1M phosphate buffer were added to each tube and sonicated for 10 m to solubilize proteins. The two homogenates were subjected to a series of antimicrobial tests (see below).
178 The first two antimicrobial tests were conducted to observe if crude or acid -derived homogenates of non -parasitized and parasitized F. fusca females negatively impacted P. ananatis. The antimicrobial tests were conducted by placing 5 l of the homogenate sample either on a 1 cm2 filter disc or in a 2 -mm plug on a TSBA plate spread with 200 l of stock P. ananatis solution. For both antimicrobial tests, streptomycin (2 mg/ml; Sigma, St. Louis, MO) served as a positive control. The second antim icrobial test was conducted to observe the antibacterial response of F. fusca when parasiti zed with T. fuscum by determination of lysozyme activity. First, standard chicken egg white lysozyme solution was made in two-fold serial dilution of stock solution (100,000,000 enzyme units (EU)/5g; Cat No. 4403, Calbiochem Merck KGaA, Darmstadt, Germany) to obtain a final protein concentration of 320, 160, 80, 40, 20, 10 and 5 g/ml or 6400, 3200, 1600, 800, 400, 200 and 100 EU/ml. Two l of each concentration wer e placed in individual 2 -mm2 plugs cut into a Micrococcus lysodeikticus plate (5 mg/ml + 0.1M phosphate buffer + 1.2 g agar; Sigma Chemical, St. Louis, MO). Two l of the acid derived sample homogenates and 2 l of a negative control (0.1M phosphate citra te buffer) were also placed in individual 2 -mm2 plugs and incubated overnight at 37C. After 24 h, the zone of inhibition for each sample was measured in mm2. A standard curve was generated by plotting the zone of inhibition (in mm2) against units of act ivity. The resulting regression equation was used to determine units of activity per thrips. The third antimicrobial test was conducted to observe the effect of lysozyme activity on P. ananatis 200 l of the bacterial stock solution were spread on a T SBA plate and 2 l or 15 l lysozyme standards were placed in plugs or on filter discs on the plate, respectively. Plates were incubated at 37C and the zone of inhibition recorded after 24 h.
179 Materials and Methods ( Tomato spotted wilt virus ) Viruliferous nonparasitized and parasitized F. fusca colonies: In June 2008, leaves displaying symptoms of TSWV were collected from A. hypogaea at the Institute of F ood and A gricultural S cience P lant and S cience Research E ducation U nit at Citra, FL (2924 N 8210 W) A 100-mg tissue subsample from each plant was tested for TSWV infection with a double antibody sandwich enzyme linked immunosorbent assay (DAS -ELISA) that uses anti -mouse antibodies to detect viral structural (nucleocapsid) proteins in sample homogena tes (SRA 39300, Agdia Inc., Elkhart, IN). Subsamples with an ELISA optical density value greater than the determined threshold value for a plate (mean of control readings + 3[standard deviations]) were considered positive. Plants whose subsample tested positive by ELISA were used to inoculate first instar F. fusca as described previously (see Chapter 4). After 72 h, larvae were transferred from TSWV infected plant material to healthy groundnut leaves that were replaced daily until pupation. Frankliniella fusca pupae were collected and transferred in groups (n=20) to 1.5 -ml microcentrifuge tubes provisioned with a groundnut leaflet. Two T. fuscum parasitized females excreting nematodes were added to half of the tubes and two non-parasitized one -day old females were added to the remaining half of the tubes. Twenty pupae obtained from healthy laboratory colonies were also placed in a 1.5 -ml microcentrifuge tube with two non-parasitized adult female F. fusca ( control ) After 24 h, all adult female F. fusca were disposed of and individual pupae were placed in tubes with a fresh groundnut disc until adult emergence. The resulting viruliferous treatments were non-parasitized females not exposed to T. fuscum non parasitized females exposed to T. fuscum and f emales parasitized by T. fuscum Non viruliferous, non -parasitized females served as the negative control. TSWV titration of F. fusca cohorts: Six -day old female F. fusca were individually dissected in 1l of Hepes buffered saline and grouped in sterile RNase free 1.5 -ml
180 microcentrifu ge tube on ice containing 0.5 ml of TRI Reagent (Sigma -Aldrich Corp., St. Louis, MO, USA) until 20 thrips had been obtained per treatment. The tubes were centrifuged at 5,000 x g for 1 minute 4C at and stored at 80C until RNA could be extracted. RNA was extracted from F. fusca following the Sigma Technical Bulletin MB 205 for TRI Reagent (Appendix C1). Reverse transcriptase PCR was conducted on 1 l RNA from each sample using the Access RT PCR System (Promega, Madison W I) following the manufacturers Technical Bulletin TB220 protocol See Appendix C2-C3 for primer information and P CR protocol and cycling profile s for RTPCR Five l of PCR product was run on a 1.5% agarose gel to confirm presence of the TSWV amplicon and t he obtained product was sent to the University of Floridas DNA Sequencing Core (ICBR) for sequencing to confirm that amplification was of the target gene. RTPCR was also used to determine suitability of thrips selected internal 28S and COI standards Reverse transcribed c DNA was synthesized from 50 ng of RNA using the iScript cDNA Synthesis Kit (Bio -Rad, Hercules, CA ) following the manufacturers protocol (Appendix C4). Quantitative real time PCR (qPCR) was conducted on the reverse transcribed cDNA using the Quantace iQ SYBR Green Supermix kit ( Bioline Taunton, MA ) according to the manufacturers protocol (Appendix C5) Following amplification, threshold cycle (CT) values for both the target gene (TSWV) and the internal standard (CO1) were recorde d for all samples. Samples whose CT values were above 30 indicated that the target gene was not amplified and were therefore considered negative. The approximate relative quantity of TSWV titer in each sample was calculated by comparative quantification using the equation 2 TT = (CTTSWV CTCO1)calibrator (CTTSWV CTCO1)unknown (Livak and Schmittgen, 2001). TSWV titration of individual F. fusca : The initial assay described above assumed the proportion of thrips in each cohort acquired TSWV equally even though each pool of 20 thrips
181 may have contained an unknown number of non-viruliferous thrips ( i.e ., the assay did not provide quantification for which thrips actually harbored virus prior to analysis). Therefore, a second assay was conducted that determined an individuals vector competence prior to conducting qPCR To determine the vector competence of individual F. fusca a leaf disc assay similar to that described by Rotenberg et al. (2009) was implemented before thri ps were dissected so that the individuals capacity to transmit TSWV was known. For the leaf disc assay, single one -day -old adult female F. fusca were placed individually into 1.5 -ml microcentrifuge tubes with a fresh peanut disc (1 -cm2). The females were given three consecutive 48 h inocul ation access periods (IAP) each with a fresh leaf disc, and the old leaf disc was immediately placed on sterile water in 24 -well plates and incubated for 5 d at 25C to amplify virus titer. Leaf discs were then stored at 80C for DAS -ELISA. After analys is, thrips from each treatment were categorized into four transmission categories: 0 (0 out of 3 transmissions), 1 (1 out of 3 transmissions), 2 (2 out of 3 transmissions), and 3 (3 out of 3 transmissions). Leaf discs were tested until a minimum of three adults from each treatment f illed a transmission category. After the last IAP, individual females were dissected and their paras itism status was recorded. Females were then transferred to individual tubes with 10 l of Tri -Reagent and stored at 80C. The Sigma RNA extraction bulletin was optimized for individual thrips and three individuals were randomly selected from each treatment (Microsoft Excel 2007, RAND Function) for analysis by q PCR. An external standard prepared from the pBluescript plasmid containing the TSWV -N gene was used to generate a standard curve for absolute quantification in the qPCR reactions. Serial dilutions ranging from 1 x 106 to 1 x 101 plasmid copies per l were made from the stock plasmid, and CT valu es generated from the standard curve were used
182 to calculate the absolute viral genome copy numbers from individual thrips. Reverse transcribed cDNA from viruliferous thrips tested by qPCR to have a threshold cycle less than 24 served as a positive control The efficiency of the COI housekeeping gene to serve as a stable internal control was determined by calculating the mean CT values as 2 -CT and comparing the foldchange in gene expression between treatments (Schmittgen and Liva k, 2008). Statistical anal yses: Mean cumulative function plots were generated for viruliferous nonparasitized and viruliferous parasitized female F. fusca to observe differences in transmission over the three IAPs (Proc RELIABILITY; SAS, 2006). A generalized linear mixed model ( Proc GLIMMIX; SAS, 2006) was used to determine if the overall incidence of transmission was dependent upon parasitism and if parasitism influenced transmission over the three IAPs. Each IAP was treated as a repeated measure, with the measure being a binar y response of an infected or non infected leaf disc. Pairwise comparisons among the treatment groups were made using the LS means option. A general linear anova model (Proc GLM, SAS 2006) was performed on log transformed copy numbers to determine if vira l copy number is dependent upon F. fusca parasitism status and/or the number of transmission events by viruliferous thrips. The model was extended for an ordinal logistic regression (Proc LOGISTIC, SAS 2006) to determine whether the probability of transmi ssion was related to copy number o r pa rasitism status. A scatter plot and line of best fit was generated to determine if a line ar correlation existed between ELISA optical density values and TSWV copy numbers. Results (Pantoea ananatis ) Surve y of bacteria associated with F. fusca : Both F. fusca laboratory and field individuals contained bacteria cultivable on TSBA (Table 5 -1). Transmission electron micrographs revealed the bacteria are located with in the midgut and hindgut of i ndividual thrips, and in cl ose
183 apposition to the midgut microvilli and the hindgut intimal lining (Figure 5 -1). The bacteria are fairly uniform in shape (1 m in diameter) with both short and long rods present. Two bacterial phenotypes were frequently found on the plated individua ls at 24 h post inoculation (Figure 5 2A). Phenotype A was identified on TSBA as smooth, yellowish, dry, and circular umbonate colonies 1.5 -mm2 in diameter with compact, raised centers and flattened smooth edges (Figure 5 2B). Phenotype B was identified on TSBA by its shiny, wet appearance and milky white texture (Figure 5 2C). The proportion/percentage of F. fusca harboring cultivable bacteria was higher for non-parasitized ( x= 78%) than for parasitized (x=61%) females collected from either the field or laboratory populations (Table 5 1) Statistically, non parasitized F. fusca collected from laboratory populations had a significantly higher frequency of 2=14.75, d.f. =1, P<.0001). Over 90% of individuals that harbored bacteria c ontained phenotype A, and the frequency of individuals with this phenotype was statistically higher for laboratory colonies of non -parasitized F. fusca 2=11.65, d.f.=1, P=.0006) than for the other populations 2=70 4.07, d.f.=1, P<.0001) (Table 5 1) Only 15% of the F. fusca tested contained phenotype B, and over half these individuals were collected from non -parasitized field populations (Table 5 1) Non -parasitized and parasitized F. fusca were rarely infected wi th any other bacterial phenotype (Table 5 1) Co infection of two or more phenotypes was not common (Table 5 1) Both the A and B bacterial phenotypes were identifie d as Gram -negative. According to MIDI analysis, t he fatty acid profile of phenotype A had a high association to P ananatis/Erwinia uredovora (Sim Index = 0.905) However, the less frequent phenotype B was not identified with MIDI analysis; the GC profile had a low association to both Ewingella americana (Sim Index = 0.224) and to Pantoea agglomerans (Sim Index = 0.150; see Appendix
184 C6 for the full MIDI report). Phenotype A ( P. ananatis ) was selected for the co infection assays with T. fuscum due to the high frequency of individuals associated with this phenotype. Coinfection assay : In an a ttempt to increase the number of F. fusca females harboring P. ananatis adults were exposed for 48 h to bacterial -coated leaf discs. T his exposure period increased the number of thrips with bacteria from 66% to 75% (Table 5 1, 52) Exposure to T. fuscu m did not affect the number of non -parasitized F. fusca harboring P. ananatis (Table 5 -2). However, there was a statistically significant reduction in the number of parasitized F. fusca acquiring P. ananatis when compared to the non-parasitized conspecifi 2=39.73, d.f. =2, P<.0001). There was a 4 0% reduction in the number of parasitized F. fusca females harboring P. ananatis when compared to the nonparasitized treatments (Table 5 2) The number of bacterial CFUs recorded for non parasitized F. fusca ranged from 0 1.7 x 108. T he number of bacterial CFUs recorded for parasitized F. fusca ranged from 0 7.0 x 106. The CFUs were log transformed prior to analysis to achieve a more normal distribution of the data. Statistically, there was a significan t difference in the CFU counts among all thrips (F2, 296=20.97, P<0.0001), with the parasitized F. fusca having a lower number of CFUs than either the non-parasitized (t=5.41, d.f. = 296, P<.0001) or the exposed, non-parasitized (t= 5.84, d.f. =296, P<.0001) (Table 5 2). There was no t a statistically significant difference in CFU counts between the control and exposed, non -parasitized F. fusca females (t= 0.34, d.f. =296, P=0.73) (Table 5 2) The length of time F. fusca incubated P. ananatis did not signi ficantly influence the number of CFUs for all three treatments (F1, 296=2.08, P=0.15) (Table 5 2) Although not statistically significant, the nonparasitized F. fusca exposed to T. fuscum had nearly twice as many CFUs when they harbored P. ananatis for five additional days (t= 2.21, d.f.=296, P=0.03)
185 Antimicrobial assays: Crude and acid-derived homogenates of non-parasitized and parasitized F. fusca did not exhibit antimicrobial activity against P. ananatis (Fig 5 3a, b). Lysozyme activity was not detected in non -parasitized female F. fusca but parasitized F. fusca females exhibited lysozyme activity against M. lysodeikticus after 24 hours that resulted i n a zone of clearance equaling 28.3 mm2. From the standard curve, the lysozyme activity of this sa mple was calculated to be equal to 2.88 EU per single thrips (Figure 5 3c, Figure 5 4, Table 5 3 ). The lysozyme standards did not show activity against P. ananatis (Fig 5 3d). Results (TSWV) TSWV titration of F. fusca cohorts : Reverse transcriptase PCR v alidated the presence of all targeted products (Figure 5 -5 ). The COI primers amplified a fragment of the expected size (143 bp) for all samples and the TSWV primers amplified a fragment of the expected size (123 bp) for all viruliferous samples. TSWV pro duct was not detected in the non -viruliferous cohorts. The 28S primers amplified negative controls and were therefore not used for further PCR (Figure 5-6). Sequencing results of the targeted gene confirmed amplification was of the TSWV N protein (Append ix 5 -7). Quantitative PCR of cDNA synthesized from total extracts of viruli ferous female F. fusca detected COI and TSWV N protein transcripts in the appropriate samples ( Figure 5 7A,B ). Amplification of the TSWV N protein was not observed in non -virulif erous samples within 30 cycles ( Figure 5 -7C). T he average CT values for the three treatments are located in Table 5-4. The average CT value s for the COI transcripts (internal control) varied by only one threshold cycle between all four treatments indica ting extraction efficiencies were similar between all sample cohorts. There was variation in viral titers both between and within all the viruliferous treatments, as indicated by the high standard deviations associated with the average TSWV CT values. Th e largest variation in viral titer was observe d with the viruliferous parasitized F. fusca
186 cohort s where average CT values ranged from 36.48 1.31 to 22.25 0.17 (data not shown ). Overall, there was a 33 -fold and 124 -fold reduction in the TSWV titers o f parasitized F. fusca females when compared to the non parasitized and T. fuscum exposed (non -parasitized) cohorts, respectively. There was a 3 -fold increase in virus titer for F. fusca exposed to T. fuscum (non parasitized) compared to those not exposed TSWV titration of individual F. fusca : Of the 980 F. fusca assayed to estimate TSWV titer 795 and 185 of the thrips eclos ed as females and males, respectively ( data not shown ). Nearly one third of the adults (n=285) died before the end of the assay t he majority of which (75%) succumbed to infection by Entomophthorales sp. during the last IAP. A total of 676 thrips survived through out all three IAPs, and dissections of these individuals revealed there were nearly twice as many nonparasitized than par asitized F. fusca Only 31 of thrips surviving through the last IAP were infected with Entomophthorales sp.. Overall, 1,218 leaf discs were analyzed by DAS -ELISA from the non-parasitized (N=262) and parasitized (N=144) F. fusca females. The DAS ELISA abs orbance values for each leaf disc were used to categorize non parasitized and parasitized F. fusca according t o thei r vectoring capabilities P arasitism by T. fuscum reduced the proportion of transmitting individuals by 20% (Table 5 -5 ). On average, 74 an d 54% of non -parasitized and parasitized female F. fusca respectively, transmitted TSWV at least one time during the three consecutive IAPs (Table 5 5) Of the 406 F. fusca individuals tested by leaf disc assay, only 47 transmitted TSWV during all three IAPs (Table 5 5) The mean cumulative frequency of TSWV transmission was significantly higher over all three IAPs for non -parasitized thrips (F1,465=19.80, P<.0001) (Figure 5 -8 Table 5 -5 ). The rate of transmissions varied over the three IAPs (F2,786=23. 77, P<.0001), with transmission being
187 significantly lower in the first IAP than in the second (t= 6.14, d.f.=755, P<.0001) and third (t= 6.27, d.f.=1195, P<.0001) IAPs for both non -parasitized and parasitized F. fusca (Table 5 -6 ). Non -parasitized individu als transmitted at a significantly higher frequency than parasitized F. fusca over the second (F1,392=9.24, P=.0025) and third ( F1,388=9.82, P=.0019) IAP (Table 5 6) Statistically, t here was no significant difference in IAP of parasitized F. fusca transmitting TSWV (F2,786=2.79, P = 0.062) (Table 5 6) However, the efficien cy of both parasitized and non -parasitized thrips transmitting TSWV to leaf discs was higher during the third IAP than any other period (Table 5 6) Optimization of m ethods used for RN A extr action from viruliferous thrips and the ge ne ration of the standard curve for qPCR is shown elsewhere ( see Appendix C8 -C9). The CT values of nonparasitized and parasitized thrips f or the COI housekeeping gene did not vary significantly between sampl es u nder conditions of the qPCR signifying its suitability as a stable internal control (Appendix 5-6 ). Despite the optimization methodologies used for extracting RNA from individual F. fusca TSWV and COI copy numbers were extremely low and variable acro ss all treatments (Table 5-7 ). The COI and TSWV CT values across treatments ranged from 12.41 to 23.58 and 21.09 to 37. 39, respectively Some comparisons in absolute quantification were able to be made between the different treatments of F. fusca despite the variable extraction efficiencies. Non -transmitting F. fusca had significantly fewer genome copy numbers of TSWV than those that transmitted TSWV regardless of parasitism status (Figure 5-9). The TSWV copy number increased with the number of transmiss ions for both nonparasitized and parasitized F. fusca (F3,79=2.78, P=.0465) Non -parasitized F. fusca ha d higher TSWV titers than parasitized thrips, but this was not found to be significant (F1,76=3.75, P=0.0567) (Figure 5 10). The logistic regression, which evaluated the number of transmissions as a function of parasitism status and
188 copy number, found copy number to be positively correlated with the number of transmissions x=6.25, d.f.=1, P=.01) and parasitism status to not affect the number of trans missions to leaf x=0.28, d.f.=1, P=.60). There was no statistically significant correlation between ELISA optical density values and TSWV viral copy numbers (R2=0.0138) (Figure 5 11). Discussion In this study, e x tracellular bacteria observed in th e mid and hindgut of individual F. fusca w ere similar to thrips gut bacteria reported by others ( McKenzie et al., 1993; Wells et al., 2002, Gitaitis et al., 2003; Ullman et al., 1989; de Vries et al., 1995, 2001, 2004, 2008; Chanbusarakum and Ullman, 2008, 2009). Pantoea ananatis was t he dominant bacterium isolated from over 90% of individual F. fusca collected from laboratory and field populations. The high association of P ananatis with individual F. fusca collected over space and time suggests a symb iotic relationship between the bacteria and host Similarly, Chanbusarakum and Ullman (2008, 2009) found related strains of Enterobacteriaceae in F. occidentalis collected from geographically isolated areas and suggested the bacteria and hosts share a fac ultative symbiotic association. In an attempt to clarify the relationship of gut bacteria on thrips host, de Vries et al. (2004) examined fitness effects of F. occidentalis with and without bacteria By feeding different diets to the thrips, they discove red thrips with gut bacteria have reduced fitness effects with a nutrient rich diet and increased fitness effects with a nutrient -depleted diet. These results suggest the gut bacteria are both parasitic and mutualistic by either competing with the host under favorable environments or benefiting the ir hosts in poor environments. Environmental conditions can also influence the phenotypic characteristics of bacteria, including behavior, growth, and biofilm formation (Brown and Barker, 1999). These character istics can impact the prevalence of bacteria growth on a host. Frankliniella fusca collected from laboratory colonies had the highest frequency of individuals infected with P.
189 ananatis and highest CFU counts (data not shown). Transmission of P ananatis occurs horizontally on the food source from feces or saliva or vertically through contamination of the egg shell (de Varies et al., 2001b; Wells et al., 2002). The environmental conditions for rearing F fusca in laboratory colonies are more suitable for P. ananatis growth and transmission than are field conditions (see Chapter 2 ). Laboratory conditions include a high humidity and temperature, a shared food substrate, and a high density of thrips maintained over subsequent generations. Individual F. fusc a collected from field pop ulations are restricted to contacting relatively few isolated thrips populations located in groundnut flowers or terminal buds. Parasitism by T. fuscum also influence d the number of individuals infected with P. ananatis and reduce s the quantity of bacteria in the host insect as demonstrated by the co inoculation assays. There was not a s tatistically significant difference in the quantity of bacteria in parasitized F. fusca individuals over time, suggesting the parasitism event by the motherworm is responsible for reducing P. ananatis in the thrips host. It is unlikely the motherworm residing in the hemocoel exerts a direct effect on bac teria in the alimentary tract I nfection by certain microbes may indirectly induce a host immune response that acts against pathogens in the insect vector. Acid -derived homogenates of parasitized F. fusca exhibited lysozyme activity against M. luteus but activity by the lysozyme standard did not inhibit P. ananatis The high concentration of P. an anatis spread onto the TSBA plates likely concealed any detectable activity by the lysozyme standard ( i.e ., the concentration of the bacteria on the plate was much higher than would naturally occur in the thrips) Lysozyme is commonly known as an antibact erial enzyme of the insect immune system synthesized by insect fat body and hemocy t es upon infection with bacteria, fungi, viruses, nematodes, etc. (Bulet et al., 1999). The detection of lysozyme in the present study may suggest immune upregulation, but parasitism by T. fuscum does not decrease
190 the lifespan (an indicator of upregulation) of F. fusca (Sims et al., 2005). Lysozyme also functions as a digestive enzyme in the midgut and salivary glands of some insects by lysing gut bacte ria and providing ins ects with additional nutrients (Callewaert and Michiels, 2010). The increased lysozyme concentrations observed in parasitized F. fusca may be a host induced response to digest and sequester nutrients from P. anana tis since T. fuscum reduces feeding of inf ected individuals (see Chapter 4). T ransmission assays in this study validated previous results that T. fuscum reduces the capacity of F. fusca to vector TSWV (see Chapter 4). D ifference s in transmission rates between non -parasitized and parasitized F. fus ca in the two experiments are likely due to the different IAPs utilized (48 vs. 24 h). Nonetheless, parasitism by T. fuscum reduced transmission rates of F. fusca even with a long er IAP. Previous results also suggested that T. fuscum may induce a direct effect on TSWV replication (see Chapter 4), and assays conducted to quantify viral genome titers found significant variation between all viruliferous F. fusca samples in both the cohort and individual qPCR assays. Viral titer in insect vectors is known to vary and is thought to reflect different physiological potentials between individuals to amplify virus in the salivary glands (Ullman et al., 1993; Wijkamp et al., 1995; Nagata et al., 1999). However, transcription of the COI housekeeping gene should not have varied between replicate samples in the individual qPCR assays. This variation, in combination with low TSWV yield, suggests possible degr a dation of thrips samples stored longterm at 80C in TRI Reagent Bravo et al. (2007) found tissue preserved in RNA L ater at 80C, followed by RNA extraction and storage at 80C, to be unstable for small RNAs (20 nt) when using Trizol reagent as an isolation method. In contrast, Mraz et al. (2009) reported that TRIReagent based isolation produced stable RNA suitable for long -term storage at 80C In this experiment, an alternative RNA
191 extraction method may have yielded higher extracti on efficiency. Boonham et al. (2002) reported detection of TSWV in virulif erous F. occidentalis to be more efficient when using a chelating resin rather than alcohol -precipitation for RNA extraction. Using this method, Rotenb e rg et al. (2009) w ere able to obtain 2 x 105 2 x 107 copies of N transcript per female F. occidentalis Regardless of the low extraction efficiency, dosedependency was found between viral copies and numbers of transmissions. These findings corroborate other report ings that TSWV present in viruliferous thrips is a quantitative factor affecting vector comp etence (Nagata, 2002, Wijkamp et al., 1995; van de Wetering et al., 1996; Rotenberg et al., 2009). Wijkamp et al. (1995) and Nagata et al. (1999) found that small amounts of virus were detected in the salivary glands of non-transmitting thrips, which sugg ests there is a threshold titer of TSWV required for thrips to transmit successfully. In this experiment, low viral titers were also detected in non transmitting F. fusca and higher titers tended to be associated with transmission. Results from this stud y demonstrate that parasitism by T. fuscum is a qua litative factor affecting vector competence of host thrips. While in the cohort assay parasitized F. fusca females had a significantly lower titer of TSWV than non -parasitized females, parasitism was foun d to be a non -significant factor impacting viral titer for the individual assay. It should be noted that the pooled thrips in the cohort samples were completely randomized and the chance of selecting a combined sample of 20 non-viruliferous from virus -exp osed thrips in the experimental pools was very unlikely. Individual female F. fusca exposed to TSWV and parasitized by T. fuscum were more likely to not transmit TSWV when compared to their non p arasitized conspecifics (Table 5 -5 ). It is more probable that parasitized individuals pooled in
192 the cohort assay had a reduced TSWV titer because they had a higher percentag e of nonviruliferous thrips. The response of the F. fusca vector to the plant pathogens P. ananatis and TSWV is modulated by its interaction with T. fuscum To my knowledge, this is the first report of infection by an obligate parasite antagonistically affecting a bacterial and viral pathogen in an insect vector. It is unknown whether the reduction in plant pathogens is induced by the F. fusca host, the parasitic T. fuscum or is simply a by -product of infection (Poulin, 1995; Adamo, 2002; Thomas et al., 2005). In both the P. ananatis and TSWV coinfection assays, F. fusca acquired the plant pathogen before acquiring the T. fuscum parasite Possibly, primary infection by the pathogen activated the host immune system (as shown by Medeiros et al., 2004) and the enhanced immune response under dual infection with the parasitic T. fuscum reduced and/or eliminated the plant pathogen but not the par asite Data from Chapter 3 suggests T. fuscum evades host immunity as demonstrated by the lack of cellula r and humoral immune factors at the host parasite interface. Alternatively, the parasitic T. fuscum may outcompete plant pathogens in the insect vect or by (1) releasing antimicrobial metabolites that are active against TSWV and P. ananatis ; (2) sequestering available host nutrients; (3) inhibiting normal host alimentary and salivary gland function; or (5) interfering with other host endosymbionts that regulate host vector capacity. As reviewed in the introduction, most of the research conducted on insect vector -pathogen microbe systems suggests immune activation is an important factor in regulating infection. It is most likely that the parasitism event by the T. fuscum motherworm activates the transcriptional upregulation of the thrips immune system, which in turn, triggers a cascade of antibacterial and antiviral peptides that act against both pathogens. Most work on insect immunity comes from
193 molecul ar studies on Drosophi l a melanogaster where researchers have discovered the induction of innate antibacterial (pattern recognition receptor proteins) and antiviral (RNA interference and silencing) mechanisms upon parasite infection ( Schmid Hempel, 2005; Ke m p and Imler, 2009; Sabin et al., 2010). Further studies like those conducted by Medeiros et al (2004), who used subtractive cDNA libraries to examine the upregulation of F. occidentalis gene activity upon TSWV infection, will be needed for validation of this hypothetical mechanism (see Chapter 6). Elucidating the mechanisms behind how T. fuscum disrupts the plant pathogens P. ananatis and TSWV within the F. fusca vector may offer novel biological -based management strategies for controlling plant disease spread by Thysanoptera (see Chapter 6)
194 Ta ble 5 1. The percentage of individual females containing bacteria cultivable on trypic soy broth agar plates from non -T. fuscum parasitized and T. fuscum parasitized Frankliniella fusca laboratory and field populations. Location F. fusca with bacteria F. fusca with bacteria harboring phenotype A F. fusca with bacteria harboring phenotype B F. fusca with bacteria harboring other phenotypes F. fusca with bacteria infected with > 1 phenotype Non parasitized F fusca Laboratory (n=39) 85% (33) 97% (32) 6% (2) 0 3% (1) Field (n=34) 71% (24) 79% (19) 33% (8) 4% (1) 13% (3) Parasitized F. fusca Laboratory (n=25) 56% (14) 100% (14) 7% (1) 0 7% (1) Field (n=16) 69% (11) 91% (10) 9% (1) 9% (1) 9% (1) The numb er in parentheses represents the number of F. fusca within each category. Table 5 2. The mean number of Pantoea ananatis colony forming unit counts standard deviation for early and late stage Frankliniella fusca plated individuals, based on Thripin ema fuscum parasitism status Treatment (314) 7 days after exposure to P. ananatis 12 days after exposure to P. ananatis % of F. fusca with P. ananatis Non parasitized F. fusca (102) 1.8 x 10 6 1.06 x 10 7 a 1.0 x 10 6 1.76 x 10 6 a 85% (87) Paras itized F. fusca (99) 2.5 x 10 5 1.05 x 10 6 b 1.5 x 10 5 2.81 x 10 5 b 52% (51) Exposed, non parasitized F. fusca (97) 1.9 x 10 6 7.5 x 10 6 a 4.5 x 10 6 2.39 x 10 7 a 86% (113) Numbers within the column followed by the same letter are not significantly different acco rding to least squares mean (p Numbers in parentheses corresponds to the number of individuals within each category.
195 Table 5 3. Measurements of the zone of inhibition (in mm) for lysozyme standards (g/ml) used for determining the un its of activity (EU/ml) in acid -derived T. fuscum parasitized Frankliniella fusca homogenates. stock (g/ml) Zone of inhibition (mm) Units of activity (EU/ml) 320 9 6400 160 8 3200 80 7 1600 40 6 800 20 5 400 10 4 200 5 3 100 Table 5 -4 The average threshold cycle (CT) values standard deviation for TSWV and COI transcripts and relative quantity of TSWV detected by quantitative PCR for viruliferous Frankliniella fusca cohorts. Treatment Average CT COI Average CT TSWV T T Relative increase* No n viruliferous non parasitized F. fusca 12.75 0.58 35.9 1.6 23.15 0.00 1 V iruliferous non parasitized F. fusca 13.11 0.53 22.06 3.53 8.95 14.20 18820 Viruliferous F. fusca exposed to T. fuscum 13.08 0.23 20.11 1.87 7.03 16.12 71275 Viruliferous parasitized F. fusca 13.77 0.63 27.76 7.64 13.99 9.16 573 *Expressed as a number -fold difference to the control.
196 Table 5 -5 Efficiency of Tomato spotted wilt virus transmission by non-T. fuscum parasitized and T fuscum parasitized Frankliniella fusc a individuals Treatment Number of transmissions 0 1 2 3 Non parasitized females (n=262) 25.6% (67) 25.9% (68) 35.9% (94) 12.60% (33) Parasitized females (n =144) 46.53% (67) 27.78% (40) 16.67% (24) 9.03% (13) N umbers in parentheses corresponds to the number of individuals within each category. Table 5 -6 The percentage standard error of viruliferous Frankliniella fusca cohorts transmitting Tomato spotted wilt virus during each inoculation period. Tr eatment Inoculation Access Period 1 (0 48 hr) 2 (49 96 hr) 3 (97 144 hr) Non parasitized females (n=263) 25.8 2.7a 52.7 3.1b 56.9 3.1b Parasitized females (n=145) 21.1 3.4ad 31.1 3.9ac 33.9 3.9ac Percentages within the columns followe d by the same letter are not significantly different according to differences of LS means (P<0.05).
197 Table 5 -7 The average threshold cycle (CT) values for TSWV and COI transcripts detected by quantitative PCR for individual v iruliferous Frankliniella fu sca Treatment Average CTCOI Average CTTSWV Non -parasitized F. fusca 0 (12) 16.72 1.89 34.27 3.36 1 (15) 17.36 2.25 30.3 3.73 2 (19) 16.35 1.66 28.91 2.95 3 (6) 16.73 3.40 28.84 4.39 Parasitized F. fusca 0 (11) 17.58 2.20 32.87 3 .88 1 (13) 17.14 1.63 30.92 3.79 2 (12) 17.42 2.05 30.81 2.53 3 (6) 17.82 3.40 30.07 5.63 The number in parentheses corresponds to the number of individuals within each category.
198 Figure 5 1. Transmission e lectron micrographs of the alim entary tract of a non -parasitized and Thripinema fuscum parasitized Frankliniella fusca female. (A) The midgut and (B) hindgut lumen of a non -Thripinema fuscum parasitized female; (C) The midgut and (D) hindgut of a T. fuscum parasitized female Note th e direct association of bacteria to the microvil li of midgut epithelial cells.
199 Figure 5 2. Bacteria isolated from Frankliniella fusca individuals. (A) Bacterial colonies of various bacteria isolated from F. fusca individuals growing on a Tryptic soy broth agar plate; (B) Phenotype A at 1.6X magnification; (C) Phenotype B at 1.6X magnification. Scale bar = 10 mm.
200 Figure 5 3. A series of assays testing for antimicrobial activity from non -Thripinema fuscum parasitized and T. fuscum parasitized F rankliniella fusca individuals against Pantoea ananatis (A) A c rude and (B) acid -derived homogenates tested against P. ananatis ; (C) Acid derived homogenates tested against M icrococcus lysodeikticus ; (D) lysozyme standards tested against P. ananatis s =streptomycin; c=buffer control; np=non -parasitized F. fusca ; p=parasitized F. fusca .
201 Figure 5 4. The s tandard curve used to generate units of activity for acid derived Thripinema fuscum parasitized Frankliniella fusca homogenates.
202 Figure 55. Reve rse transcriptase PCR product of Frankliniella fusca cohort extractions using TSWV and COI primers. Lane 1=Marker Hyperladder II (5 l), lanes 2,6 = nonviruliferous nonThripinema fuscum parasitized F. fusca lanes 3,7 = viruliferous nonT. fuscum parasi tized F. fusca lane 4,8 = viruliferous nonT. fuscum parasitized F. fusca exposed to T. fuscum lane 5,9 = viruliferous T. fuscum parasitized F. fusca lane 10 = TSWV negative control, lane 11 = COI negative control. Figure 5 6. PCR product testin g the suitability of using 28S as an internal control for Frankliniella fusca RNA extraction s Lane 1=Marker Hyperladder II (5 l), lane 2=viruliferous non -Thripinema fuscum parasitized F. fusca lane 3=viruliferous parasitized F. fusca lane 4=healthy A. hypogaea plant tissue, lane 5=TSWV+ A. hypogaea plant tissue, lane 6=negative control (TSWV primer), lane 7=negative control (28S primer). Loading v olume was 5 l of PCR product.
203 Figure 57. An amplification plot from a cohort qPCR reaction. The graph shows the detection of (A) COI and the TSWV N -protein for (B) viruliferous and (C) non viruliferous Frankliniella fusca 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1 2 0.258 0.211 0.572 0.326 0.526 0.354 IAP 3 IAP 2 IAP 1 Parasitized females Non parasitized females MCF of TSWV Transmission Figure 5 8. The m ean cumulative frequency (MCF) of Tomato spotted wilt virus (TSWV) transmissions for non -Thripinema fuscum pa rasitized and T. fuscum parasitized Frankliniella fusca females for each in oculation access period (IAP).
204 Figure 5 -9. A box plot of the number of Tomato spotted wilt virus (TSWV) copy numbers (data log transformed) according to the Thripinema fuscum p arasitism status of viruliferous Frankliniella fusca females. Figure 5 -10 A s catter plot of the correlation between Tomato spotted wilt virus (TSWV) copy number and ELISA optical density (OD405) values for Frankliniella fusca fema les.
205 CHAPTER 6 DA TA SYNOPSIS AND FUTU RE RESEARCH The F. fusca / T. fuscum system is an ideal model for examining the interface between a host insect and its obligate parasite. Thripinema has a direct life cycle that does not require an intermediate host; therefore, all physi ological development occurs inside the thrips. Unlike vertebrate -parasitizing nematodes, which develop within host tissues, all Thripinema life stages exist in the insect hemocoel and can be readily collected. Thripinema, unlike other entomopathogenic nematodes, depends upon its host for survival and transmission and has negligible effects on thrips longevity or mortality. It is important to emphasize that all species of thrips known to be parasitized by Thripinema are considered pests, and all Thripinema spp. appear to induce similar impacts on their host thrips; thus, the results of this project can be extended to these systems. In Chapter 2, I used Wolbachia specific 16S rRNA primers to identify whether Wolbachia strain(s) are associated with non -parasit ized female and male F. fusca T. fuscum parasitized F. fusca females, and free -living T. fuscum nematodes collect ed over time and space. I proceeded with a multigene approach ( wsp gatB, coxA, hcpA, ftsZ, fbpA ) to strain type the Wolbachia(s) associated with six select population cohorts of non parasitized F. fusca females. All populations tested were associated with one or more Wolbachia strains. I additionally determined Wolbachia impacted the arrhenotokous F. fusca population biology through a series of developmental and reproductive bioassays involving Wolbachiainfected and Wolbachiacured individuals. Transmission electron micrographs documented Wolbachia infection in the reproductive tissues of non -parasitized and T. fuscum parasitized F. fusca femal es. However, the discovery of Wolbachia in this system leaves many unanswered questions regarding the tritrophic interaction between the endosymbiont, thrips, and nematode parasite. For example, what
206 are the effects of Wolbachia on the fitness parameter s of F. fusca and T. fuscum ? Results from the mating bioassays suggest Wolbachia alters host reproduction. Further studies involving mating experiments between tetracycline treated females and males (e.g., Wolbachiacured females mated with infected and non -infected males and vice versa) are warranted to elucidate the full impact of Wolbachia on host reproduction. Studies evaluating the presence of Wolbachia on host vector competence are also needed. In order to determine the effects of Wolbachia on T. fuscum the parasite must survive in the absence of Wolbachia; however Wolbachia is an ob ligate symbiont of filarial nematodes and antibiotic treatment results in mortality of the filarial parasite It would be interesting to observe whether antibioticall ytreated parasitized F. fusca induced host mortality or cleared the host of the T. fuscum parasites and/or restored their reproductive potential. One potential pitfall for these experiments is the need to develop geneticallyidentical colonies of F. fusc a Wolbachia and T. fuscum so the researcher knows the exact Wolbachia strain(s) utilized in the laboratory (vs. genetically diverse field collections) Regardless, manipulati ng Wolbachia in F. f us ca may affect vector competence, and a s a re su lt offers potential as a biological control agent ( e.g ., removing Wolbachia in F fusca females results in t he production of all male progeny Wolbachia ma y upregulate the immune system in infected thrips t hus reduc ing in vivo plant pathogens etc. ). In Chapter 3, I conducted a thorough histological examination of non -parasitized female F. fusca T. fuscum parasitized female F. fusca and T. fuscum nematodes. Light and electron microscopy revealed significant internal damage to the fat body and reproductive structures of parasitized F. fusca Based on observations of the host system s and tissues, I hypothesize that the invasive motherworm releases effector molecules that block egg formation in the thrips panoistic ovary, down regulates vitellogenesis in the fat body, destructs the follicular epithelium
207 (apoptosis), and/or or induces h ormonal dysfunction. Results from data in Chapter 3 also suggest T. fuscum either evades or suppresses the host thrips immune system In Chapters 4 and 5, I report that T. fuscum reduces host feeding and suppresses the vector competency their thrips host by altering host behaviors and by reducing their ability to maintain plant pathogens in vivo. The major impacts of parasitism affecting host function ( i.e., reductions in feeding and vector competency) are induced by the single invasive female prior to pr oduction of first -generation progeny nematodes. Therefore I hypothesize that the parasitic motherworm interferes with host physiology by producing effector molecules, or metabolites that disrupt host metabolism are detrimental to viruses and bacteria (e.g., antimicrobials) disturb pathways involved in host behaviors, and/or signal the up regulation of the thrips innate immune system,. Again data from these two chapters support the hypothesis that an upregulation of the host immune system upon dual i nfection is active a gainst plant pathogens but not the T. fuscum parasite. Data from this project suggest the upregulation of the thrips immune system is a key factor in regulating entomoparasites in the F. fusca vector when parasitized by T. fuscum. New technologies involving high throughput sequencing technology in combination with microarray analysis would elucidate the interplay between the thrips and its obligate parasite T. fuscum and identify key gene and pathways that are specifically up and down regulated by T. fuscum The genes and/or pathways associated with host tissues altered by parasitism may provide additional targets for designing experiments that address TSW V acquisition and transmission. Presently, it is unknown the mechanisms Thri pinema utilizes to affect the behavior of F. fusca Lack of such knowledge precludes understanding as to how this host -parasite system interfaces with TSWV disease epidemiology and hinders successful implementation of this
208 important biological control age nt. I hope the research I have conducted during my time at the University of Florida opens the door to a new, exploratory avenue for controlling Thysanoptera and the pathogen s they spread.
209 APPENDIX A WOLBACHIA 16S RRNA, COI, AND M LST SEQUENCES A 1. Wolb achia specific 16S rRNA nucleotide sequences for 28 nonparasitized Frankliniella fusca parasitized F. fusca and Thripinema fuscum cohort populations >HF1_UF_09 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTACCATCAGGTAAT GCTGGGGACTTTAAGGAAACTG CCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGG AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGC TAAGCCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTA CACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >HF2_UF_09 ACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAA ACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTT ATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCG AGGCTAAACCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAG TGCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTC TCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >HF3_UF_09 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGC GCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGGACTTTAAGGAAACTG CCAGTGATAAACTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCCTTATGA AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAGGTCGCGAGGC TAACCTAATCCCTTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >PF4_CIT_09 TGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTCC CAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTTATGAA GTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGCC TAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCAT GAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAGG TCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAA >PF5_CIT_09 TGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTCC CAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTTATGAA GTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGCC TAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCAT GAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAGG TCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAA >PF6_CIT_09
210 TGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTCC CAGTGATAAGCTGGAGGAAGATGGGGA TGATGTCAAGTCATCATGGCCTTTATGAA GTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGCC TAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCAT GAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAGG TCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >HM7_UF_09 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTC CCAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTTATGA AGTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGC CTAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >TF8_CIT_09 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACT G CCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGG AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGC TAAGCCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATG GGAATTGGTTTCACTCGAA >TF9_CIT_09 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTG CCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGG AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGC TAAGCCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAA >TF10_CIT_08 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGT TACCATCAGGTAATGCTGGGGACTTTAAGGAAACTG CCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGG AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGC TAAGCCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGT TCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAA >TF11_UF_08 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTG CCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGG AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGC TAAGCCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA
211 TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAA >HF13_CIT_09 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTC CCAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTTATGA AGTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGC CTAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >HF14_CIT_09 TGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGC CAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCAT CATGGCCCTTATGGA GTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCT AAGCCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCAT GAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTA >PF17_CIT_09 TGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGGACTTTAAGGAAACTGC CAGTGATAAACTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCCTTATGAA GTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGGCGAGGCT TAACCTAACCCTTAAAAGACATCTCAGTTCGGATTG TACTCTGCAACTCGAGTGCAT GAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTA >HM18_CIT_09 TGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTC C CAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTTATGAA GTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGCC TAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCAT GAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAGG TCTTGTACACACTGCCCGTCACGCCATGG GAATTGGTTTCACTCGAAGCTA >PM19_CIT_09 GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAG TTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGA AGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTA CAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCCAATCCCTTAAAAGC CATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTA ATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGT CACGCCATGGGAATTGGTTTCACTCGAAGCTA >PM21_CIT_09 GCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAG TTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGA AGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTA
212 CAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCCAATCCCTTAAAAGC CATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTA ATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGT CACGCC ATGGGAATTGGTTTCACTCGAAGCTA >PP23_CIT_09 GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCC TTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGA GGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGT GCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCC AATCCCTTAA AAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCT AGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGC CCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTA >TF24_CIT_09 GGCTGTCAGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTCATCCTTAGTTACCATCAGGTA ATGCTGGGGACTTTAAGGAAACTGCCAGTGAT AAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCT ACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCCAA TCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTG GAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTA CACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTA >TF25_CIT_09 GGCTGTCAGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGAT AAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCT ACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCCAA TCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTG GAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTA CACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTA >HF30_CIT_08 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTC CCAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTTATGA AGTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGC CTAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGG ATCAGCATGCCACGGTGAATACGTTCTCAG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >HF31_CIT_08 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTC CCAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTT ATGA AGTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGC CTAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >HF34_UF_08
213 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTC CCAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTTATGA AGTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGC CTAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAG TGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >HF37_UN_07 CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTT AGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAG GAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTG CTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCCAATCCCTTAAA AGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTA GTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCC CGTCACGCCATGGGAATTGGTTTCACTCGAAGCTA >HF40_UF_08 TGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGAT AAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCT ACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCCAA TCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCT GCAACTCGAGTGCATGAAGTTG GAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTA CACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTA >PF41_UF_08 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTG CCAGTG ATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGG AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGC TAAGCCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCT >HF42_CIT_08 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTGCCATCAGGTAATGCTGAGCACTTTAAGGAAACTC CCAGTGATAAGCTGGAGGAAGATGGGGATGATGTCAAGTCATCATGGCCTTTATGA AGTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGCTGCAAGGTGCGCAAGC CTAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCAG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCG >PF44_CIT_08 GTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACG AGCGCAACCCTCATCCTTAGTTACCA TCAGGTAATGCTGGGGACTTTAAGGAAACTG CCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGG AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGC TAAGCCAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCA
214 TGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAA
215 A 2. Wolbachia specific 16S rRNA nucleotide sequences used for phylogenetic reconstruction of Frankliniella fusca and Thripinema fuscum populations. Supergroup designation is listed after the host name of each Wolbachi a strain. >Diaeacircumlita_A GGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTATTGCTATTAGA TGAGCCTATATTAGATTAGCTTAGTTGGTGGGGTAATGGCCTACCAAGGCAATGATC TATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACT CCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCTTGATCCAGCC AT GCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATA ATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAAT ACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCGGAT TAGTAAGTTAAAAGTGAAATCCCAAGGCTCAACCTTGGAATTGCTTTTAAAACTGCT AATCTAGAGATTGAAAGAGGATAGAGGAATTC CTAGTGTAGAGGTGAAATTCGTAA ATATTAGAGGAACACCAGTGGCGAAGCGTCTATCTGGTTCAAATCTGACGCTGAGG CGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAAC GATGAATGTTAATATGGGGA >16Spartial_A GGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAGT AATAGCCTACCAAGGCAATGATCTATAG CTGATCTGAGAGGATGATCAGCCACACT GGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACA ATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGT AAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAA CTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGG GCG TAAAGGGCGCGTAGGCGGATTAGTAAGTTAAAAGTGAAATCCCAAGGCTCAAC CTTGGAATTGCTTTTAAAACTGCTAATCTAGAGATTGAAAGAGGATAGAGGAATTCC TAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCT ATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGA TACCCTGGTAGTCCACGCTGTAAACGATGAATGT TAAATATGGGAAGTTTTACTTTC TGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAA ACTCAAAGGAATTGACG >Drosophilamelanogaster_A CATGCAAGTCGAACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGAC GGGTGAGTAATGTATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGC AACTAATACCGTATACGCC CTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTAT ATTAGATTAGCTAGTTGGTGGAGTAATAGCCTACCAAGGCAATGATCTATAGCTGAT CTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAG GCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATG AGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTA CTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGG GCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCGGATTAGTAAGTTA AAAGTGAAATCCCAAGGCTCAACCTTGGAATTGCTTTTAAAACTGCTAATCTAGAGA TTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAG GAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGG CGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATG TTAAATATGGGAAGTTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTG GGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCG
216 GTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGA CATG GAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGTCCGGGTTTCACACAGGTGTT GCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC AACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGT GATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGG GCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGC TAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAG TTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTT GTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTAACGACCTAAC CGCAAGGAGGGAG >Muscidifuraxuniraptor_A AGAGTTTGATCCTGG CTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTC GAACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAA TGTATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCG TATACNCCCTACGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCT AGTTGGTGGAGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATG ATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGG GAATATTGGACAATGGGCGAAANNNTGATCCAGCCATGCCGCATGAGTGAAGAAGG CCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAA GTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTC GGAATTATTGGGCGTAAAGGGCGCGTAGGCGGATTAGTAAGTTAAAAGTGAGATCC CAAGGCTCAACCTTGGAATTGCTTTTAAAACTGCTAATCTAGAGATTGAGAGAGGAT AGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGG CGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAA ACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGA AGTTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTC GCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTG GTTTAACTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCT ATTCGAAGGGATAGGGTCGGTTCGGCCGGGTTTCACACAGGTGTTGCATGGCTGTCG TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCT TAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAGACTGCCAGTGATAAACTGGA GGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGT GCTACAATGGTGGCTACAATGGGCTGCAAAGTGCGCGAGGCTAAGCCAATCCCTTA AAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGC TAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTG CCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTAACGACCTAACCGCAAGGAG GGAGTTATTTAAAGTGGGATCAGTGACTGGGGTG >Drosophilasechellia_A GCTCAGAATGAACTGTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATAT TGTAGCCTGCTATGGTATAACTTA GTGGCAGACGGGTGAGTAATGTATAGGAATCTA CCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGG GGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAGTA ATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAACCACACTG GAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAA TGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTA AAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAAC
217 TCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGG GCGTAAAGGGTGCGTAGGCGGATTAGTAAGTTAAAAGTGAAATCCCAATGCTTAAC CTTGGAATTGCTTTTAAAACTGCTAATCT ATAGATTGAAAGAGGATAGAGGAATTCC TAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGGGAAGGCGTCT ATCTGGTTCAAATCTGACGCTGAGGGGCGAAGGCGTGGGGAGCAAACAGGATTAGA TACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTTACTTTC TGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAA AC TCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGA TGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGA TAGGGTCGGTTCGTCCGGGTTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGT CGTGAGATGTTGGGTTAAGTCCCGCAACGATCGCAACCCTCATCCTTAGTTACCATC AGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGG ATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTG GCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGT TCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGTGGAT TAGCACGCCACGGTGAATACGTTCTCGGGTCTTGTACTCACTGCCCGTCACGCCATG GGAATTGGTTTCACTCGAAGCTAACGACCTAACCGCAAGGAGGGAGTTATTTAAAG TGGGATCGGTGACT >16Scomplete_B TGGCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTAT GTTATAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATC TACCTAGTAGTACGGAATAATTGCTGGAAACGGCAGCTAATACCGTATACGCCCTAC GGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAG TAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACT GGAACTGAGATACGGCCCAGACTTCTACGGGAGGCAGCAGTGGGGAATATTGGACA ATGGGCGAAAGCCTGATCCAGCCATGTCGCATGAGTGAAGAAGGCCTTTGGGTTGY AAAGCTCTTTTAGTGAGGAAAGATAATGACGGNACTCACAGAAGAAGTCCNGGGTT AACTCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNGGGCGTAAAGGGCGCGTAGGCTGATTAATAAGTTAAAAGTGAAATCCCGAGGC TTAACCTTGGAATTGCTTTTAAAACTATTAATCTAGAGATTGAAAGAGGATAGAGGA ATTCCTGATGTAGAGGTAAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGG CGTCTATCTG GTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGA TTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTAC TTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGAT TAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAGW TCGATGCAACGCGAAAAACTTACCACTTCTTGACATGGAAATCATACCTATTCGAAG GGATAGGGTCGGTTCTGCCGGATTTTACACAGGTGTTGCATGGCTGTCGTCAGCTCG TGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCTTCCTTAGTTGSC ATCAGGTAATGCTGAGTACTTTAAGGAAACTGCCAGTGATAAGCTGGAGGAAGGTG GGGATGATGTCAAGTCATCATGGSCTTTATGAAGTGGGCTACACACGTGCTACAATG GTGTCTACAATG GGCTGCAAGGKGCGCAAGCCTAAGCTAATCCCTAAAAGACATCT CAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGT GGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACRC CATGGGAATTGGTTTCACTCGAAGCTAATGGCCTAACCRCAAGGAAGGAGTTATTTA AAGTGGGATCAGTGACTGGGGTG >Bryobiasarothamni_B
218 AATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGC TTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGT AGTACGGAATAATTGTTGGAAACGACAACTAATACCGTATACGCCCTACGGGGGAA AAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGC CTACCAAGGTAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACT GAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGC GAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCT CTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTG CCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAA A GGGCGCGTAGGCTGGTTAATAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGA ATTGCTTTTAAAACTATTAATCTAGAGATTGAAAGAGGATAGAGGAATTCCTGATGT AGAGGTAAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGG TTCAAATCTGACGCTGAAGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCT GGTAGTCCACGCTGTAAACGATGAATGTTAAA TATGGGGAGTTTACTTTCTGTATTA CAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAA AGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAAC GCGAAAAACCTTACCACTTCTTGACATGAAAATCATACCTATTCGAAGGGATAGGGT CGGTTCGGCCGGATTTTACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTGCCATCAGGTAA TGCTGAGTACTTTAAGGAAACTGCCAGTGATAAGCTGGAGGAAGGTGGGGATGATG TCAAGTCATCATGGCCTTTATGGAGTGGGCTACACACGTGCTACAATGGTGTCTACA ATGGGCTGCAAGGTGCGCAAGCCTAAGCTAATCCCTAAAAGACATCTCAGTTCGGA TTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCA TGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAAT TGGTTTCACTCGAAGCTAATGGCCTAACCGCAAGGAAGGAGTTATTTAAAGTGGGAT CAGTGACTGGGGTG >Bryobiapraetiosa_B AATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGC TTGCTATGGTATAGCTTAGTGGCAGACGG GTGAGTAATGTATAGGAATCTACCTAGT AGTATGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAA AAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAGTAATAGC CTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACT GAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGC GAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCT CTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTG CCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAA AGGGCGCGTAGGCTGGTTAATAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGA ATTGCTTTTAAAACTATTAATCTAGAGATTGAAA GAGGATAGAGGAATTCCTGATGT AGAGGTAAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGG TTCAAATCTGACGCTGAAGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCT GGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTA CAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAA AGGAATTG ACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAAC GCGAAAAACCTTACCACTTCTTGACATGGAAATCATACCTATTCGAAGGGATAGGGT CGGTTCGGCCGGATTTTACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTGCTATCAGGTAA
219 TGCTGAGTACTTTAAGGAAACTGCCAGTGATAAGCTGGAGGAAGGTGGGGATGATG TCAAGTCATCATGGCCTTTATGGAGTGGGCTACACACGTGCTACAATGGTGTCTACA ATGGGCTGCAAGGTGCGCAAGCCTAAGCTAATCCCTAAAAGACATCTCAGTTCGGA TTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCA TGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAAT TGGTTTCACTCGAAGCTAATGGCCTAACCGCAAGGAAGGAGTTATTTAAAGTGGGAT CAGTGACTGGGGTG >Bryobiaspec_B AATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGC TTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGT AGTATGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAA AAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAGTAATAGC CTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACT GAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGC GAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCT CTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGT CCTGGCTAACTCCGTG CCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAA AGGGCGCGTAGGCTGGTTAATAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGA ATTGCTTTTAAAACTATTAATCTAGAGATTGAAAGAGGATAGAGGAATTCCTGATGT AGAGGTAAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGG TTCAAATCTGACGCT GAAGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCT GGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTA CAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAA AGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAAC GCGAAAAACCTTACCACTTCTTGACATGGAAATCATACCTATTCGAAGGGATAGGGT CGGTTCGGCCGGATTTTACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTGCTATCAGGTAA TGCTGAGTACTTTAAGGAAACTGCCAGTGATAAGCTGGAGGAAGGTGGGGATGATG TCAAGTCATCATGGCCTTTATGGAGTGGGCTACACACGTGCTACAATGGTGTCTACA ATGGGCTGCAAGGTGCGCAAGCCTAAGCTAATCCCTAAAAGACATCTCAGTTCGGA TTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCA TGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAAT TGGTTTCACTCGAAGCTAATGGCCTAACCGCAAGGAAGGAGTTATTTAAAGTGGGAT CAGTGACTGGGGTG >Tetranychusurticae_B CAGGCCTAAC ACATGCAAGTCGAACGGAGTTATATTGTAGCTTGCTATGGTGTAACT TAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTACGGAATAATTG TTGGAAACGACAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTA GATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGTAATGAT CTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGAC TCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGC CATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGAT AATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAA TACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGG TTAATAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAATTGCTTTTAAAACTAT TAATCTAGAGATTGAAAGAGGATAGAGGAATTCCTGATGTAGAGGTAAAATTCGTA
220 AATATTAGGAGGAACACCGGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTG AAGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTA AACGATGAATGTTAAATATGGGGAGTTTACTTTCTGTATTACAGC TAACGCGTTAAA CATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGAC CCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCA CTTCTTGACATGAAAATCATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTT TACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC CGCAACGAGCGCAACCCT CATCCTTAGTTGCCATCAGGTAATGCTGAGTACTTTAAG GAAACTGCCAGTGATAAGCTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCC TTTATGGAGTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGTTGCAAGGTGC GCAAGCCTAAGCTAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCG AGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACG TTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGC TAATGGCCTAACCGCAAGGAAGGAGTTATTTAAAG >Nasoniagiraulti_B CTAACACATGCAAGTCGAACGGAGTTGTACTGTAGCTTGCTATGGTATAACTTGGTG GCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGA AACGGCAACTAATACCGTATAG TCCCTANNGGGGAAAAATTTATTGCTATTAGATGA GCCTATATTAGATTAGCTAGTTGGTGGAGTAATAGCCTACCAAGGCAATGATCTATA GCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTA CGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAANNNTGATCCAGCCATGC CGCATGAGTGAAGAAGGCCTATGGGTTGTAAAGCTGTTTTAGTGAGGAAGAT AGTG ACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACG GAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGNNTGATTAG TAAGTTAAAAGTGAAATCCCAAGGCTCAACCTTGGAATTGCTTTTAAAACTGTTAAT CTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTAAAATTCGTAAATA TTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAAGC GCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACG ATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATT CCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGC ACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCT T GACATGGAAATCATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACAC AAGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTCATCCTTAGTTGCTATCAGGTAATGCTGAGTACTTTAAGGAAAC TGCCAGTGATAAGCTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCTTTAT GGAGTGGGCTACACACGTGCTACAATGGTGTCTACAATGGGTTGCAAGGTGCGCAA GCCTAAGCCAATCCCTAAAAGACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTA CATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTC GGGTCTTGTACACACTGCCCGTCACGCCATGGGAATCGGTTTCACTCGAAGCTAATG GCCTAACCGCAAGAAGGAGTTATTTAAAGTGGGATCAGTGACTGGGGTG >16Sparti al_B GCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATAT TATAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTA CCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGG GGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAGTA ACAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTG
221 GAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAA TGGGCGAAAGCCTGATCCAGCCATGCCGCATGGGTGAAGAAGGCCTTTGGGTTGTA AAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAAC TCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGG GCGTAAAGGGCGCGTAGGCTGGTTAATAAGTTAAAAGTGAAATCTCGAGGCTTAAC CTTGGAATTGCTTTTAAAACTATTAATCTAGAGATTGAAAGAGGATAGAGGAATTCC TGATGTAGAGGTAAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCT ATCTGGTTCAAATCTGACGCTGAAGCGCGAAGGCGTGGGGAGCAAACAGGATTAGA TACCCTGGTAGTCCACGCTGTAAACGATGAA TGTTAAATATGGGAAGTTTACTTTCT GTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAA CTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAT GCAACGCGAAAAACCTTACCACTTCTTGACATGGAAATCATACCTATTCGAAGGGAT AGGGTCGGTTCGGCCGGATTTTACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTC GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTGCCATCA GGTAATGCTGAGTACTTTAAGGAAACTGCCAGTGATAAGCTGGAGGAAGGTGGGGA TGATGTCAAGTCATCATGGCCTTTATGGAGTGGGCTACACACGTGCTACAATGGTGT CTACAATGGGTTGCAAGGTGCGCAAGCCTAAGCTAATCCCTAAAAGACATCTCAGTT CGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATC AGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGG GAATTGGTTTCACTCGAAGCTAATGGCCTAACCGCAAGGAAGGAGTTATTTAAAGTG GGATCAGTGACTGGGGTG >Onchoceraochengi_C GCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATGGTATAGCTTGCTAT AGTATAACTTAGTGGCAGACGG GTGAGTAATGTATAGGAATCTACCTAGTAGTACG GAACAATTGCTGGAAACGACAACTAATACCGTATACGCCCTACGGGGGAAAGATTT ATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTAGGGTAATGGCCTGCCA AGGCTATGATCTATAGCTGATCTGAGAGGATGGTCAGCCACACTGGAACTGAGATA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGC TTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTCAG TGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCA GCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGAGCA CGTAGGCTGGTTAGTAAGTTAAAAGTGAAATTCCAAAGCTTAACTTTGGAATTGCTT TTAAAACTGCTGATCTAGAGGTTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGT GAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAA TCTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGT CCACGCTGTAAACGATGAATGTTAAATATGGGGAGATTACTTTCTGTGTTACAGCTA ACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAAT TG ACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAA AACCTTACCACTCCTTGACATGGAAATTATATCTATTCGAAGGGATAGAGTCGGTTC GGCCGGGTTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTA GGTTAAGTCCCGCAACGAGCGTACCCTCATTCCTTAGTTACCATCAGGTAATGCTGG GGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGT CATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGC TGCAAAGTCGTGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGCACT CTGCAACTCGAGTGTATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACG GTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTC
222 ACT CGAAGCTAATGACCTAACCGTAAGGAAGGAGTTATTTAAAGTGGGATCAGTGA CTGGGGTGAAG >Onchocercagutturosa_C GAAGTGGCGGCAGGCCTAACACATGCAAGTCGAACGGGGTTATGGTATAGCTTGCT ATAGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTAC GGAACAATTGCTGGAAACGACAACTAATACCGTATACGCCCTACGGGGGAAAGAT T TATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTAGGGTAATGGCCTACC AAGGCTATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGAT ACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGGAAG CTTGATCCAGCCATGCCGCGTGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTCA GTGAGGAAGATAATGACGGTACTCACAGA AGAAGTCCTGGCTAACTCCGTGCCAGC AGCCGCGGTAATACGGAGAGGGCTGCCGTTATTCGGAATTATTGGGCGTAAAGAGC ACGTAGGCTGGTTAGTAAGTTAAAAGTGAAATTCCAAAGCTTAACTTTGGAATTGCT TTTAAAACTGCTGACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGG TGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAA ATCT GACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAG TCCACGCTGTAAACGATGAATGTTAAATATGGGGAGATTACTTTCTGTGTTACAGCT AACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAA TTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAA AAACCTTACCACTCCTTGACATGGAAATTATATCT ATTCGAAGGGATAGAGTCGGTT CGGCCGGGTTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTT AGGTTAAGTCCCGCAACGAGCGTAACCCTCATCCTTAGTTACCATCAGGTAATGCTG GGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAG TCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGG CTGCAAAGTCGTGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGCAC TCTGCAACTCGAGTGTATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCAC GGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTT CACTCGAAGCTAATGACCTAACCGTAAGGAA >Dirofilariarepens_C AGAACGCTGGCGGCAAGCCTAACACATGCAAGTCGAACGGGGTTATGTTATAGCTT ATGCTATAATGTACCTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTA GTACGGAATAATTGCTGGAAACGGCAGCTAATGCCGTATACGCCCTATGGGGGAAA GATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTAAGGTAATGGCT TACCAAGGCTATGATCTATAGCTGGTCTGAGAGGATGATCAGCCACACTGGAACTG AGATACGGTCCAG ACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGAGCG GAAGCTTGATCCAGCTATGCCGCGTGAGTGAAGAAGGCCCTTGGGTTGTAAAGCTCT TTCAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGC CAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAA GAGCACGTAGGCCGATCAGTAAGTTAAAAGTGAAATTCCAAAGC TTAACTTTGGAA TTGCTTTTAAAACTGCTGACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTA GAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGT TCAAATCTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTG GTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGGCTACTTTCTGTGTTAC AGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAA GGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACG CGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGAATAGGGTC
223 GGTTCAGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTAGGTTAAGTCCCGCAACGAGCGTAACCCTCATCCTTAGTTACCA TCAGGTGAT GCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGT CAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAA TGGGCTGCAAAGTTGTGAAGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATT GCACTCTGCAACTCGAGTGTATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATG CCACGGTGAATACGTTCTCG GGTCTTGTACACACTGCCCGTCACGCCATGGGAATTG GTTTCACTCGAAGCTAATCACCCAACCGTAAGGAGGGA >Brugiamalayi_D AATGAACGCTGGCGGCAGGCCTAACACATGCAAGTTGAACGGAGTTATATTATAAC GAGTTATAGTATAACTGAGTAGCAGACGGGTGAGTAATGTATAGGAATCTACCTAG TAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGA AAGATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTAGGGTAATGG CCTACCAAGGCAGTAATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAAC TGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGG CGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGC TCTTTCAGTGAGGAAGATAATGACGGT ACTCACAGAAGAAGTCCTGGCTAACTCCGT GCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTA AAGGGCGCGTAGGCTGATTAGTAAGTTAAAAGTGAAATCCCAAAGCTTACTTTGGA ATTGCTTTTAAAACTGTTAATCTAGAGGTTGAAAGAGGATAGAGGAATTCCTAGTGT AGAGGTGATATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGG T TCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCT GGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTA CAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGATCGCAAGATTAAAACTCAA AGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAAC GCGAAAAACCTTACCACTCCTTGACATGGGAATTATTCCTATTCGAAGGAATAGGGT CGGTTCGGCCGAATTTCACGCAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGTAACCCTCATCCTTAGTTACCATCAGGTAA TGCTGGGGACTTTAAGGAAACTGCTAGTGATAAACTGGAGGAAGGTGGGGATGATG TCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACA ATGG GCTGCAAAGTCGCGAGGCCAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGAT TGTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCAT GCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATT GGTTTCACTCGAAGCTAATGACCTAACCGTAAGGAGGGAGTTATTTAAAGTGGGATC AGTGACTGGGGTGAAGA >Litomosoidess igmodontis_D TGTTTGATCCTGGCTAGAATGAACGCTGGCCGGCAGGCCTAACACATGCAAGTTGAA CGTGGTTATATTATAGCTTACTATAATATAGCTAAGTAGCAGATGGGTGAGTAATAT ATAGGAATCTACCTGGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTAT ACGCCCTACGGGGGAAAGATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAG TTGGTGGGGTAATAG CTTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATC AGCCACACTGGAACTGAGATACAGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAA TATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCT TTGGGTTGTAAAGCTCTTTCAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGT CCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTA GCGTTATTCG GAATTATTGGGCGTAAAGGGCGCGTAGGCTGATTAGTAAGTTAAGAGTGAAATCCC
224 AAAGCTCAACTTTGGAATTGCTTTTAAAACTGCTAATCTAGAGGTTGAGAGAGGATA GAGGAATTCCTAGTGTAGAGGTGATATTCGTAAATATTAGGAGGAACACCAGTGGC GAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAA CAGGATTAGATACCCTGGTAG TCCACGCTGTAAACGATGAATGTTAAATATGGGAA GTTTACTTTCTGTATTACAGCTAACGCGTTAAGCATTCCGCCTGGGGACTACGGTCG CAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGG TTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTA TTCGAAGGAATAGGGTCGGTTAGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGTAACCCTCATCCTT AGTTACCATCAGGTAATGCTGGGGACTTTAATAAAACTGCTAGTGATAAACTGGAG GAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTG CTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCCAAGCCAATCCCTTAAA AGCCATCTCAGTTCGGATTGTACTC TGCAACTCGAGTGCATGAAGTTGGAATCGCTA GTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCC CGTCACGCCATGGGAATTGGTTTCACTCGAAACTAATGACCTAACCGTAAGGAAGG AGTTATTTAAAGTGGGATCAGTGACTGGGGTGAAGTCGTAACAAGGTAGCAGTAGG GGAATCTGCAGCTGG >16Spartial_E AATGAACGCTGGCGGCAGGCCTAAC ACATGCAAGTCGAACGGAGTTATATTGTAGC TTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGT AGTACGGAATAATTGCTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAA AAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTTGTTGGTGGGGTAATGGC CTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAAC T GAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGC GAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTCGGGTTGTAAAGCT CTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTG CCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATCATTGGGCGTAA AGGGCGCGTAGGCGGATTAGTAAGTTAAAAGTGAAATCCCAAGGCTCAACCTTGGA ATTGCTTTTAAAACTGCTAGTCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGT AGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGG TTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCT GGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTA CAGC TAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAA AGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAAC GCGAAAAACCTTACCACTTCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGT CGGTTCGGCCGGGTTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT CATCCTTAGTTACCATCAGGTAA TGCTGGGGACTTTAAGGAAACTGCTAGTGATAAACTGGAGGAAGGTGGGGATGATG TCAAGTCATCATGGCCCTTATGAAGTGGGCTACACACGTGCTACAATGGTGGCTACA ATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGAAT TGCACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCAT GCCACG GTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATT GGTTTCACTCGAAGCTAACGACCTAACCGCAAGGAGGGAGTTATTTAAAGTGGGAT CGGTGACTGGGGTGAAGTCGTAACAAGGTAGCGGTAGGGGAATCA >Mesaphoruramacrochaeta_E GGCAGGCCTAACACATGCAAGTCGAACGGAGTTATGTTGTAGCTTGCTATGATGTAA
225 CTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTACGGAATAATT GTTGGAAACGACAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATT AGATGAGCCTATATTAGATTAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCAATGA TCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGA CTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCT GATCCAG CCATGCCGCATGAGTGAAGAAGGCCTTCGGGTTGTAAAGCTCTTTTAGTGAGGAAG ATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGT AATACGGAGAGGGCTAGCGTTATTCGGAATCATTGGGCGTAAAGGGCGCGTAGGCG GATTAGTAAGTTAAAAGTGAAATCCCAAGGCTCAACCYTGGAATTGCTTTTAAAACT GCTAGTCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCG TAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCT GAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGT AAACGATGAATGTTAAATATGGGAAGCTTGCTTTCTGTATTACAGCTAACGCGTTAA RCATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGA CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACC ACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGGTT TCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAATC CCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAA GGAAACTGCTAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGC CCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAACGAGCTGCAAAGT CGCGAGGCTAAGCTAATCTCTTAAAAGCCATCTCAGTTCGAATTGCACTCTGCAACT CGAGTGCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATA CGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAA G CTAACGACCTAACCGCAAGGAGGGAGTTATTAAAGTGGGATCGGTGACTGGGGTG >Microcerotermessp_F GCAAGTCGAACGGGGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGG TGAGTAATGTATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACT AATACCGTATACGCCCTACGGGGGAAAGATTTATCGCTATTAGATGAGCCTATATTA GATTAGCT AGTTGGTAAGGTAATGGCTTACCAAGGCAATGATCTATAGCTGATCTGA GAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCTTACGGGAGGCAG CAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTG AAGAAGGCCTTTGAGTTGTAAAGCTCTTTCGGTGAGGAAGATAATGACGGTACTCAC AGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAG CGTTATTCGGAATTATTGGGCGTAAAGAGCGCGTAGGCTGGTTAGTAAGTTAAAAGT GAAATCCCAAAGCTCAACTTTGGAATTGCTTTTAAAACTGCTAACCTAGAGATTGAA AGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACA CCAGTGGCGAAGGCGTCTATCTAGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGG GGAGCAAACAGG ATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAA TATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGAC TACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGA GCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAAT CATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATG GCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGTAACCC TCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCTAGTGATAA ACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTAC ACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATC
226 CCTTAAAAGCCATCT CAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGA ATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACA CACTGCCCGT >Rhinocyllusconicus_F CAUGGCUCAGAUUGAACGCUGGCGGCAGGCUUAACACAUGCAAGUCGAACGGGG UUAUAUUGUAGCUUGCUAUGGUAUAACUUAGUGGCAGACGGGUGAGUAAUGUAU AGGAAUCUACCUAGUAGUACGGAAUAAUUGUUGGAAACGGCAACUAAUACCGUA UACGCCCUACGGGGGAAAGAUUUAUUGCUAUUAGAUGAGCCUAUAUUAGAUUAG CUAGUUGGUAAGGUAAUGGCUUACCAAGGCAAUGAUCUAUAGCUGAUCUGAGAG GAUGAUCAGCCACACUGGAACUGAGAUACGGUCCAGACUCCUACGGGAGGCAGCA GUGGGGAAUAUUGGACAAUGGGCGAAAGCCUGAUCCAGCCAUGCCGCAUGAGUG A AGAAGGCCUUUGGGUUGUAAAGCCCUUUCGGUGAGGAAGAUAAUGACGGUACU CACAGAAGAAGUCCUGGCUAACUCCGUGCCAGCAGCCGCGGUAAUACGGACAGGG CUAGCGUUAUUCGGAAUUAUUGGGCGUAAAGAGCGCGUAGGCUGGUUAGUAAGU UAAAAGUGAAAUCCCAAAGCUCAACUUUGGAAUUGCUUUUAAAACUGCUAACCU AGAGAUUGAAACAGGAUAGAGGAAUUCCUAGUGUAGAGGUGAAAUUCGUAAAUA UUAGGAGGAACACCAGUGGCGAAGGCGUCUAUCUGGUUCAAAUCUGACGCUGAG GCGCGAAGGCGUGGGGAGCAAACAGGAUUAGAUACCCUGGUAGUCCACGCUGUA AACGAUGAAUGUUAAAUAUGGGAAGUUUACUUUCUGUAUUACAGCUAACGCGUU AAACAUUCCGCCUGGGGACUACGGUCGCAAGAUUAAAACUCAAAGGAAUUGACG GGAACCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAUGCAACGCGAAAAAC CUUACCACUUCUUGACAUGGAAAUCAUACCUAUUCGAAGGGAUAGGGUCGGUUC GGCCGAUUUCACACAGGUGUUGCACGGCUGUCGUCAGCUCGUGUCGUGAGAUGU UGGGUUAAGUCCCGCAACGAGCGUAACCCUCAUCCUUAGUUACCAUCAGGUAAUG CUGGGGACUUUAAGGAAACUGCUAGUGAUAAACUGGAGGAAGGUGGGGAUGAUG UCAAGUCAUCA UGGCCCUUAUGGAGUGGGCUACACACGUGCUACAAUGGUGGCU ACAAUGGGCUGCAAAGUCGCGAGGCUAAGCUAAUCCCUUAAAAGCCAUCUCAGUU CGGAUUGUACUCUGCAACUCGAGUGCAUGAAGUUGGAAUCGCUAGUAAUCGUGG AUCAGCAUGCCACGGUGAAUACGUUCUCGGGUCUUGUACACACUGCCCGUCACGC CAUGGCCCUUGGUUUCACUCGAAGCUAAUGACCUAACCGCAAGGAGGGAGUUAU UUAAAGUGGGAUCAGUGACUGGGGUGAAGUCGUAACAAGGUAACCGUAGGGGGA A >Kalotermesflavicollis_F GCAAGTCGAACGGGGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGG TGAGTAATGTATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACT AATACCGTATACGCCCTACGGGGGAAAGATTTATTGCTATTAGATGAGCCTATATT A GATTAGCTAGTTGGTAAGGTAATGGCTTACCAAGGCAATGATCTATAGCTGATCTGA GAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAG CAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTG AAGAAGGCCTTTGGGTTGTAAAGCTCTTTCGGTGAGGAAGATAATGACGGTACTCAC AGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAG CGTTATTCGGAATTATTGGGCGTAAAGAGCGCGTAGGCTGGTTAGTAAGTTAAAAGT GAAATCCCAAAGCTCAACTTTGGAATTGCTTTTAAAACTGCTAACCTAGAGATTGAA AGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACA CCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGG GGA GCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAA
227 TATGGGAAGTTTACTTTCTGTATTACAGCTAACACGTTAAACATTCCGCCTGGGGAC TACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGA GCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAAT CATACCTATTCGAAGGGATAGGGTCGGTTCGGC CGGATTTCACACAGGTGTTGCATG GCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGTAACCC TCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCTAGTGATAA ACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCCAC ACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATC CCTTAA AAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTGCATGAAGTTGGA ATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACA CACTGCCCGT >Zootermopsisangusticollis_H GGAGAGGGCTAGCGTTATTCGGAATCATTGGGCGTAAAGGGCGCGTAGGCGGATTA GTAAGTTAAAAGTGAAATCTCAAGGCTTAACCTTGGAATTGCTTTTAAAACT GCTAG TCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAAT ATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGG CGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTGAAC GATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACAT TCCGCCTGGGGATTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCG CACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCC TTGACATGGAAATTATACCTGTCCGAAGGGATAGGGTCGGTTCGGCCAGATTTCACA CAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA ACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAA CTGCTAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTAT GGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAG GCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGAATTGTACTCTGCAACTCGAGTG CATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTC GGGTCTTGTACACACTGCCCGTA >Zo otermopsisnevadensis_H GGAGAGGGCTAGCGTTATTCGGAATCATTGGGCGTAAAGGGCGCGTAGGCGGATTA GTAAGTTAAAAGTGAAATCTCAAGGCTTAACCTTGGAATTGCTTTTAAAACTGCTAG TCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAAT ATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGG CGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTGAAC GATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACAT TCCGCCTGGGGATTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCG CACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCC TTGACATGGAAATTATACCTGTCCGAAGGGATAGGGTC GGTTCGGCCAGATTTCACA CAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA ACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAA CTGCTAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTAT GGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAG GCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGAATTGTACTCTGCAACTCGAGTG CATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTC GGGTCTTGTACACACTGCCCGT >Ctenocephalidesfelis_I
228 GGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTACG GAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAATTTA TTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAGTAACAGCCTACCAA GACAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATAC GGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAGGCC TGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGT GAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAG CCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGC GTAGGCTGATTAGTAAGTTAAAAGTGAAATCCCAAGGCTCAACCTTGGAATTGCTTT TAAAACTGCTAATCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGGCTGA AATTCGTAATATTAGGAGGAACACCCAGTGGCGAAGGCGTCTATCTGGTTCAAA TCT GACGCTGAGCCGCCAAGGCCTGGGGAGCAAACAAGGATTAGATACCCTGGTAGTCC ACGCTGTAAACGATGAATGTTAAATATGGGGAGTTTACTTTCTGTATTACACCTAAC GCGTTAACCATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTG ACGGGGACCCGCACAAGTGGTGGAGCATGTAGTTTAATTAGATGCAACACGAAAAA CCTTACCATTCCTTGACATGGAAATTAT ACGTATTGGAAGGGATAGGGTCGGTTCGG CCGGGTTTCACACAGGTGTTGCATGGCTGTCGTCAGCTAGTGTCGTGAGATGTTGGG TTAAGTCCCGCAATGAGGGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGG ACTTTAAGGAGACTGCCAGTGATGAACTGGAGGAAGGTGGGGATGATGTCAAGTCC TCATGACCCTTACGGGCTGAGCTACACACGTGCTACAATGGTGGCTACAATGGGCTG CAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCA GCAACTCGAGTGCATGAAGTAGGAATCGCTAGTAATCGTGGATCAGCACGCCCCGG TGAATACGTTCTCGGGTTTTGTACACGCTGAATGTCACCCCTTGGTTAACCTTGTTAC CACAY >Orchopeasleucopus_I GGTATAACTTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTACG GAATGATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAATTTA TTGCTATTAGATGAGCCTATATTTGGATTAGTTGGTTGGTGGAGTAATAGCCTACCA AGACAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATA CGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAGGC CTGATCCAGCCATGCCGCATGAGTGAAGAAGGC CTTTGGGTTGTAAAGCTCTTTTAG TGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCA GCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCG CGTAGGCTGATTAGTAAGTTAAAAGTGAAATCCCAAGGCTCAACCTTGGAATTGCTT TTAAAACTGCTAATCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGCGTG AAATTCGTAAATATTAGGAGGAACACCCAGTGGCGAAGGCGTCTATCTGGTTCAAA TCTGACGCTGAGCCGCCAAGGCCTGGGGAGCAAACAAGGATTAGATACCCTGGTAG TCCACGCTGTAAACGATGAATGTTAAATATGGGGAGTTTACTTTCTGTATTACACCT AACGCGTTAACCATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAA TTGACGGGGACCCGCACAAGTGGTGGAGCATGTAGTTT AATTAGATGCAACACGAA AAACCTTACCATTCCTTGACATGGAAATTATACGTATTGGAAGGGATAGGGTCGGTT CGGCCGGGTTTCACACAGGTGTTGCATGGCTGTCGTCAGCTAGTGTCGTGAGATGTT GGGTTAAGTCCCGCAATGAGGGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTG GGGACTTTAAGGAGACTGCCAGTGATGAACTGGAGGAAGGTGGGGATGATGTCAAG TCCTCATGACC CTTACGGGCTGAGCTACACACGTGCTACAATGGTGGCTACAATGGG CTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTAC
229 TCAGCAACTCGAGTGCATGAAGTAGGAATCGCTAGTAATCGTGGATCAGCACGCCC CGGTGAATACGTTCTCGGGTTTTGTACACGCTGAATGTCACCCCTTGGTTAACCTTGT TACCAC >16Spartial_J GAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAACGGGATTTGTTATAGCTTGCT ATAATATAATCTAGTGGCAGACGGGTGAGTAATGTATAGGAATCTACCTAGTAGTAC GGAATAATTATTGGAAACGATAACTAATACCGTATACGCCCTACGGGGGAAAGATT TATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTAGGGTAATGGCCTGCC AAGGCTATAATCTATAGCTGATCTGAGAGGATGGTCAGCCACACTGGAACTGAGAT ACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGAGCGAAAG CTTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTCA GTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGC AGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGAGC ACGTAGGCTGGTTAGTAAGTTAAAAGTGAAATTCCAAAGCTTAACTTTGGAATTGCT TTTAAAACTGTTAATCTAGAGGTTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGG TGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAA ATCTGACGCTGAGGTGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAG TCCACGCTGTAAACGATGAATGTTAAATATGGGGAGGATACTTTCTGTATTAT AGCT AACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAA TTGACGGGGACCTGCACAAGCGGTGGACCATGTGGTTTAATTCGATGCAACGCGAA AAACCTTACCACTCCTTGACATGGAAATTGTACCTATTCGAAGGGATAGGGTCGGTT TGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTT AGGTTAAGTCCCGCAACGAGCGTAAC CCTTATCCTTAATTGCCATCGGGTGATGCTG GGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAG TCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGTTACAATGGG CTGCAAAGTCGTGAGGCTAAGCTAATCTCTTAAAAGCCATCTCAGTTCGGATTGCAC TCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCC AC GGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTT CACTCGAAGCTAATGACCTAACCGCAAGGAGGGA >Bryobiaspec_K AATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACAGAGTTATATTGTAGC TTGCTATAGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGT AGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAA AGATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTAAGGTAATTGC TTACCAAGGCAGTGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACT GAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGC GAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTCGGGTTGTAAAGCT CTTTTAGTG AGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTG CCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAA AGAGCGCGTAGGCTGATTAGTAAGTTAAAAGTGAAATCCCAGGGCTTAACTTTGGA ATTGCTTTTAAAACTGCTAGTCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGT AGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGG TTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCT GGTAGTCCACGCTGTCAACGATGAATGCTAAATATGGGAAGGTTACTTTCTGTATTA CAGCTAACGCGTTAAGCATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAA AGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAAC
230 GCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGT CGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCACCAGGTAA TGCTGGGGACTTTAAGGAAACTGCTAGTGATAAACTAGAGGAAGGTGGGGATGATG TCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGTTACA ATGGGCTGCAAGGTTGTAAGACTGAGCTAACCCCTTAAAAGCCATCTCAGTTCGGAT TGTTCTCTGCAACTCGAGAGCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCAT GCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATT GGTTTCACTCGAAGCTAACGACCTAACCGCAAGGAGGGAGTTATTTAAAGTGGGAT CGGTGACTGGGGTG
231 A 3. Nucleotide sequence and amino acid profile for Wolbachia surface coat protein ( wsp ) non-parasitized Frankliniella fusca cohort populations. Italicized nucleotides represent the forward primer. Sample #11 > wsp TGCAGCAGGTGAATTTTTACCTCTTTTCACAAAAGTTGAT GGTATTACCTATAA GAAAGACAAGATTGATTACAGTCCATTAAAACCGTCTTTTATAGCTGTGGTGGG GGCGTTTGGTTACAAAATGGACGACATCAGGGTTGATGTTGAAGGAGTTTATT CATACCTAAACAAAAATGATTTTAAAGGTGTAACATTTCACCCAGCAAATACTA TTGCAGACAGTGTAACAGCAATTTCAGGATTAGTGAACGTGTATTACGATATAG CAATTGAAGATATGCCTATCACTCCAT ACATTGGTGTTGGTGTTGGTGCGGCGT ATATTAGCACTCCTCTGGAACCCGCTGTAAATAATCAAAAAAATAAATTTGGTT TTGCTGGTCAAGTAAAAGCTGGTGTTAGTTATGATGTAACTCCAGAAGTCAAAC TTTATGCTGGAGCTCGTTATTTCGATTCTTTTGGTTCTAACTTTGATAAAAGTAA AGAAGTAGATAAAGTGGGTGGTGGAAAAGAAATAAAAGTCACTAAAGACGCAT ATAAAGTTT TTTACAGCGCTATTGGTGCAG E F L P L F T K V D G I T Y K K D K I D Y S P L K P S F I A V V G A F G Y K M D D I R V D V E G V Y S Y L N K N D F K G V T F H P A N T I A D S V T A I S G L V N V Y Y D I A I E D M P I T P Y I G V G V G A A Y I S T P L E P A V N N Q K N K F G F A G Q V K A G V S Y D V T P E V K L Y A G A R Y F D S F G S N F D K S K E V D K V G G G K E I K V T K D A Y K V Sample #20 > wsp GAATTTTTACCTTTTTATACAAAAGTTGATGGTATTACACTATACGAAGGAAAG ATTGATTACAGTCCATTAACAACGTCTTTTACAGCGCTGGTGGTGCGGATTGGT TATAAAATGGATGACATTAGAGTTGATGTTGAAGGGCTTTACTCACAATTGGCT AAAGATACAGCTGTAGTAAATACTTCTGAAACAAATGTTGCAGACAGTTTAACA GCATTTTCAGGATTGGTTAACGTTTATTACGATATAGCGATTGAAGATATGCCT ATCACTCCATACCTTGGTGTTGGTGTTGGTGCAGCATATATCAGCAATCCTTCA AAAGCTGATGCAGTTAAAGATCAAAAAGGATTTGGTTTTGCTTATCAAGCAAAA GCTGGTGTTAGCTATGATGTAACTCCAGAAATCAAACTCTTTGCTGGAGCTCGT TACTTCGGTTCTTATGGTGCTAGTTTTGATAAGGCAACTAAGGATGATAATGGT ATCAAAAATGTTG TTTACAGCGCTATTGGTGCAG E F L P F Y T K V D G I T L Y E G K I D Y S P L T T S F T A L V V R I G Y K M D D I R V D V E G L Y S Q L A K D T A V V N T S E T N V A D S L T A F S G L V N V Y Y D I A I E D M P I T P Y L G V G V G A A Y I S N P S K A D A V K D Q K G F G F A Y Q A K A G V S Y D V T P E I K L F A G A R Y F G S Y G A S F D K A T K D D N G I K N V Sample #7 > wsp GAAATTTTACCTTTTT ATACAAAAGTTGATGGTATTACACTATGCGCA:GGA:AA GGTAAAGGACAGTCCATTAACAAGATCTTTTATAGCTGGGGTGGGGCCATTTG GTTATAAAATGGATGACATTAGAGTTGATGTTGAAGGGCTTTACTCACAATTGG CTAAAGATACAGCTGTAGTAAATACTTCTGAAACAAATGTTGCAGACAGTTTAA CAGCATTTTCAGGATTGGTTAACGTTTATTACGATATAGCGATTGAAGATATGC
232 CTATCACTCCATACCTTGGTGTTGGTGTTGGTGCAGCATATATCAGCAATCCTT CAAAAGCTGATGCAGTTAAAGATCAAAAAGGATTTGGTTTTGCTTATCAAGCAA AAGCTGGTGTTAGCTATGATGTAACTCCAGAAATCAAACTCTTTGCTGGAGCTC GTTACTTCGGTTCTTATGGTGCTAGTTTTGATAAGGCAACTAAGGATGATAATG GTATCAAAAATGTTG TTTACAGCGCTATTGGTGCAG E I L P F Y T K V D G I T L C A G K V K D S P L T R S F I A G V G P F G Y K M D D I R V D V E G L Y S Q L A K D T A V V N T S E T N V A D S L T A F S G L V N V Y Y D I A I E D M P I T P Y L G V G V G A A Y I S N P S K A D A V K D Q K G F G F A Y Q A K A G V S Y D V T P E I K L F A G A R Y F G S Y G A S F D K A T K D D N G I K N V Sample #8 > wsp GAAATTTTACCTTTTTATACAAAAGTTGATGGTATTACACTATGCGCA:GGA:AA GGTTAATTACAGTCCATTAACAAGATCTTTTATAGCTGTGGTGGGGCCATTTGG TTATAAAATGGATGACATTAGAGTTGATGTTGAAGGGCTTTACTCACAATTGGC TAAAGATACAGCTGTAGTAAATACTTCTGAAACAAATGTTGCAGACAGTTTAAC AGCATTTTCAGGATTGGTTAACGTTTATTACGATATAGCGATTGAAGATATGCC TATCACTCCATACCTTGGTGTTGGTGTTGGTGCAGCATATATCAGCAATCCTTC AAAAGCTGATGCAGTTAAAGATCAAAAAGGATTTGGTTTTGCTTATCAAGCAAA AGCTGGTGTTAGCTATGATGTAACTCCAGAAATCAAACTCTTTGCTGGAGCTCG TTACTTCGGTTCTTATGGTGCTAGTTTTGATAAGGCAACTAAGGATGATAATGG TATCAAAAATGTTG TTTACAGCGCTATTGGTGCAG E I L P F Y T K V D G I T L C A G K V N Y S P L T R S F I A V V G P F G Y K M D D I R V D V E G L Y S Q L A K D T A V V N T S E T N V A D S L T A F S G L V N V Y Y D I A I E D M P I T P Y L G V G V G A A Y I S N P S K A D A V K D Q K G F G F A Y Q A K A G V S Y D V T P E I K L F A G A R Y F G S Y G A S F D K A T K D D N G I K N V Sample #2 > wsp GAAATTTTACCTTTTTATACAAAAGTTGATGGTATTACAATATGCACA:GGT:AA AGAAAAGGACAGTCCATTAACAAGATCTTTTATAGCTGGTGGGGGGGCATTTG GTTATAAAATGGATGACATTAGAGTTGATGTTGAAGGGCTTTACTCACAATTGG CTAAAGATACAGCTGTAGTAAATACTTCTGAAACAAATGTTGCAGACAGTTTAA CAGCATTTTCAGGATTGGTTAACGTTTATTACGATATAGCGATTGAAGATATGC CTATCACTCCATACCTTGGTGTTGGTGTTGGTGCAGCATATATCAGCAATCCTT CAAAAGCTGATGCAGTTAAAGATCAAAAAGGATTTGGTTTTGCTTATCAAGCAA AAGCTGGTGTTAGCTATGATGTAACTCCAGAAATCAAACTCTTTGCTGGAGCTC GTTACTTCGGTTCTTATGGTGCTAGTTTTGATAAGGCAACTAAGGATGATAATG GTATCAAAAATGTTG TTTACAGCGCTATTGGTGCAG E I L P F Y T K V D G I T I C T G K E K D S P L T R S F I A G G G A F G Y K M D D I R V D V E G L Y S Q L A K D T A V V N T S E T N V A D S L T A F S G L V N V Y Y D I A I E D M P I T P Y L G V G V G A A Y I S N P S K A D A V K D Q K G F G F A Y Q A K A G V S Y D V T P E I K L F A G A R Y F G S Y G A S F D K A T K D D N G I K N V Sample #6
233 > wsp GAAATTTTACCTTTTTATACAAAAGTTGATGGTATTACAATATGCACAGGTAAA GAAAAGGATAGTCCCTTAACAAGATCTTTTATAGCTGGTGGGGGGGCATTTGG TTATAAAATGGATGACATTAGAGTTGATGTTGAAGGGCTTTACTCACAATTGGC TAAAGATACAGCTGTAGTAAATACTTCTGAAACAAATGTTGCAGACAGTTTAAC AGCA TTTTCAGGATTGGTTAACGTTTATTACGATATAGCGATTGAAGATATGCC TATCACTCCATACCTTGGTGTTGGTGTTGGTGCAGCATATATCAGCAATCCTTC AAAAGCTGATGCAGTTAAAGATCAAAAAGGATTTGGTTTTGCTTATCAAGCAAA AGCTGGTGTTAGCTATGATGTAACTCCAGAAATCAAACTCTTTGCTGGAGCTCG TTACTTCGGTTCTTATGGTGCTAGTTTTGATAAGGCAACTAAGG ATGATAATGG TATCAAAAATGTTG TTTACAGCGCTATTGGTGCAG E I L P F Y T K V D G I T I C T G K E K D S P L T R S F I A G G G A F G Y K M D D I R V D V E G L Y S Q L A K D T A V V N T S E T N V A D S L T A F S G L V N V Y Y D I A I E D M P I T P Y L G V G V G A A Y I S N P S K A D A V K D Q K G F G F A Y Q A K A G V S Y D V T P E I K L F A G A R Y F G S Y G A S F D K A T K D D N G I K N V V Y S A I G A
234 A4 Nucleotide sequences of six non parasitized Frankliniella fusca cohort populations at the provided loci for the MLST. Italicized nucleotides represent the forward primer and u nderlined nucleotides represent regions of polymorphism Sample #11 > gatB GAAGCTGCAGAATGCATGAAAAAATTGAGGCAGATTTTGCGTTATATTGGTTCA TGTGATGGTGATATGGAAAAGGGATCACTGCGTTGTGATGCAAATGTTTCTGTC CGCCTAAAAGGC AA TAACACATTTGGCACTCGTTGTGAGATAAAAAATCTGAAC TCGATACGTTATATTGTGCAAGCTATAGAA TATGAAATACAAAGACAAATTGAA ATTTTAGAAAGTGGG A AAGAAATAAGTCAAGATACCTTATT A TTTGAT GTTGCT TCGGGAAAAACAAAAGTGATGCGAAACAAAGAAGATGCAAGCGACTATAGATA CTTCCCTGAGCCTGATTTATTACCTGTT A AGGTAAGCCAGGATAAAATCGATT CT ATTCAA TCATCTTTACCCGAGTTACCA >fbpA GCTGGT ATGCTCCC G CTTATTTTGAAACTTAATAGT G CCAACTCCTTACATTCG AAGAATCTGACTTCTGATCAAGCAATAACCTCTTCTGTGAAAGATGCACTGCGT TTGGGCTGCTTGGCTGTTGGATTTACTATATATCCTGGTTCTGCTAAGTGTTTC GATATGATGGAAGAAGCCCGTGAAATCGTAGCTGAAGCCAAATCTTATGGGCT TGCAGTG GTGTTATGGTCTTATCCACGCGGTGAG GGAATTTCCAAAGAAGGTG AAACAGCGGTTGATGTTATTGCCTATGCTGCACACATGGCAGCTTTGCTTGGCG CTAATATAATAAAAGTAAAACTTCCAACTAAATATTTGGAAAGGGAGAAAATAG AAACAGAAAATATTGAG TCATTATCTAAAAGAATTGAATATGTTAAAAGATCT T GTTTTGCAGGAAAAA GAATAGTAATTTTCTCTGGCGG >c oxA AT GCGCCAAAGGTATGTTATTAACTAAGATGCCACTGTTTGTTTGGTCTAGTCT TAGCTAACAGCATTTATGTTGATTGTTGCCTTACCAGTGCTTGCCGGTGCTATA ACTATGCTTCTTACTGATCGCAATGTTGGTACTTCCTTTTTTGATCCTGCAGGT GGTGGTGACCCTGTGTTATTTCAACATTTATTTTGGTTTTTTGGTCATCCAGAA GTTTACGTAATTATTTTTCCTGCATTTGGCATCATAAGTCAGGTTGTATCAACTT TTTCTCACAGACCTGTATTTGGTTACATAGGGATGGTTTATGCAATGATAGGTA TAGCAGTATTTGGCTTTATGGTTTGGGCTCATCATATGTTCACTGTTGGGCTTA GTGCTGACGCTGCTACATTTTTTAGCACTACCACAATTTTTATCGGTGTTATA ACT GGCGTCAAAGTCTTTAG >ftsZ GG C GGTACTGGAACCGGTGCAGCACCGGTAATTGCAAAAGCAGCT AGAGAAGC AAGAGCCGCAGTTAAGGATAGAGCGCCAAAAGAAAAAAAGATATTGACTGTTG GAGTTGTAACTAAACCGTTCGGTTTTGAAGGTGTGCGCCGTATGCGCATTGC G GAGCTTGGACTTGAAGAACTGCAAAAATA T GTGGATACACTTATTGTCATTCCA AATCAGAATTTATTTAGAATTGCAAATGAAAAAACTACATTTTCTGATGCATTTA AACTTGCTGATAATGTTCTGCACATTGGCATCAGAGGAGTAACTGACTTGATGG TCATGCCAGGGCT G ATCAATCTTGACTTCGCTGATATAGAAACAGTAATGAGCG AGATGGGCAAAGCGATGATCGGCACCGGAGA G GCAGAAGGAGAGGATAGA T C AATTAGCGCTGCAGAGGCTGCA ATATCTAATCCATTGCTTGAT Sample #20 > gatB
235 GAAGCTGCAGAATTCATGAAAAAATTGAGGCAGATTTTGCGTTACATCGGTTCA TGTGATGGTGATATGGAAAAGGGGTCACTTCGCTGTGATGCAAATGTTTCTGTT CGCCCAAAGGGCAGTAGCACATTTGGCACTCGTTGTGAAATAAAAAACTTAAAT TCAATACGTTATATTGTACAAGCTATAGATTATGAAGCACAAAGGCAGATCAAA ATTTTGGAAAGCGGAGGAGAAATAAGTCAAGATACCTTATTGTTTGATGTCACT TTAGGAAAAACAAAAGTGATGAGAAGCAAAGAAGATTCAAGTGACTATAGATA TTTCCCTGAACCTGATTTGCTACCTGTTGAAATAAGCCAAGACAAAATTGATTCT ATTAAATCATCTTTACCCGAGTTACCA >hcpA GATCCCGAACTCAATCCACGTCTTCGCTCTGCTATCTTTGCTGCGCGAAAGGAA AATCTACCAAAAGATAAAATAGAAACAGCAATAAAAAATGCAGCTGGTAACGTT GCTGGAGAAAGTTATGAAGAAATACAATATGAAGGCTGCGGACCTTCTGGTGC TGCACTTATTGTCCAT GCTCTGACAAATAATCGCAACCGAACTGCTTCTGAGAT ACGTTATATCTTTTCTCGCAAAGGCGGTAATTGGGGAGAAACAGGATGTGTGA GTTACCTTTTCGATCATGTAGGCTTAATTGTCTATAAAGTAGAGGGTATAAATT TTGAAGATTTATTTAACTATGGAATTGAATTAGAAGTATTGAATGTTGAGGAAA ATAACAAAGAAGAATTATATGTTATAACTTGTGAAGTAAAAGACTTTGGTAAAG TAC GTGACGCTTTC TATACAAAATTTGGAGAAC CAGAACTTGCTCAACTTTC >c oxA ATGC GTGCAAA AGGTATGTCACTAACTAAGATACCACTATTTGTTTGGTCTATT TTACTCACGTCATTTATGTTAATTGTTGCCTTACCGGTACTTGCTGGTGCTATA ACTATGCTGCTCACTGATCGTAATATAGGTACCTCCTTTTTTGATCCTGCTGGT GGTGGTGATCCTGTGTTATTTCAACATCTGTTTTGGTTTTTTGGTCATCCAGAA GTTTACATAATTATTTTTCCTGCATTTGGTATCATCAGTCAGATTGTGTCAACTT TTTCTCATAGGCCAGTATTTGGTTATATGGGAATGGTTTATGCCATGATAGGAA TAGCAACGTTTGGCTTCATGGTTTGGGCTCACCATATGTTTACTGTTGGGCTTA GCGAG A ATGCTGCTATATTTTTTAGCACTAGCACAATTTTCATTGGTGTTATA ACT GGCGTCAAAGTCTTTAG >ft sZ GGTGGTACTGGAACAGGTGCTGCACCGGTAATTGCAAAAGCAGC A AGAGAAGC AAGAGCGGTAGTTAAAGATAAAGGAGCAAAAGAAAAAAAGATACTGACTGTTG GAGTTGTAACTAAGCCGTTCGGTTTTGAAGGTGTGCGACGTATGCGCATTGCA GAGCTTGGACTTGAAGAGTTGCAAAAATACGTAGATACACTTATTGTCATTCCC AATCAAAATTTATTTAGAATTGCTAACGAGAAAACTACAT TTGCTGACGCATTT CAACTCGCCGATAATGTTCTGCATATTGGCATAAGAGGAGTAACTGATTTGATG ATCATGCCAGGACTGATTAATCTTGATTTTGCTGATATAGAAACAGTAATGAGT GAGATGGGTAAAGCAATGATTGGTACTGGAGAGGCAGAAGGAGAAGATAGGG CAATTAGT GCTGCAGAGGCTGCGATATCTAATCCATTGCTTGAT Sample #7 > gatB GAAGCTGCAGAATTCATGAAAAAATTGAGGCAGATTTTGCGTTACATCGGTTCA TGTGATGGTGATATGGAAAAGGGGTCACTTCGCTGTGATGCAAATGTTTCTGTT CGCCCAAAGGGCAGTAGCACATTTGGCACTCGTTGTGAAATAAAAAACTTAAAT TCAATACGTTATATTGTACAAGCTATAGATTATGAAGCACAAAGGCAGATCAAA ATTTTGGAAAGCGGAGGAGAAATAAGTCAAGATACCTTATTGTTTGATGTCACT
236 TTAGGA AAAACAAAAGTGATGAGAAGCAAAGAAGATTCAAGTGACTATAGATA TTTCCCTGAACCTGATTTGCTACCTGTTGAAATAAGCCAAGACAAAATTGATTCT ATTAAATCATCTTTACCCGAGTTACCA >hcpA AAATACAGTTGCTGCAAA ACAAGGGCTGCCT GATCCCGAACTCAATCCACGTCTTCG CTCTGCTATCTTTGCTGCGCGAAAGGAAAATCTACCAAAAGATAAAATAGAAAC AGCAATAAAAAATGCAGCTGGTAACGTTGCTGGAGAAAGTTATGAAGAAATAC AATATGAAGGCTGCGGACCTTCTGGTGCTGCACTTATTGTCCATGCTCTGACAA ATAATCGCAACCGAACTGCTTCTGAGATACGTTATATCTTTTCTCGCAAAGGCG GTAATT G GGGAGAAACAGGATGTGTGAGTTACCTTTTCGATCATGTAGGCTTAA TTGTCTATAAAG T AGAGGGTATAAATTTTGAAGATTTATTTAACTATGGAATTG AATTAGAAGTATTGAATGTTGAGGAAAATAACAAAGAAGAATTATATGTTATAA CTTGTGAAGTAAAAGACTTTGGTAAAGTACGTGACGCTTTCTATACAAAATTTGG AGAAC CAGAACTTGCTCAACTTTC >fbpA GCTGCTCCGCTTGGTTTGATCGAAGCTGGTGCTTCAACTTAT GCTGGAATGCTACCA CTTATTTTGAAGCTTAATAGTGCTAACTCCTTGCACTCGAAAAACTTAACTTCT G ATCAAGCAATAACTGCTTCTGTAAAAGATGCGCTGCGTTTGGGTTGCGTAGCT GTTGGGTTTACTATATATCCTGGTTCTGCTAAGTGCTTCGATATGATGGAAGAA GCTCG A GAAATTATAGCTGAGGCTAAGTCTTGCGGCCTTGCCGTAGTGCTATG GTCTTATCCACGCGGTGAAGGAATTTCCAAAGAAGGC A AAACAGCAGTTGATG TTATTGCCTATGCTGCGCACATAGCAGCTTTACTTGGCGCTAATATAATCAAAG TAAAACTTCCAATTAACTATTTGGAAAGGGAGAAAATAGAAACAGAAAATATTG AGTCATTATCTAAAAGAATTGAATATGTTAAAAGATCT TGTTTTGCAGGGAAAA G AATAGTAATTTTCTCTGGCGG >c oxA ATGCGTGCAAAAGGTATGTCACTAACTAAGATACCACTATTTGTTTGGTCTATT TTACTCACGTCATTTATGTTAATTGTTGCCTTACCGGTACTTGCTGGTGCTATA ACTATGCTGCTCACTGATCGTAATATAGGTACCTCCTTTTTTGATCCTGCTGGT GGTGGTGATCCTGTGTTATTTCAACATCTGTTTTGGTTTTTTGGTCATCCAGAA GTTTACATAATTATTTTTCCTGCATTTGGTATCATCAGTCAGATTGTGTCAACTT TTTCTCATAGGCCAGTATTTGGTTATATGGGAATGGTTTATGCCATGATAGGAA TAGCAACGTTTGGCTTCATGGTTTGGGCTCACCATATGTTTACTGTTGGGCTTA GCGAGGATGCTGCTATATTTTTTAGCACTAGCACAATTTTCATTGGTGTTATA ACT GGCGTCAAAGTCTTTAG >ftsZ GGTGGTACTGGAACAGGTGCTGCACCGGTAATTGCAAAAGCAGC A AGAGAAGC AAGAGCGGTAGTTAAAGATAAAGGAGCAAAAGAAAAAAAGATACTGACTGTTG GAGTTGTAACTAAGCCGTTCGGTTTTGAAGGTGTGCGACGTATGCGCATTGCA GA GCTTGGACTTGAAGAGTTGCAAAAATACGTAGATACACTTATTGTCATTCCC AATCAAAATTTATTTAGAATTGCTAACGAGAAAACTACATTTGCTGACGCATTT CAACTCGCCGATAATGTTCTGCATATTGGCATAAGAGGAGTAACTGATTTGATG ATCATGCCAGGACTGATTAATCTTGATTTTGCTGATATAGAAACAGTAATGAGT GAGATGGGTAAAGCAATGATTGGTACTGGAGAGGCAGAAGGAGAAGATAGGG CAATTAGT GCTGCAGAGGCTGCGATATCTAATCCATTGCTTGAT Sample #8
237 > gatB GAAGCTGCAGAATTCATGAAAAAATTGAGGCAGATTTTGCGTTACATCGGTTCA TGTGATGGTGATATGGAAAAGGGGTCACTTCGCTGTGATGCAAATGTTTCTGTT CGCCCAAAGGGCAGTAGCACATTTGGCACTCGTTGTGAAATAAAAAACTTAAAT TCAATACGTTATATTGTACAAGCTATAGATTATGAAGCACAAAGGCAGATCAAA ATTTTGGAAAGCGGAGGAGAAATAAGTCAAGATACCTTATTGTTTGATGTCACT TTAGGAAAAACAAAAGTGATGAGAAGCAAAGAAGATTCAAGTGACTATAGATA TTTCCCTGAACCTGATTTGCTACCTGTTGAAATAAGCCAAGACAAAATTGATTCT ATTAAATCATCTTTACCCGAGTTACCA >hcpA GATCCCGAACTCAATCCACGTCTTCGCTCTGCTATCTTTGCTGCGCGAAAGGAA AATCTACCAAAAGATAAAATAGAAACAGCAATAAAAAATGCAGCTGGTAACGTT GCTGGAGAAAGTTATGAAGAAATACAATATGAAGGCTGCGGACCTTCTGGTGC TGCACTTATTGTCCATGCTCTGACAAATAATCGCAACCGAACTGCTTCTGAGAT ACGTTATATCTTTTCTCGCAAAGGCGGTAATTG GGGAGAAACAGGATGTGTGA GTTACCTTTTCGATCAT GTAGGCTTAATTGTCTATAAAG T AGAGGGTATAAATT TTGAAGATTTATTTAACTATGGAATTGAATTAGAAGTATTGAATGTTGAGGAAA ATAACAAAGAAGAATTATATGTTATAACTTGTGAAGTAAAAGACTTTGGTAAAG TACGTGACGCTTTC TATACAAAATTTGGAGAAC CAGAACTTGCTCAACTTTC >fbpA GCTGCTCCGCTTGGTTTGATTGAAGCTGGTGCTTCAACTTAT GCTGGAATGC TCCC G CTTATTTTGAAACTTAATAGT G CCAACTCCTTACATTCGAAGAATCTGACTTCT GATCAAGCAATAACCTCTTCTGTGAAAGATGCACTGCGTTTGGGCTGCTTGGCT GTTGGATTTACTATATATCCTGGTTCTGCTAAGTGTTTCGATATGATGGAAGAA GCCCGTGAAATCGTAGCTGAAGCCAAATCTTATGGGCTTGCAGT G GTGTTATG GTCTTATCCACGCGGTGA G GGAATTTCCAAAGAAGGTGAAACAGCGGTTGATG TTATTGCCTATGCTGCACACATGGCAGCTTTGCTTGGCGCTAATATAATAAAAG TAAAACTTCCAACTAAATATTTGGAAAGGGAGAAAATAGAAACAGAAAATATTG AG TCATTATCTAAAAGAATTGAATATGTTAAAAGATCT TGTTTTGCAGGAAAAA G AATAGTAATTTTCTCTGGCGG >coxA ATGCGTGCAAAAGGTATGTCACTAACTAAGATACCACTATTTGTTTGGTCTATT TTACTCACGTCATTTATGTTAATTGTTGCCTTACCGGTACTTGCTGGTGCTATA ACTATGCTGCTCACTGATCGTAATATAGGTACCTCCTTTTTTGATCCTGCTGGT GGTGGTGATCCTGTGTTATTTCAACATCTGTTTTGGTTTTTTGGTCATCCAGAA GTTTACATAATTATTTTTCCTGCATTTGGTATCATCAGTCAGATTGTGTCAACTT TTTCTCATAGGCCAGTATTTGGTTATATGGGAA TGGTTTATGCCATGATAGGAA TAGCAACGTTTGGCTTCATGGTTTGGGCTCACCATATGTTTACTGTTGGGCTTA GCGAGGATGCTGCTATATTTTTTAGCACTAGCACAATTTTCATTGGTGTTATA ACT GGCGTCAAAGTCTTTAG >ftsZ GGTGGTACTGGAACAGGTGCTGCACCGGTAATTGCAAAAGCAGC A AGAGAAGC AAGAGCGGTAGTTAAAGATAAAGGAGCAAAAGAAAAAAAGATACTGAC TGTTG GAGTTGTAACTAAGCCGTTCGGTTTTGAAGGTGTGCGACGTATGCGCATTGCA GAGCTTGGACTTGAAGAGTTGCAAAAATACGTAGATACACTTATTGTCATTCCC AATCAAAATTTATTTAGAATTGCTAACGAGAAAACTACATTTGCTGACGCATTT
238 CAACTCGCCGATAATGTTCTGCATATTGGCATAAGAGGAGTAACTGATTTGATG ATCATGCCAGGACTGATTAATCTTGATTTTGCTGATATAGAAACAGTAATGAGT GAGATGGGTAAAGCAATGATTGGTACTGGAGAGGCAGAAGGAGAAGATAGGG CAATTAGT GCTGCAGAGGCTGCGATATCTAATCCATTGCTTGAT Sample #2 > gatB GAAGCTGCAGAATTCATGAAAAAATTGAGGCAGATTTTGCGTTACATCGGTTCA TGTGATGGTGATATGGAAAAGGGGTCACTTCGCTGTGATGCAAATGTTTCTGTT CGCCCAAAGGGCAGTAGCACATTTGGCACTCGTTGTGAAATAAAAAACTTAAAT TCAATACGTTATATTGTACAAGCTATAGATTATGAAGCACAAAGGCAGATCAAA ATTTTGGAAAGCGGAGGAGAAATAAGTCAAGATACCTTATTGTTTGATGTCACT TTAGGAAAAACAAAAGTGATGAGAAGCAAAGAAGATTCAAGTGACTATAGATA TTTCCCTGAACCTGATTTGCTACCTGTTGAAATAAGCCAAGACAAAATTGATTCT AT TAAATCATCTTTACCCGAGTTACCA >hcpA GAAATACAGTTGCTGCAAA ACAAGGGCTGCCT GATCCCGAACTCAATCCACGTCTTC GCTCTGCTATCTTTGCTGCGCGAAAGGAAAATCTACCAAAAGATAAAATAGAAA CAGCAATAAAAAATGCAGCTGGTAACGTTGCTGGAGAAAGTTATGAAGAAATA CAATATGAAGGCTGCGGACCTTCTGGTGCTGCACTTATTGTCCATGCTCTGACA AATAATCGCAACCGAACTGCTTCTGAGATACGTTATATCTTTTCTCGCAAAGGC GGTAATTG GGGAGAAACAGGATGTGTGAGTTACCTTTTCGATCATGTAGGCTT AATTGTCTATAAAG T AGAGGGTATAAATTTTGAAGATTTATTTAACTATGGAAT TGAATTAGAAGTATTGAATGTTGAGGAAAATAACAAAGAAGAATTATATGTTAT AACTTGTGAAGTAAAAGACTTTGGTAAAGTACGTGACGCTTTCTATA CAAAATTT GGAGAAC CAGAACTTGCTCAACTTTC >fbpA GCTGCTCCGCTTGGTTTGATCGAAGCTGGTGCTTCAACTTAT GCTGGAATGCTACCA CTTATTTTGAAGCTTAATAGTGCTAACTCCTTGCACTCGAAAAACTTAACTTCT GATCAAGCAATAACTGCTTCTGTAAAAGATGCGCTGCGTTTGGGTTGCGTAGCT GTTGGGTTTACTATATATCCTGGTTCTGCTAAGTGCTTCGATATGATGGA AGAA GCTCG A GAAATTATAGCTGAGGCTAAGTCTTGCGGCCTTGCCGTAGTGCTATG GTCTTATCCACGCGGTGAAGGAATTTCCAAAGAAGGC A AAACAGCAGTTGATG TTATTGCCTATGCTGCGCACATAGCAGCTTTACTTGGCGCTAATATAATCAAAG TAAAACTTCCAATTAACTATTTGGAAAGGGAGAAAATAGAAACAGAAAATATTG AGTCATTATCTAAAAGAATTGAATATGTTAAAAGATCT TGTTTTGCAGGGAAAA G AATAGTAATTTTCTCTGGCGG >coxA ATGCGTGCAAAAGGTATGTCACTAACTAAGATACCACTATTTGTTTGGTCTATT TTACTCACGTCATTTATGTTAATTGTTGCCTTACCGGTACTTGCTGGTGCTATA ACTATGCTGCTCACTGATCGTAATATAGGTACCTCCTTTTTTGATCCTGCTGGT GGTGGTGATCCTGTGTTATTTCAACATCTGTTTTGGTTTTTTGGTCATC CAGAA GTTTACATAATTATTTTTCCTGCATTTGGTATCATCAGTCAGATTGTGTCAACTT TTTCTCATAGGCCAGTATTTGGTTATATGGGAATGGTTTATGCCATGATAGGAA TAGCAACGTTTGGCTTCATGGTTTGGGCTCACCATATGTTTACTGTTGGGCTTA
239 GCGAGGATGCTGCTATATTTTTTAGCACTAGCACAATTTTCATTGGTGTTATA ACT GGCGTCAAAGTCTTTAG >ftsZ GGTGGTAC TGGAACAGGTGCTGCACCGGTAATTGCAAAAGCAGC A AGAGAAGC AAGAGCGGTAGTTAAAGATAAAGGAGCAAAAGAAAAAAAGATACTGACTGTTG GAGTTGTAACTAAGCCGTTCGGTTTTGAAGGTGTGCGACGTATGCGCATTGCA GAGCTTGGACTTGAAGAGTTGCAAAAATACGTAGATACACTTATTGTCATTCCC AATCAAAATTTATTTAGAATTGCTAACGAGAAAACTACATTTGCTGACGCATTT CAACTCGCCGATAATGTTCTGCATATTGGCATAAGAGGAGTAACTGATTTGATG ATCATGCCAGGACTGATTAATCTTGATTTTGCTGATATAGAAACAGTAATGAGT GAGATGGGTAAAGCAATGATTGGTACTGGAGAGGCAGAAGGAGAAGATAGGG CAATTAGT GCTGCAGAGGCTGCGATATCTAATCCATTGCTTGAT Sample #6 > gatB GAAGCTGCAGAATTCATGAAAAAATTGAGGC AGATTTTGCGTTACATCGGTTCA TGTGATGGTGATATGGAAAAGGGGTCACTTCGCTGTGATGCAAATGTTTCTGTT CGCCCAAAGGGCAGTAGCACATTTGGCACTCGTTGTGAAATAAAAAACTTAAAT TCAATACGTTATATTGTACAAGCTATAGATTATGAAGCACAAAGGCAGATCAAA ATTTTGGAAAGCGGAGGAGAAATAAGTCAAGATACCTTATTGTTTGATGTCACT TTAGGAAAAACAAAAGTGATGAGAAGCAAAGAAGATTCAAGTGACTATAGATA TTTCCCTGAACCTGATTTGCTACCTGTTGAAATAAGCCAAGACAAAATTGATTCT ATTAAATCATCTTTACCCGAGTTACCA >hcpA GATCCCGAACTCAATCCACGTCTTCGCTCTGCTATCTTTGCTGCGCGAAAGGAA AATCTACCAAAAGATAAAATAGAAACAGCAATAAAAAATGCAGCTGGTAACGTT GCTGGAGAAAGTTATGAAGAAATACAATATGAAGGCTGCGGACCTTCTGGTGC TGCACTTATTGTCCATGCTCTGACAAATAATCGCAACCGAACTGCTTCTGAGAT ACGTTATATCTTTTCTCGCAAAGGCGGTAATTG GGGAGAAACAGGATGTGTGA GTTACCTTTTCGATCATGTAGGCTTAATTGTCTATAAAG T AGAGGGTATAAATT TTGAAGATTTATTTAACTATGGAATTGAATTAGAAGTATTGAATGTTGAGGAAA ATAACAAAGAAGAATTATATGTTATAACTTGTGAAGTAAAAGACTTTGGTAAAG TACGTGACGCTTTC TATACAAAATTTGGAGAAC CAGAACTTGCTCAACTTTC >fbpA GCTGCTCCGCTTGGTTTGATCGAAGCTGGTGCTTCAACTTAT GCTGGAATGCTACCA CTTATTTTGAAGCTTAATAGTGCTAACTCCTTGCACTCGAAAAACTTAACTTCT GATCAAGCAATAACTGCTTCTGTAAAAGATGCGCTGCGTTTGG GTTGCGTAGCT GTTGGGTTTACTATATATCCTGGTTCTGCTAAGTGCTTCGATATGATGGAAGAA GCTCG A GAAATTATAGCTGAGGCTAAGTCTTGCGGCCTTGCCGTAGTGCTATG GTCTTATCCACGCGGTGAAGGAATTTCCAAAGAAGGC A AAACAGCAGTTGATG TTATTGCCTATGCTGCGCACATAGCAGCTTTACTTGGCGCTAATATAATCAAAG TAAAACTTCCAATTAACTATTTGGAAAGGGAGAAAATAGAAACAGAAAATATTG AGTCATTATCTAAAAGAATTGAATATGTTAAAAGATCT TGTTTTGCAGGGAAAA G AATAGTAATTTTCTCTGGCGG >coxA
240 ATGCGTGCAAAAGGTATGTCACTAACTAAGATACCACTATTTGTTTGGTCTATT TTACTCACGTCATTTATGTTAATTGTTGCCTTACCGGTACTTGCTGGTGCTATA ACTATGCTGCTCACTGATCGTAATATAGGTACCTCCTTTTTT GATCCTGCTGGT GGTGGTGATCCTGTGTTATTTCAACATCTGTTTTGGTTTTTTGGTCATCCAGAA GTTTACATAATTATTTTTCCTGCATTTGGTATCATCAGTCAGATTGTGTCAACTT TTTCTCATAGGCCAGTATTTGGTTATATGGGAATGGTTTATGCCATGATAGGAA TAGCAACGTTTGGCTTCATGGTTTGGGCTCACCATATGTTTACTGTTGGGCTTA GCGAG A ATGCTGCTATATTTTTTAGCA CTAGCACAATTTTCATTGGTGTTATA ACT GGCGTCAAAGTCTTTAG >ftsZ GGTGGTACTGGAACAGGTGCTGCACCGGTAATTGCAAAAGCAGC A AGAGAAGC AAGAGCGGTAGTTAAAGATAAAGGAGCAAAAGAAAAAAAGATACTGACTGTTG GAGTTGTAACTAAGCCGTTCGGTTTTGAAGGTGTGCGACGTATGCGCATTGCA GAGCTTGGACTTGAAGAGTTGCAAAAATACGTAGATACACTTAT TGTCATTCCC AATCAAAATTTATTTAGAATTGCTAACGAGAAAACTACATTTGCTGACGCATTT CAACTCGCCGATAATGTTCTGCATATTGGCATAAGAGGAGTAACTGATTTGATG ATCATGCCAGGACTGATTAATCTTGATTTTGCTGATATAGAAACAGTAATGAGT GAGATGGGTAAAGCAATGATTGGTACTGGAGAGGCAGAAGGAGAAGATAGGG CAATTAGT GCTGCAGAGGCTGCGATATCTAATCCATTGCTTGAT
241 APPENDIX B RECIPES FOR ELECTRON MICROSCOPY 2.5% Gluteraldehyde Fixative 10 ml 8% Gluteraldehyde 16 ml 0.2M Cacodylate buffer 32 mg CaCl2 1% Osmium Tetroxide Fixative 1 ml 4% Osmium tetroxide 1 ml 0.3M Sucrose 2 ml 0.2M Cacodylate buffer Wrap in foil Epon Araldite Plastic (in order) 4.5 gm DDSA 2 gm 812 1 gm 502 Warm to 60C and mix well. Add 4 drops of activator after cool and Z 60 40 (100 l/10 ml) 0.2M Cacodylate Buffer 50 ml of Cacodylate buffer stock 6 ml of 0.2M HCl Double distilled w ater to make 100 ml 0.1M Cacodylate buffer/Sucrose 5 ml 0.2M Cacodylate buffer 5 ml double distilled water 0.1 gm sucrose 0.4M Cacodylate buffer 42.8 gm of Cacodylate acid (sodium salt) and double distilled water to make 500 ml Sucrose 5.1 gm sucrose 50 ml double distilled water 4% Osmium tetroxide 1 gm Osmium tetroxide 25 ml double distilled water Wrap in foil; keep dark; store at 4C 0.2M HCl 1.6 ml of HCL Double distilled water to make 100 ml
242 APPENDIX C RNA EXTRACTION, BACT ERIAL IDENTIFICATION AND MOLECULAR PROTOCOLS C 1. RNA extraction protocol for Frankliniella fusca cohorts (modified from Sigma Technical Bulletin for TRI -Reagent). 1. Homogenize tissue samples in TRI Reagent (20 thrips/250 l). 2. Centrifuge homogenate at 12,000 g for 10 minutes at 4C t o remove insoluble material. 3. Transfer supernatant to a fresh tube and allow samples to stand for 5 minutes at room temperature (RT). 4. Add 100 l of chloroform, vortex for 15 seconds, and allow samples to stand for 15 minutes at RT. 5. Centrifuge at 10,000 g for 15 minutes. 6. Transfer upper aqueous phase to a fresh tube and add 250 l of isopropanol, invert gently, and allow samples to stand for 10 minutes at RT. 7. Centrifuge tubes at 16,000 g for 10 minutes at 4C. 8. Remove supernatant and wash the RNA pellet wit h 0.5 ml of 75% ethanol. 9. Centrifuge sample at 10,000 g for 5 minutes and remove supernatant. 10. Dry the pellet for 10 minutes at room temperature. 11. Resuspend RNA in 20 l of nuclease -free water and incubate at 37C for 5 minutes.
243 C 2. Primer sequence inform ation for PCR. Primer Expected product size (in bp) Sequence TSWV N -protein 123 F(5 CATTAGGATTGCTGGAGCTGAG 3) R(5 GACACCAGAGAAGCCTTAGGAA 3) 28S 124 F(5 -GACCCGAAAGATGGTGAACTATG -3) R(5 CGATTAGTCTTTCGCCCCTATAC 3) COI 143 F(5 GTCGATTCTCGGAGCCTTAAAC 3) R(5 CCCGCTAGAACTGGAAGAGATA 3) C3a Recipe for a 20 -l volume one -step RT PCR reaction (modified from Promega Access RT -PCR System Technical Bulletin TB220). Components Volume (in l) Nuclease free water 11.6 AMV/Tfl 5X reaction buffer 4 dNT P mix (10 mM) 0.4 Forward primer (100 M) 0.5 Reverse primer (100 M) 0.5 MgSO 4 (25 mM) 1 AMV reverse transcriptase (5u/l) 0.5 T Fl DNA polymerase (5u/ l) 0.5 RNA template 1 C3b PCR thermal cycling profile used for one-step RT -PCR (modified f rom Promega Access RT -PCR System Technical Bulletin TB220). Cycle Time Temperature Step 1 60 minutes 45C Reverse transcription 1 15 minutes 70C AMV RT inactivation 1 3 minutes 94C cDNA/primer denaturation, polymerase activation 40 1 minute 94C Dena turation 1 minute 55C Annealing 2 minutes 72C Extension 1 7 minutes 72C Final extension 1 4C Short term storage
244 C 4a. Recipe for a 10 -l volume cDNA synthesis reaction (from BioR ad iScript cDNA Synthesis Kit). Components Volume (in l) 5X iScript reaction mix 2 iScript reverse transcriptase 0.5 RNA template 7.5 C 4b. PCR thermal cyc ling profile used for cDNA synthesis reactions (from iScript cDNA Synthesis Kit). Cycle Time Temperature Step 1 5 minutes 25C Activation 1 30 minutes 42C Synthesis 1 5 minutes 85C Inactivation 1 4C Short term storage C 5a. Recipe for a 20 -l volume reaction for quantitative real -time PCR (qPCR) ( modified from Quantace SensiMixPlus SYBR & Fluorescein Kit). Components Volume (in l) SensiMixPlus SYBR & Fluroescein 10 Forward primer 1 Reverse primer 1 Template 1 Nanopure water 7 C5b Thermal cycling profile used for quantitative real -time PCR (qPCR) (modified from Quantace SensiMixPlus SYBR & Fluorescein Kit). Cycle Time Temperature Step 1 10 minutes 95C Enzyme activation 40 30 seconds 95C Denaturing 30 seconds 55C Annealing 3 0 seconds 72C Extension 1 10 minutes 72C Final extension >80 10 seconds 50C, increasing each by 0.5C/cycle Melt curve
245 C6 a. MIDI analysis results for phenotype A. ECL Deviation: 0.001 Reference ECL Shift: 0.002 Number Ref erence Peaks: 7 Total Response: 323347 Total Named: 322966 Percent Named: 99.88% Total Amount: 302646 Matches: Library Sim Index Entry Name TSBA50 5.00 0.905 Pantoea ananatis/Erwinia uredovora (E.ananatis ) 0.670 Cedecea davisae RT Response Ar/Ht RFact ECL Peak Name Percent Comment1 Comment2 1.697 3.653E+8 0.028 ---7.021 SOLVENT PEAK ---< min rt 3.232 381 0.027 ---10.085 ---4.766 9860 0.030 1.075 12.000 12:0 3.54 ECL deviates 0.000 Reference 0.000 5.907 514 0.036 1.023 13.001 13:0 0.18 ECL deviates 0.001 Reference 0.001 6.147 425 0.035 1.015 13.178 12:0 2OH 0.14 ECL deviates 0.001 6. 523 979 0.034 1.003 13.455 12:0 3OH 0.33 ECL deviates 0.001 7.208 2911 0.045 0.983 13.959 unknown 13.957 0.95 ECL deviates 0.002 7.262 7037 0.037 0.981 13.998 14:0 2.30 ECL deviates -0.002 Reference -0.002 8.033 2844 0.044 0.963 14.502 unknown 14.50 2 0.91 ECL deviates 0.000 8.794 3229 0.038 0.948 14.999 15:0 ---ECL deviates 0.001 9.130 7935 0.045 0.943 15.202 14:0 2OH 2.50 ECL deviates 0.001 9.604 25392 0.042 0.935 15.488 Sum In Feature 2 7.93 ECL deviates 0.000 14:0 3OH/16:1 ISO I 9.70 5 1120 0.040 0.934 15.550 16:0 N alcohol 0.35 ECL deviates 0.000 10.150 79180 0.042 0.928 15.818 Sum In Feature 3 24.52 ECL deviates 0.004 16:1 w7c/15 iso 2OH 10.452 106073 0.042 0.924 16.000 16:0 32.71 ECL deviates 0.000 Reference -0.001 11.982 14 311 0.044 0.908 16.889 17:0 CYCLO 4.34 ECL deviates 0.001 Reference 0.001 12.174 2917 0.044 0.907 17.000 17:0 0.88 ECL deviates 0.000 Reference 0.002 12.888 2564 0.047 0.902 17.407 17:0 10 methyl 0.77 ECL deviates 0.002 13.619 57722 0.046 0.898 17.824 18:1 w7c 17.30 ECL deviates 0.001 13.928 1180 0.039 0.896 17.999 18:0 0.35 ECL deviates 0.001 Reference 0.004 ---25392 --------Summed Feature 2 7.93 12:0 ALDE ? unknown 10.928 ------------------16:1 ISO I/14:0 3OH 14:0 3OH/16:1 ISO I ---79180 --------Summed Feature 3 24.52 16:1 w7c/15 iso 2OH 15:0 ISO 2OH/16:1w7c
246 C6 b. MIDI analysis results for phenotype B RT Response Ar/Ht RFact ECL Peak Name Percent Comment1 Comment2 1.696 3.623E+8 0.028 ---7.023 SOLVENT PEAK ---< min rt 2.332 470 0.021 ---8.304 ---< min rt 3.173 4031 0.024 1.213 10.000 10:0 1.41 ECL deviates 0.000 Reference -0.001 3.857 779 0.026 1.138 11.001 11:0 0.26 ECL deviates 0.001 Reference 0.000 4.241 254 0.025 1.110 11.423 10:0 3OH 0.08 ECL deviates 0.001 4.581 18628 0.029 1.087 11.798 unknown 11 .799 5.85 ECL deviates 0.001 4.764 26685 0.030 1.075 11.999 12:0 8.29 ECL deviates 0.001 Reference 0.002 5.767 559 0.033 ---12.880 ---5.903 665 0.032 1.023 13.000 13:0 0.20 ECL deviates 0.000 Reference 0.002 6.144 3595 0.034 1.015 13.177 12:0 2OH 1.05 ECL deviates 0.000 6.521 611 0.032 1.003 13.455 12:0 3OH 0.18 ECL deviates 0.001 7.010 5235 0.036 ---13.815 ---7.206 2541 0.043 0.983 13.959 unknown 13.957 0.72 ECL deviates 0.002 7.261 13635 0.036 0.981 13.999 14:0 3.87 ECL d eviates -0.001 Reference -0.002 7.921 803 0.041 0.965 14.431 15:1 ISO G 0.22 ECL deviates 0.009 8.032 2816 0.039 0.963 14.504 unknown 14.502 0.78 ECL deviates 0.002 8.793 1660 0.038 0.948 15.001 15:0 ---ECL deviates 0.001 9.130 10041 0.045 0.94 3 15.204 14:0 2OH 2.74 ECL deviates 0.001 9.602 28767 0.042 0.935 15.489 Sum In Feature 2 7.78 ECL deviates 0.001 14:0 3OH/16:1 ISO I 10.148 27728 0.041 0.928 15.818 Sum In Feature 3 7.43 ECL deviates -0.004 16:1 w7c/15 iso 2OH 10.450 104722 0.041 0.924 16.000 16:0 27.96 ECL deviates 0.000 Reference 0.002 10.607 2010 0.060 ---16.091 ---11.983 63094 0.044 0.908 16.890 17:0 CYCLO 16.56 ECL deviates 0.002 Reference 0.000 12.175 1447 0.044 0.907 17.001 17:0 0.38 ECL deviates 0.001 Referen ce 0.001 13.083 1450 0.067 0.901 17.518 16:0 3OH 0.38 ECL deviates 0.001 13.494 1413 0.047 ---17.752 ---13.618 35817 0.046 0.898 17.822 18:1 w7c 9.29 ECL deviates -0.001 13.926 1135 0.045 0.896 17.997 18:0 0.29 ECL deviates 0.003 Reference 0.005 14.704 2198 0.055 ---18.443 ---15.066 1324 0.046 ---18.651 ---15.231 521 0.042 ---18.745 ---15.505 16592 0.046 0.892 18.903 19:0 CYCLO w8c 4.28 ECL deviates 0.001 Reference 0.001 15.620 920 0.046 ---18.969 ---16.488 673 0.052 ---19.470 ------28767 --------Summed Feature 2 7.78 12:0 ALDE ? unknown 10.928 ------------------16:1 ISO I/14:0 3OH 14:0 3OH/16:1 ISO I ---27728 --------Summed Feature 3 7.43 16:1 w7c/15 iso 2OH 15:0 ISO 2OH/16:1w7c ECL Deviation: 0.002 Reference ECL Shift: 0.002 Number Reference Peaks: 10 Total Response: 380693 Total Named: 365839 Percent Named: 96.10% Tot al Amount: 347592 Matches: Library Sim Index Entry Name TSBA50 5.00 0.224 Ewingella americana 0.150 Pantoea agglomerans GC subgroup C (Enterobacter)
247 C 7. Sequencing of TSWV amplicon revealed 100% homology to Tomato spotted wilt virus isolate T992 nucleocapsid protein gene (Accession number AY848922). AAGCAAGTTCTGCGAGTTTT GCCTGTTTTTTAACCCCGAA CATTTCATAGAACTTGTT AAGAGTTTCACTGTAATGTT CCATAGCAATACTTCCTTTA GCATTAGGATTGCTGGA GCTGAGTATAGCAGCATACT CTTTCCCTTTCTTCACCTGA TCTTCATTCATTTCAAAT GCTTTGCTTTTCAGCACAGTGCAAAC TTTTCCTAAGGCTTCTCTGGTGTCATACTTCT TTGGGTCAATCCCGAGGTCT TTGTATTTTGCATCCTGATA TATAGCCAAGACAACAC TGATCATCTCAAAGCTATCA ACTGAAGCAATAAGAGGTAAACTACCTCCCAGCATTA TGGCAAGCCTCACAGACTTTGCATCATCAAGAGGTAATCC ATAGGCTTGAATCAAAG GGTGGGAAGCAATCTTAGAT TTGATAGTATTGAGATTCTC AGAATTCCCAGTT TCCT CTACAAGCCTGACCCTGATC AAGCTATCAAGCCTTCTGAA GGTCATGTCAGTGGCTC CAATCCTGTCTGAAGTTTTCTTTATGGTAATTTTACCAAA AGTAAAATCACTTTGTTT AATAACCTTCATTATACTCTGACGATTCTTCAGGAATGTC AGACATGAAATAATGCT CATCTTCTTGATCTGGTCGA GGTTTTCCAGACAAAAAGTCTTGAAGTTGAATGCTAC CAGATTCTGATCTTCCTCAA ACT CAAGGTCTTTGCCTTGTGTC AACAAAGCAACAAT GCTTTCCTTAGTGAGCTTAA CCTTAGACATGATG
248 C8 a Standard Curve Titration of Tomato spotted wilt virus nucleocapsid gene (TSWV -N) for quantitative real -time PCR. An external standard was prepared from a plasmid containing the TS WV N gene (pBS N2C4.5, see C8b for nucleotide sequence information) constructed by Kim et al. (1994) and sent by AnnaWhitfield (Kansas State University). A map of the plasmid construct is provided below (Appendix C8c ). To prepare the standard, purified p lasmid DNA was transformed with the DH5 alpha strain of E. coli and digested with the restriction endonuclease BAM HI (Promega Corp., Madison, WI). The 40 l reaction included 21.6 l of water, 4.0 10X RE of buffer, 0.4 l of acetylated BSA, 10.0 l of plasmid DNA and 4.0 l of REN BAM HI. Contents were inoculated overnight at 37C, and 4 l of product was run on a 0.7% agarose gel at 90 V for 1 hour (Appendix C8d) Digestion resulted in a product of 4.5 kb in size. The digested product was purified ( QIAquick PCR purification kit, Qiagen Sciences, MD) and the concentration of plasmid determined with a spectrophotometer (Thermo Scientific Nanodrop 1000 v3.7.1) to be 39 ng/l. Based on the genome size (4,500 bp), the mass per copy was estimated to be 4.93 x 1018 g using the formula m=n*1.096*(10-21), where m = mass and n=size of genome. The mass (g) of plasmid DNA needed was determined by multiplying the desired copy number by the mass of haploid genome plasmid (g). Serial dilutions were made using th e equation C1V1= C2V2, where C1= initial concentration (g/ l), V1=volume of plasmid DNA, V2=final volume (l), and C2=final concentration (g/l).
249 C8b Nucleotide sequence for the Tomato spotted wilt virus mRNA for the nucleocapsid (TSWV-N) protein (acces sion number X61799.1). ATGTCTAAGGTTAAGCTCACTAAGGAAAGCATTGTTGCTTTGTTGACACAAGGCAAAGACCTTGAGTTTG AGGAAGATCAGAATCTGGTAGCATTCAACTTCAAGACTTTTTGTCTGGAAAACCTTGACCAGATCAAAAA GATGAGCATTATTTCATGTCTGACATTCCTGAAGAATCGTCAGAGCATAATGAAGGTTATTAAGCAAAGT GATTTTACTTTTGGTAAAATTACCATAAAGAAAACTTCAGACAGGATTGGAGCCACTGACATGACCTTCA GAAGGCTTGATAGCTTGATCAGGGTCAGGCTTGTTGAGGAAACTGGGAATTCTGAGAATCTCAATACTAT CAAATCTAAGATTGCTTCCCACCCTTTGATTCAAGCCTATGGATTACCTCTTGATGATGCAAAGTCTGTG AGGCTTGCCATAATGCTGGGAGGTAGCTTACCTCTTATTGCTTCAGTTGATAGCTTTGAGATGATCAGTG TTGTCTTGGCTATATATCAGGATGCAAAATACAAGGACCTCGGGATCGACCCAAAGAAGTATGACACCAG GGAAGCCTTAGGAAAAGTTTGCACTGTGCTGAAAAGCAAAGCATTTGAAATGAATGAAGATCAGGTGAAG AAGGGGAAGGAGTATGCTGCTATACTTAGCTCCAGCAATCCTAATGCTAAAGGAAGTATTGCTATGGAAC ATTACAGTGAAACCCTTAACAAGTTCTATGAAATGTTTGGGGTTAAAAAACAGGCAAAACTCACAGAACT TGCTTAA C8 c. Map construct of the pBluescript plasmid used in generating the standard curve for quantitative real -time PCR.
250 C8 d. Plasmid digestion gel. Lane 1 =5 l of a 1 kb ladder, lane 2 = undigested plasm id, and lane 3 = plasmid digested by BAMHI. Note the multiple plasmids joined together at the digestion site in lane 3 (arrows).
251 C8e Serial dilution calculations used for generating the Tomato spotted wilt virus standard curve for quantitative real -time PCR. Dilution # Source of plasmid DNA for dilution Initial concentration (g/l) Volume of plasmid DNA (l) Volume of water (l) Final volume (l) Final c oncentration (g/ l) Resulting copy # of plasmid ( per l ) 1 Stock (39 ng/ l) 3.90 x 10 8 2. 5 17.5 20 4.93 x 10 9 1.0 x 10 9 2 Dilution 2 4.93 x 10 9 2.5 22.5 25 4.93 x 10 10 1.0 x 10 8 3 Dilution 3 4.93 x 10 10 2.5 22.5 25 4.93 x 10 11 1.0 x 10 7 4 Dilution 4 4.93 x 10 11 2.5 22.5 25 4.93 x 10 12 1.0 x 10 6 5 Dilution 5 4.93 x 10 12 2.5 22.5 25 4.93 x 10 13 1.0 x 10 5 6 Dilution 6 4.93 x 10 13 2.5 22.5 25 4.93 x 10 14 1.0 x 10 4 7 Dilution 7 4.93 x 10 14 2.5 22.5 25 4.93 x 10 15 1.0 x 10 3 8 Dilution 8 4.93 x 10 15 2.5 22.5 25 4.93 x 10 16 1.0 x 10 2 9 Dilution 9 4.93 x 10 16 2.5 22.5 25 4.93 x 1 0 17 1.0 x 10 1
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285 BIOGRAPHICAL SKETCH Kelly Renee Sims was born in 1979 in Royal Oak, M ichigan. She is a graduate of Troy High School in Troy, Michigan. Kelly obtained a Bachelor of Science degree in resource ecology and management from the University of Michigans Department of Natural Resources and Environment in 2001. Kelly pursued her education and in 2003 graduated with a m aster s degree from the University of Floridas Department of Entomology and Nematology under the guidance of Dr. Joseph Funderburk. Kelly was awarded with an Alumni Fellowship, the highest graduate student award available, to pursue a Doctor of Philosophy at the University of Florida. In 2004, she continued her research project under the guidance of Dr. Joseph Funderburk, Dr. Drion Boucias, Dr. Stuart Reitz, Dr. James Becnel, and Dr. Timur Momol She has received numerous scholarships and awards, and was a recipient of a National Science Foundation Science Partners in Inquiry -based Collaborative Education grant. Kelly is currently continuing her career as an entomologist with her husband in Atlanta, Geor gia