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Spirorchiid Trematodes of Sea Turtle in Florida

Permanent Link: http://ufdc.ufl.edu/UFE0024049/00001

Material Information

Title: Spirorchiid Trematodes of Sea Turtle in Florida Associated Disease, Diversity, and Life Cycle Studies
Physical Description: 1 online resource (248 p.)
Language: english
Creator: Stacy, Brian
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: caretta, carettacola, chelonia, fluke, hapalotrema, learedius, loggerhead, mydas, neospirorchis, pathology, spirorchiid
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Spirorchiid trematodes or blood flukes of sea turtles have been known to science for nearly 150 years. Like related parasites, such as schistosomes, of mammals, birds, and other taxa, spirorchiids primarily inhabit the vascular system where they cause injury to the host by damaging vessels, impeding circulation, and eliciting an inflammatory response. Many genera and species have been described from sea turtles, although their taxonomy has been complicated by multiple revisions, an inadequate specimen base, and incomplete descriptions. Likewise, studies of the effects of these parasites on sea turtle health have been limited in both numbers of hosts examined and geographical regions studied. There have been no large scale studies of spirorchiid trematodes of sea turtles of Florida, although these parasites have been a health issue of concern for many years. In addition, none of the life cycles have been discovered for any marine spirorchiid species or for any digenean trematode parasite of sea turtles, despite the rich diversity of Digenea in these animals. As a result, the emergent understanding of the spirorchiids and their impact on sea turtles is incomplete in many fundamental areas. Our study investigated three different, but highly inter-related aspects of spirorchiids and their relationship with sea turtles in Florida including: characterization of associated disease in stranded loggerhead (Caretta caretta) and green (Chelonia mydas) turtles; study of genetic diversity and correlation with parasite microhabitat and host usage; and development and application of a molecular screening technique for life cycle discovery. Spirorchiids were found in high prevalence in stranded turtles and were associated with pathological lesions of concern in a significant number of animals. Differences in infection among size classes were identified, which have implications on disease studies and clinical management. Furthermore, we were able to demonstrate substantial previously unrecognized diversity among marine spirorchiids that correlated in many instances with microhabitat/host organ usage, and thus is relevant to associated disease and diagnosis. Lastly, the molecular tools and genetic database developed during this study were successfully applied to life cycle discovery efforts in the field and yielded the first evidence of intermediate host species utilized by sea turtle trematodes.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Brian Stacy.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Jacobson, Elliott R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0024049:00001

Permanent Link: http://ufdc.ufl.edu/UFE0024049/00001

Material Information

Title: Spirorchiid Trematodes of Sea Turtle in Florida Associated Disease, Diversity, and Life Cycle Studies
Physical Description: 1 online resource (248 p.)
Language: english
Creator: Stacy, Brian
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: caretta, carettacola, chelonia, fluke, hapalotrema, learedius, loggerhead, mydas, neospirorchis, pathology, spirorchiid
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Spirorchiid trematodes or blood flukes of sea turtles have been known to science for nearly 150 years. Like related parasites, such as schistosomes, of mammals, birds, and other taxa, spirorchiids primarily inhabit the vascular system where they cause injury to the host by damaging vessels, impeding circulation, and eliciting an inflammatory response. Many genera and species have been described from sea turtles, although their taxonomy has been complicated by multiple revisions, an inadequate specimen base, and incomplete descriptions. Likewise, studies of the effects of these parasites on sea turtle health have been limited in both numbers of hosts examined and geographical regions studied. There have been no large scale studies of spirorchiid trematodes of sea turtles of Florida, although these parasites have been a health issue of concern for many years. In addition, none of the life cycles have been discovered for any marine spirorchiid species or for any digenean trematode parasite of sea turtles, despite the rich diversity of Digenea in these animals. As a result, the emergent understanding of the spirorchiids and their impact on sea turtles is incomplete in many fundamental areas. Our study investigated three different, but highly inter-related aspects of spirorchiids and their relationship with sea turtles in Florida including: characterization of associated disease in stranded loggerhead (Caretta caretta) and green (Chelonia mydas) turtles; study of genetic diversity and correlation with parasite microhabitat and host usage; and development and application of a molecular screening technique for life cycle discovery. Spirorchiids were found in high prevalence in stranded turtles and were associated with pathological lesions of concern in a significant number of animals. Differences in infection among size classes were identified, which have implications on disease studies and clinical management. Furthermore, we were able to demonstrate substantial previously unrecognized diversity among marine spirorchiids that correlated in many instances with microhabitat/host organ usage, and thus is relevant to associated disease and diagnosis. Lastly, the molecular tools and genetic database developed during this study were successfully applied to life cycle discovery efforts in the field and yielded the first evidence of intermediate host species utilized by sea turtle trematodes.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Brian Stacy.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Jacobson, Elliott R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0024049:00001


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1 SPIRORCHIID TREMATODES OF SEA TURTLES IN FLORIDA: ASSO CIATED DISEASE, DIVERSITY, AND LIFE CYCLE STUDIES By BRIAN ADAMS STACY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Brian Stacy

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3 To my family

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4 ACKNOWLEDGMENTS This project would not have been possibl e without the substantial contributions and support of many agencies and individuals. I am grateful for the encouragement and assistance provided by my committee members: Elliott Jaco bson, Ellis Greiner, Alan Bolten, John Dame, Larry Herbst, Rick Alleman, and Paul Klein. The sea turtle community, both government agencies and private, were essential contributors to the various aspect s of this work. I greatly appr eciate the contributions of my colleagues in the Florida Fish and Wildlife Conservation Commission (FWC), both present and past, including Allen Foley, Karrie Minch, Rhonda Bailey, Susan Shaf, Kim Sonderman, Nashika Brewer, and Ed DeMaye. Furthermore, I thank the many participants in the Sea Turtle Stranding and Salvage Network. I also am gratef ul to members of the turtle rehabilitation community, including the faculty an d staff of The Turtle Hospital, Mote Marine Laboratory and Aquarium, Marinelife Center at Juno Beach, the Marine Scien ce Center, Clearwater Marine Aquarium, and the Georgia Sea Turtle Center. Sp ecifically, I would like to thank Richie Moretti, Michelle Bauer, Corrine Rose, Sandy Fournies Nancy Mettee, Charles Manire, Terry Norton, Ryan Butts, Janine Cianciolo, Douglas Mader, and their invaluable s upport staff. Essential to the life cycle aspects of this projec t were the critical input and co llaboration of the members and volunteers of the In-water Research Group, including Michael Bresse tte, Blair Witherington, Shigatomo Hirama, Dean Bagley, Steve Traxler, Ri chard Herren, and Carrie Crady. I also thank the staff and board of the Cayman Turtle Farm, Li mited for their hospitality and collaboration in life cycle studies. Joe Parsons, Catherine Bell, Brian Andrysak, and Nancy Andrysak were essential for field efforts on Grand Cayman. In addition, I am grateful to Mario Santoro and Terry Work for providing input and specimens from their considerable proj ects in the Costa Rica and Hawaii, respectively.

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5 I am deeply indebted to Tom Frankovich w ho has been an essent ial collaborator and good friend throughout this experience. None of the field studies w ould have been possible without his expertise, enthusiasm, and field savvy. I al so thank our volunteer di vers Carlos, Mike, and Benny for their time and effort. Friends and colleagues at the University of Fl orida, College of Vete rinary Medicine have provided support and productive collaboration for ma ny aspects of this work. I thank Hendrik Nollens, Jim Wellehan, April Childress, Toni McIn tosh, Michael Sapper, Charlie Courtney and Ruth Francis-Floyd. My family has been their throughout th e long journey through undergraduate school, veterinary school, residency, a nd graduate school. Each has provided their own unique contributions, for which I am ever grateful. I thank my parents, James and Sandy, and my siblings, Jim, Jennifer, Chad, Brad, Marc, and Stacy for their friendship and unwavering encouragement. Furthermore, the achievements over the last four years would not have been possible without the commitment and dedication of my wife Nicole. Portions of this research were funded by the Florida Sea Turtle Tag Grants Program (2006 and 2007) and Awards 2003-0206-011 and 2006-0087-004 from the National Fish and Wildlife Foundation using funding from the Nationa l Oceanic and Atmospheric Administration. All sea turtle-related studies were performed under the authorization of the NMFS permit No. 086 and under University of Florida IACUC Proj ect No. D994. Gastropod collections from protected areas were conducted under permi ssions and permits granted by the Marine Conservation Commission (Grand Cayman Island) U.S Fish and Wildlife Service (Key West National Wildlife Refuge), and National Park Service (Everglades National Park, permit no. EVER-2007-SCI-0055).

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........9 LIST OF FIGURES.......................................................................................................................11 ABSTRACT...................................................................................................................................17 CHAP TER 1 INTRODUCTION..................................................................................................................19 Background.............................................................................................................................19 Goals and Objectives........................................................................................................... ...20 Review of the Literature.........................................................................................................21 Sea Turtle Biology: Florida Species Records, Life History, and Diet ........................... 21 Spirorchiid Trematodes................................................................................................... 22 Family diagnosis......................................................................................................22 Taxonomy and phylogeny........................................................................................ 23 Marine spirorchiids.................................................................................................. 24 Prevalence, microhabitat, and host pathology.......................................................... 27 Spirorchiids and serological studies......................................................................... 36 Pathology associated with freshwater spirorchiids .................................................. 37 Fibropapillomatosis and spirorchiids....................................................................... 38 Life cycles................................................................................................................ 39 Molecular studies of digeneans and a pplications to spirorchiid research ................ 44 2 SPIRORCHIID TREMATODES OF STRA NDED SEA TURTLES IN FLORIDA AND ASSOCIATED PATHOLOGY .................................................................................... 57 Introduction................................................................................................................... ..........57 Materials and Methods...........................................................................................................58 Necropsies and Parasite Collection................................................................................. 58 Parasitological Methods..................................................................................................59 Laboratory Methods........................................................................................................ 60 Categorization of Duration of Illness.............................................................................. 60 Primary Diagnosis / Cause of Death Designations.......................................................... 61 Spirorchiid Impact Rating............................................................................................... 62 Body Condition Calculations for Immature C. mydas ....................................................62 Statistical Methods..........................................................................................................63 Results.....................................................................................................................................63 Caretta caretta .................................................................................................................63 Study population demographics, duration of illness categories ...............................63 Primary diagnosis or cause of death by duration of illness ...................................... 64

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7 Prevalence by genus and identifiable species (category 1 & 2 data). ...................... 66 Site predilection and associated pathological lesions ..............................................67 Spirorchiid impact rating.......................................................................................... 75 Chelonia mydas ...............................................................................................................77 Study population demographics and duration of illness categ ories......................... 77 Primary diagnosis or cause of death by duration of illness ...................................... 77 Prevalence by genus and identifiable species (category 1 & 2 data). ...................... 78 Site predilection and associated pathological lesions ..............................................78 Spirorchiid impact rating.......................................................................................... 82 Body condition indices in hypothermic-stunned immature C. mydas .....................83 Discussion...............................................................................................................................83 Conclusions...........................................................................................................................100 3 GENETIC DIVERSITY OF MARINE SPI RORCHIIDS AND CORREL ATION WITH MORPHOLOGY, MICROHABITAT USAGE, AND HOST SPECIES............................ 136 Introduction................................................................................................................... ........136 Methods and Materials.........................................................................................................137 Parasite Specimens........................................................................................................ 137 PCR and Sequencing.....................................................................................................138 Sequence Alignments and Comparisons....................................................................... 140 Phylogenetic Analyses...................................................................................................140 Results...................................................................................................................................141 ITS2 and MCOI Sequencing.........................................................................................141 Hapalotrema species .............................................................................................. 141 Learedius learedi ....................................................................................................143 Carettacola bipora .................................................................................................144 Neospirorchis species .............................................................................................144 Phylogenetic Analysis...................................................................................................145 Genotypes by Microhabita t and Host Species ............................................................... 146 Mixed Infections and Embolized Egg Data................................................................... 152 Discussion.............................................................................................................................153 Application of Genetic Fi ndings and Im plications........................................................ 153 Comparison of Genetic Diversity wi th Other Studies of Digenea ................................ 155 Diversity of Spirorchiids of Sea turtles in Florida......................................................... 156 Hapalotrema Learedius and Carettacola .............................................................156 Neospirorchis species .............................................................................................159 Genotypes and Microhabitat..........................................................................................162 Genotypes and Host Species......................................................................................... 165 Coevolution and Colonization: Potentia l Influences on Parasite Diversity .................. 166 Conclusions...........................................................................................................................169

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8 4 MOLECULAR DETECTION OF MARINE SPIRORCHIIDS IN INTERMEDIATE HOSTS AND APPLICATION IN LIFE CYCLE STUDIES ............................................... 204 Introduction................................................................................................................... ........204 Methods and Materials.........................................................................................................207 Gastropod DNA Extraction........................................................................................... 207 Polymerase Chain Reaction Protocol for Detection of Trematode DNA......................207 Detection of Spirorch iids in Gastropods .......................................................................209 Field Studies: Gastropod Collecti ons and Synopsis of Study Sites .............................. 210 The Turtle Hospital, Marathon Key, Florida.......................................................... 211 Cayman Turtle Farm, Limited (CTFL), Grand Cayman Island, British West Indies................................................................................................................... 211 Marquesas Keys region.......................................................................................... 211 Florida Bay, Everglades National Park..................................................................212 Field Studies: Ancillary Data........................................................................................ 212 Cayman Turtle Farm, Limited................................................................................ 212 Marquesas Keys..................................................................................................... 213 Results...................................................................................................................................213 Method for Screening Gastropods fo r Spirorchiid Trem atodes by PCR.......................213 Field Studies..................................................................................................................214 The Turtle Hospital, Marathon, Florida................................................................. 214 Cayman Turtle Farm, Limited, Grand Cayman Island, British West Indies.......... 215 Marquesas Keys..................................................................................................... 216 Florida Bay............................................................................................................. 218 Discussion.............................................................................................................................218 Conclusions...........................................................................................................................222 LIST OF REFERENCES.............................................................................................................238 BIOGRAPHICAL SKETCH.......................................................................................................248

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9 LIST OF TABLES Table page 1-1 Reported marine spirorchiid trematodes by genus, host species, geographic location, anatom ic location, associated pathol ogy, reported prevalence, and citation.....................50 1-2 Marine spirorchiid trematodes by turtle definitive hos t spec ies with locality and reference citation............................................................................................................. ...55 2-1 Criteria for categorization of the duration of illn ess........................................................ 123 2-2 Criteria for determining spiror chiid trem atode impact score........................................... 123 2-3 Criteria for grading spirorchiid infec tion and associated pathological lesions. ............... 124 2-4 Causes of death in C. car etta categorized by duration of illness (category 1 & 2 data)..................................................................................................................................125 2-5 Prevalence of Hapaltrema-associated larg e vessel arteritis in C. caretta by grade, necropsy data category, duration of illness, and size class.............................................. 126 2-6 Anatomic locations from which adult specimens of Hapalotrema species were collected fro m C. caretta .................................................................................................127 2-7 Prevalence of Neospirorchis sp. by anatom ic location (a dults and/or localized egg deposition) and necropsy data category........................................................................... 127 2-8 Prevalence of neurospirorchiidiasis (Neospirorchis sp.) in C. caretta by grade, necropsy data catego ry, duration of illness, and size class.............................................. 128 2-9 Prevalence of thyroid gland parasitism ( Neospirorchis sp.) in C. caretta by grade, necropsy data catego ry, duration of illness, and size class.............................................. 129 2-10 Prevalence of parasitism (Neospirorchis sp.) of the thym us in C. caretta by grade, necropsy data category, duration of illness, and size class.............................................. 130 2-11 Prevalence of enteric N eospirorchis sp. in C. caretta by grade, necropsy data category, duration of illness, and size class..................................................................... 131 2-12 Spirorchiid impact ratings (SIR) in C. caretta categorized by duration of illness. .........132 2-13 Causes of death in necropsied C. mydas by duration of insult. .......................................132 2-14. Prevalence of Neospirorchis sp. in C. mydas by anatom ic location (adults and/or localized egg deposition) and necropsy data category.....................................................132 2-15 Prevalence of thyroi d gland parasitism ( Neospirorchis sp.) in C. mydas by grade, necropsy data catego ry, duration of illness, and size class.............................................. 133

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10 2-16 Spirorchiid impact ratings (SIR) in C. mydas categorized by duration of illness............ 134 2-17 Body condition indices (BCIs) and Neospirorchis-related findings in hypotherm icstunned immature C. mydas St. Joseph Bay, Florida.................................................... 135 3-1 Hapalotrema Learedius learedi and Carettacola bipora specim ens from by locality, anatomic location, host species, sample number, and gene sequence obtained.............. 193 3-2 Internal transcribed spacer 2 pairwise distances between Hapalotrema Learedius and Caretta cola specimens..............................................................................................194 3-3 Mitochondrial cytochrome oxidase I ge ne and pairwise distances (nucleotide) between Hapalotrema Learedius, and Carettac ola specimens...................................... 195 3-4 Neospirorchis specim ens by host, anatomic location, sample type, gene sequence obtained, and genotype.................................................................................................... 196 3-5 Neospirorchis genotypes by organ system and host species........................................... 202 3-6 Mitochondrial cytochrome oxidase I gene and internal transcribe d spacer 2 pairwise distan ces (nucleotide) between Neospirorchis specimens...............................................203 4-1 Gastropod DNA extr action protocol. ...............................................................................231 4-2 Recipe for CTAB tissue lysis buffera.............................................................................. 231 4-3 PCR reaction conditions for trem atode detection primers............................................... 232 4-4 Marine gastropods collecte d and screened for spirorchii d trematode infection from The Turtle Hospital, Marathon, Florida and surrounding area........................................232 4-5 Marine gastropods collecte d and screened for spirorchii d trematode infection from the Cayman Turtle Farm, Limited, Grand Cayman Island, British West Indies............. 233 4-6 Digenean genera detected by fecal floata tion and sedimentation for samples collected from wild C. mydas in the Marquesas Keys region.........................................................234 4-7 Gastropods collected from the Marquesa s Keys region and Florida Bay exam ined by microdissection................................................................................................................ 235 4-8 Marine gastropods collecte d and screened for spirorchii d trematode infection from Marquesas Keys region, Florida...................................................................................... 236 4-9 Marine gastropods collecte d and screened for spirorchii d trematode infection from Florida Bay (Twin Key Basin, Rabbit Key Basin), Everglades National Park............... 237

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11 LIST OF FIGURES Figure page 2-1 Examined C. caret ta by geographical region and categ ory of necropsy data. Light gray areas are counties in which necropsied turtles were found stranded....................... 102 2-2 Histogram of size class frequencies of necropsied Caretta car etta Fresh necropsies are category 1 turtles and frozen necr opsies include all other categories........................ 103 2-3 Photomicrographs of three Hapalotrema species collected fro m C. caretta ...................104 2-4 Arteritis associated with Hapalotrema infection in C. caretta The top image depicts extensive thickening of the left aorta (LA) celiac artery (CA), superior m esenteric artery (SMA) and a congested, irregular endothelial surface (chronic arteritis). Multifocal lesions also are apparent within the right aorta (RA). In the lower left image, chronic arteritis of the left aorta (L A) is contrasted with a normal right aorta (RA). A cluster of Hapalotrema mistroides (white arrowhead) are attached within an area of endarteritis in the lower right image.................................................................... 105 2-5 Photomicrographs of arteritis associated with H apalotrema infection in C. caretta The upper left image shows marked, diffuse thickening of the artery wall due to proliferation of the intima and a severe inflammatory infiltrate. A spirorchiid ( Hapalotrema mistroides) is visible within the lumen (black arrowhead). At higher magnification (upper right image), an intens e heterophilic infiltrate surrounds the acetabulum (black arrowhead) of a H. mistroides More chronic lesions are shown in the lower images, which illustrate fibromus cular intimal proliferation and chronic granuloma formation with pigmented debris (left lower image), and a transmural infiltrate with intralesional spirorchiid egg granulomas (white arrowhead) and a cuff of mononuclear cells within the ad ventitia (lower right image)...................................... 106 2-6 Necrosis of the tunica media in arteritis lesions associated with Hapalotrema inf ection in C. caretta In the left image, there is extensive, well-demarcated necrosis of the tunica media (black arro whead) underlying an ar ea of endarteritis (H&E). In the right image, disruption of the reticulin fibe rs is visible at the margin of the necrotic segment (black arrowheads) (reticulin stain)........................................... 107 2-7 Histopathological lesions of small arteries (g astrointestinal tract) in C. caretta with spirorchiidiasis. The upper left image shows a small thrombosed artery with a mononuclear and granulocytic inflammatory in filtrate. The artery in the upper right image is similarly inflamed and has a small cluster of spirorchiid eggs ( Hapalotrema sp.) within the lumen, as well as perivasc ular multinucleated giant cell formation. The lower left image demonstrates an ar ea of microvascular proliferation in an animal that had other lesions, including arteritis and medial h ypertrophy. The lower right image shows severe medial hypertophy of two small arteries................................ 108

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12 2-8 Masses of spirorchiid eggs ( Hapalotrema sp.) w ithin subserosal vessels of a C. caretta This lesion is the typical appearance of subserosal egg masses observed in turtles infected with Hapalotrema mistroides or H. pambanensis ..................................109 2-9 Extensive enteric lesions associated with Hapalotrema inf ection in a C. caretta There are large transmural egg masses and th rombosis with ischemic necrosis of the enteric mucosa (right image) in this C. caretta infected with a novel species of Hapalotrema These lesions are more extensiv e that those shown in Figure 2-8...........109 2-10 Colonic polyp in a C. ca retta associated with granul omatous inflammation and intralesional Hapalotrema eggs.......................................................................................110 2-11 Granulomatous cholecystitis with pa pillary mucosal proliferation in a C. caretta associated with Hapalotrema eggs ( Hapalotrema nov. sp.)............................................ 110 2-12 Comparison of grades of neurospirorchiidiasis (N eospirorchis sp.) in C. caretta A) Only two small egg masses, both less than 3 mm in diameter. B) Approximately eight to ten distinct egg ma sses, all of which are less than 3 mm in diameter. C) Greater than 10 egg masses, including seve ral large, coalescing masses that distend meningeal vessels............................................................................................................. 111 2-13 Severe infection of the thyroid gland by Neospirorchis sp. in C. caretta Large black m asses of spirorchiid egges efface the gla nd and distend blood vessels within the surrounding cervical tissues............................................................................................. 112 2-14 Occlusion of thymic blood vessels by eggs of Neospirorchis sp. in C. car etta Large black spirorchiid egg masses are seen within thymic vessels and egg emboli are demonstrated within the higher magnification inset........................................................ 112 2-15 Abundant submucosal egg masses ( Neospirorchis sp) in th e small intestine of a C. caretta Numerous black, serpiginous egg mass es are visible within the submucosa....113 2-16 Enteric ulceration secondary to spirorchiidiasis ( Neospirorchis sp.) in C. caretta In this im age, an ulcer is forming along the pa th of a large egg ma ss (EM). The filiform adult spirorchiid can be seen migrating th rough a submucosal vessel and the anterior portion has been freed from the tissue a nd is laying on the mucosal surface.................. 113 2-17 Images illustrating variation in adult and egg m orphology among observed specimens of Neospirorchis from C. caretta The upper (A) and middle (C) images show Neospirorchis specimens adjacent to egg masses within the enteric submucosa. Example A is more petite and white as compared to the larger and yellow example C. The eggs in the adjacent images (B and F) correspond to the adult morphological types and demonstrate the smaller egg size for the a dult in A (both bars = 50 m). In addition, the Neospirorchis in the leptomeninges (E) have prominent pigmented cecae, which are not observed in the two gastrointestinal examples. Figure F demonstrates variation in Neospirorchis egg morphology in a squash preparation from the lung of an infected C. caretta (bar = 100 m). Two Hapalotrema eggs with bipolar processes also are present.................................................................................... 114

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13 2-18 Spirorchiid egg masses ( Neospirorchis sp.) within cloacal su bm ucosal vessels of a C. caretta Each cluster (black arrowheads) measures approximately 1.0 mm in diameter....................................................................................................................... .....115 2-19 Cytology and scanning electron microscopy (SEM) of cloacal Neospirorchis egg m asses from a C. caretta Cytological impressions (left image) of the egg masses from Figure 2-18 reveal fine surface proj ections on the shell surface (bar = 25 m). These projections are readily observed by scanning electron microscopy (lower right).................................................................................................................................115 2-20 Atrial thrombosis associated with Neospirorch is pricei infection in a C. caretta The left atrium has been opened and a thrombus (white arrowheads) is adhered to the endocardium. The higher magnificantion in set shows the dens e aggregates of Neospirorchis eggs underlying the thrombus.................................................................. 116 2-21 Severe verminous orchitis in a C. car etta infected with Neospirorchis sp.. Numerous Neospirorchis eggs and adults (not visible) are distribute d throughout the testis. Shown are the testis in situ (top) in which eggs are observed within adjacent vessels. Individual eggs are more easily obser ved in the closer image (bottom).......................... 116 2-22 Examined C. mydas by geographical region and cate gory of necropsy data. Light gray areas are counties in which necropsied turtles were found stranded. ...................... 117 2-23 Histogram of size class frequencies of necropsied C. mydas. Fresh necropsies are category 1 turtles and frozen necropsi es include all other categories. ............................. 118 2-24 Endocarditis in C. mydas associated with L. learedi infection The atria have been opened in both photographs exposing the s upravalvular endocardium. Both cases have well-demarcated, raised areas of e ndocarditis (white arrows). An adult spirorchiid ( Learedius learedi) (black arrowhead) can be seen attached to the lesion in closer image (inset)...................................................................................................... 119 2-25 Severe arteritis in a C. mydas associ ated with L. learedi infection. There is severe thickening of the left brachiocephalic ar tery (BCA), left aorta (LA), and left pulmonary artery (PA). The lumina are seve rely reduced, especially in the left aorta and brachiocephalic artery (ins et), due to intimal prolifer ation and chronic arteritis...... 120 2-26 Arteritis in a C. mydas associated with H. postorchis inf ection. The artery wall is severely thickened and th ree adult spirorchiids ( Hapalotrema postorchis ) are seen protruding from the vessel lumen.................................................................................... 120 2-27 Thyroiditis in C. mydas associated w ith infection by Neospirorchis sp: Numerous spirorchiid eggs ( Neospirorchis sp.) and associated gra nulomatous inflammation are distributed throughout the thyroid gland..........................................................................121

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14 2-28 Eggs of Neospirorch is species within the gastric submucosa of a C. mydas Two morphologies of Neospirorchis eggs are observed. The la rger, dark brown eggs are embolized deep within the submucosa. Th e smaller, golden eggs are dispersed into loosely organized clusters with in the superficial submucosa.......................................... 121 2-29 Numerous embolized Neospirorchis eggs in the lungs a nd gastric m ucosa of a C. mydas Numerous Neospirorchis eggs are embolized th roughout the lungs (A), stomach (B), and other organs in these C. mydas that died during a hypothermic stunning event. Note the multifocal mu cosal erosion and inflammation (white arrowheads) in the lower image....................................................................................... 122 3-1 Primer sequences and binding locations for am plification of the internal transcribed spacer regions of th e ribosomal gene............................................................................... 171 3-2 Alignment of internal tran scribed spacer 2 sequences of Hapalotrema species. Base pair differences between Hawaiian an d Atlantic-C aribbean specimens of H. postorchis are shaded.......................................................................................................172 3-3 Alignment of partial nucle otide sequences of the m itoc hondrial cytochrome oxidase I gene of Hapalotrema species. Shaded positions ar e differences between specimens identified as the same species by morphology................................................................. 173 3-4 Alignment of partial predicted amino acid sequence of m itochondrial cytochrome oxidase I gene of Hapalotrema species. Shaded positions are differences between specimens identified as the same species by morphology............................................... 175 3-5 Alignment of internal transcri bed spacer 2 (ITS2) sequences of Learedius learedi ......176 3-6 Mitochondrial cytochrome oxidase I gene partial nucleotide sequence for Learedius learedi .............................................................................................................................177 3-7 Alignment of partial amino acid sequences of the m itochondrial cytochrome oxidase I gene of Learedius learedi..............................................................................................179 3-8 Box and whiskers plot of pairwise distances in the m itochondrial cytochrome oxidase I gene within and be tween genotype variants of L.learedi from Florida and Hawaii. Outliers are denoted by a plus sign. Note the significantly lower pairswise distances between Hawaii vari ant A and Florida variant B as compared to genotypes from the same locality...................................................................................................... 179 3-9 Alignment of internal transcri bed spacer 2 (ITS2) sequences of Carettacola bipora. ....180 3-10 Mitochondrial cytochrome oxidase I gene partial nucleotide sequence and predicted am ino acid sequence for Carettacola bipora ...................................................................181 3-11 Alignment of internal tran scribed spacer 2 sequences of Neospirorchis specim ens....... 182

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15 3-12 Alignment of partial nucleotide sequences of the m itoc hondrial cytochrome oxidase I gene for Neospirorchis specimens. Shaded positions are differences between like genotypes based on predicted amino acid (mitochondrial cytochrome oxidase I) sequence and ribosomal ITS2 sequence.......................................................................... 184 3-13 Alignment of partial predicted amino aci d sequences of the m itochondrial cytochrom oxidase I gene for Neospirorchis specim ens. Shaded positions are differences between like genotypes based on predicte d amino acid (mitoch ondrial cytochrome oxidase I) sequence and ribosomal ITS2 sequence.......................................................... 189 3-14 Gel electrophoresis of products obtained by PCR am plif ication of the ITS1. A forward primer within the 3 end of the 18s and a reverse primer within the ITS2 produced amplicons of multiple sizes. Side-b y-side lanes represent replicates of the same sample. Differences in band sizes are due to variable numbers of tandem repeats. With the exception of some varia tion in intensity, band patterns are nearly identical between like genotypes. Samples 1 and 2 = Neogen-9, Sample3 = Neogen10, Sample 4, 5, and 6 = Neogen-11................................................................................ 190 3-15 Bayesian phylogenetic tree of 270 to 306 nucleotides of the internal transcribed spacer regio n 2 of the ribosomal gene of Neospirorchis based on T-Coffee alignment. Bayesian posterior probabilitie s (italics) as percentages are given below branch nodes. Only branches with a proba bility of greater than 60% are shown. The ML bootstrap values are based on 200 re-sam plings and are given above each node. Other marine spirorchiids, H. mistroides and L. learedi were used as an outgroup. Brackets indicate the anatomic locations from which specimens within groups were collected...................................................................................................................... .....191 3-16 Bayesian phylogenetic tree of 142 partia l am ino acid sequence of the mitochondrial cytochrome oxidase I gene of Neospirorchis based on ClustalW alignment. Bayesian posterior probabilities (italics) as percentages are given below branch nodes. Only branches with a probability of greater than 60% are shown. The ML bootstrap values are based on 100 re-samplings and are given above each node. Other marine spirorchiids, H. mistroides and L. learedi were used as an outgroup. The anatomic locations from which specimens within groups were collected are indicated by brackets........................................................................................................192 4-1 Marine system adjoining The Turtle Ho spital, Marathon, F lorida. The predominant gastropod habitat at this site is seagrass beds ( Thalassia testudinum ) (inset)................. 223 4-2 Satellite image of the Cayman Turtle Farm, Limited, Grand Cayman Island. The intake and effluent channels are labeled and are approxim ately 215 meters apart. The side-by-side round and square structures are tanks used for rearing C. mydas ........224 4-3 The limestone shore (ironshore) adjoining the Caym an Turtle Farm, Limited. Several large tide pools are visible..................................................................................225 4-4 Burrows of rock-boring sea urchin ( E chinometra sp.) pocket the subtidal surface of the ironshore ....................................................................................................................225

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16 4-5 The Marquesas Keys and surrounding region. Global positional system waypoints of areas from which gastropods were collected included a large grazi ng area west of the islands, a small near-shore site on the outer shore of the southwestern-most islands, and Mooney Harbor lagoon. The scale bar equals 1 kilometer...................................... 226 4-6 Grazing plot created by foraging C. mydas in seagrass beds w est of the Marquesas Keys. The Thalassia and Syringodium have been cropped by grazing turtles...............226 4-7 The collection areas within Florida Bay ar e sho wn here as three shaded areas, which include Twin Key Basin, Rabbit Key Basin, and Arsniker Basin................................... 227 4-8 Gel electrophoreses of products produced by PCR a mplification of the partial ITS2 of Neospirorchis species using specific primers. Each numbered sample lane pair represents 1.5 grams of Pomacea bridgesii tissue spiked with the following egg equivalents of Neospirorchis DNA: samples 1 and 2 (1 egg); samples 3, 4, and 5 (3 eggs); samples 6 and 7 (5 eggs), samples 8 and 9 (10 eggs). Sample 10 only contains snail tissue and sample 11 was extracted with a complete adult Neospirorchis species. The positive control (+) is Neospirorchis DNA and the negative control (-) is PCR reagents only. Lanes labeled A have been processed using a QIAquick spin column, whereas lanes B are have been si mply diluted in TE buffer to equivalent template concentrations. The ITS2 is detected in one of the samples spiked with a single egg equivalent and all of the sample s containing 3 or more egg equivalents. Brighter amplicons are observed in samp les processed using the QIA quick column as compared the TE dilutions........................................................................................... 228 4-9 Gel electrophoresis of products obtained from PCR amp lification of the trematode ITS2 from DNA extracted from the limpet Fissurella nodosa. The brightest band in the ladder lanes is 500 base pairs. The brig ht bands in lane six reflect the detection of the 300 base pair complete ITS2 using consensus primers and the smaller 175 base pair indicates specific amplification of the 5 region of the ITS2. Products of similar size are present in the pos itive control lane (+), which is Hapalotrema mistroides DNA. Direct sequencing of the produc ts excised from lane six confirmed the sequence to be that of Learedius learedi ITS2........................................................... 229 4-10 Two Fissurella nodosa a re adhered to the intertidal zone of the ironshore at the Cayman Turtle Farm, Limited......................................................................................... 229 4-11 Gel electrophoresis of products resulting from hemi-nested PCR amplification of the trematode ITS2 region from the gastropod Modulus modulus The R1 gel reflects amplification of trematode ITS2 using cons ensus primers in the first reaction. The two hemi-nested reactions R2A and R2B use spirorchiid-specific reverse primers and the bright smaller bands (black circles) in the positive controls reflect specific amplification of the 5 region of the Neospirorchis ITS2 (R2A) and Hapalotrema/Learedius ITS2 (R2B). The positive controls are Neospirorchis sp. (+A) and Hapalotrema mistroides (+B). Note that the specific primers do not amplify the non-target control......................................................................................... 230

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17 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SPIRORCHIID TREMATODES OF SEA TURTLES IN FLORIDA: ASSOCIATED-DISEASE, DIVERSITY, AND LIFE CYCLE STUDIES By Brian Adams Stacy December 2008 Chair: Elliott R Jacobson Major: Veterinary Medical Sciences Spirorchiid trematodes or blood flukes of sea tu rtles have been known to science for nearly 150 years. Like related parasite s, such as schistosomes, of mammals, birds, and other taxa, spirorchiids primarily inhabit the vascular sy stem where they cause injury to the host by damaging vessels, impeding circulation, and eliciti ng an inflammatory response. Many genera and species have been described from sea turt les, although their taxonomy has been complicated by multiple revisions, an inadequate specimen ba se, and incomplete descriptions. Likewise, studies of the effects of these parasites on sea turtle health have been limited in both numbers of hosts examined and geographical regions studied. There have been no large scale studies of spirorchiid trematodes of sea turtles of Florida, although these paras ites have been a health issue of concern for many years. In addition, none of the life cycles have been discovered for any marine spirorchiid species or for any digenean trematode parasite of sea turtles, despite the rich diversity of Digenea in these animals. As a result, the emerge nt understanding of the spirorchiids and their impact on sea turtles is incomplete in many fundamental areas. Our study investigated three di fferent, but highly inter-relate d aspects of sp irorchiids and their relationship with sea turtle s in Florida including: characteri zation of associated disease in stranded loggerhead ( Caretta caretta ) and green ( Chelonia mydas ) turtles; study of genetic

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18 diversity and correlation with parasite microhabitat and host usage; and development and application of a molecular scr eening technique for life cycle di scovery. Spirorchiids were found in high prevalence in stranded tur tles and were associated with pathological lesions of concern in a significant number of animals. Differences in infection among size classes were identified, which have implications on disease studies and clinical management. Furthermore, we were able to demonstrate substantial previously unrecogni zed diversity among mari ne spirorchiids that correlated in many instances with microhabitat/host organ usage, and thus is relevant to associated disease and diagnosis. Lastly, the molecular tools and gene tic database developed during this study were successfully applied to life cycle discovery efforts in the field and yielded the first evidence of intermediate host sp ecies utilized by sea turtle trematodes.

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19 CHAPTER 1 INTRODUCTION Background Many aspects of sea turtle biology, ecology, health, and disease are poorly understood due to study limitations associated with the marine environment and the complex life histories. Ontogenic shifts and migrations occur over thousa nds of kilometers of ocean and are set in a diverse array of marine habitats. Life stages of sea turtle s literally are distributed across the globe, often resulting in profound knowledge gaps. Thus, like many s ea turtle studies, much of the information about diseases of these animals is obtained from land, wher e all turtles begin and a small number end their lives. A glimpse at heal th problems in w ild sea turtles is obtained by examining stranded animals, which although a somewhat biased population and representing only a fraction of at-sea mortality, provi de a wealth of information. All sea turtle species currently are classified as endangered or threatened. Anthropogenic causes of population declines include over-harves ting, incidental take by various fisheries, destruction of nesting beaches, and deaths associ ated with other human activities. In addition, a number of diseases of sea turtles, includi ng infectious diseases, various syndromes, and toxicoses, have been documented and some have been associated with mass mortality events. Of the reported infectious diseases, studies on fibr opapillomatosis (FP) and parasites, especially spirorchiid trematodes or spirorchiids, comprise a large percentage of the sea turtle disease literature. Both are regarded as important heal th problems in wild turtles based on prevalence, associated pathology, and implications on survival. Although well-recognized as pathogens of concern in sea tu rtles, spirorchiids had never been intensively studied in Florida turtles beyond initial descrip tions and reports. In late 2000 and early 2001, an unusual mortality event char acterized by neurological signs affecting Caretta

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20 caretta occurred in south Florida (Jacobson et al., 2006). During this event, neurological infection by unidentified spir orchiids within the genus Neospirorchis was implicated as a possible contributing cause of ne urological signs observed in the affected turtles. Several deficiencies in the general understanding of thes e parasites were identifie d or highlighted during the investigation of this event ranging from inabili ty to specifically identify the parasites in many instances to the lack of knowle dge of prevalence of parasites, intensities, and associated pathological lesions in Florida turtles. Furthermore, all of th e marine spirorchiid life cycles, which have fundamental implications on the epidemiology and disease ecology of spirorchiidiasis, are unknown. The 2000/2001 C. caretta mortality event was a primary impetus for further study of spirorchiidi asis in Florida sea turtles. Goals and Objectives The aim of these studies was to gain a bett er understanding of spir orchiid trematodes in several key areas: 1) characterize spirorchiidiasis in stranded Florida sea turtles with regard to prevalence, associated pathology, and significance in terms of cau se of death based on necropsy and parasitology findings; 2) investigate the pot ential for parasite diversity using molecular prospecting techniques in combination with obs ervations in the turtle host and morphological studies; 3) develop a means of molecular iden tification and detection; and 4) apply these molecular techniques to life cycle discovery in field study sites. The first objective contributes to a basic tenant of population health assessment, which is to estab lish a pathologic database. This information is especially sparse for Caretta caretta These objectives encompass a broad range of topics including basic pa thology and disease surveillanc e, classical and molecular parasitology, and marine biology. Thus, a review of the literature includes relevant sea turtle biology, an in-depth review of spirorchiids and a ssociated disease, and ap plication of molecular techniques in parasitology studies.

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21 Review of the Literature Sea Turtle Biology: Florida Species Records, Life History, and Diet Six species of sea turtle are found in Florida waters including the loggerhead ( Caretta caretta ), green turtle ( Chelonia mydas ), hawksbill turtle ( Eretmochelys imbricata ), Kemps ridley ( Lepidochelys kempi), olive ridley ( Lepidochelus olivacea ), and leatherback ( Dermochelys coriacea ). All sea turtles are classi fied as threatened or endange red and are protected under state and federal laws. The most frequently encountered species in Florida are C. caretta and C. mydas which were the focus of these studies. The life histories of sea turtles are co mplex and three major patterns have been characterized (reviewed by Bolte n, 2003a). The first (Type 1) pattern includes complete development and maturation entirely within the in shore or neritic zone. The only example of a living sea turtle that exhibits the Type 1 pattern is the flatback turtle ( Natator depressus). The Type 2 pattern is characterized by initial development in the oceanic zone followed by recruitment into the neritic zone. These ontogenetic shifts in habitat uti lization are hypothesized to provide maximum growth potential and minimi ze predation risk (Werner and Gilliam, 1984). The Type 2 pattern is the most common and is characteristic of the life histories of C. caretta C. mydas E. imbricata L. kempi and some populations of L. olivacea In Atlantic C. caretta these shifts appear to be complex and reversible rather than discrete transitions (Witzell 2002; McClellan and Read, 2007). The size and apparent ag e of recruitment into the neritic zone varies among species. The oceanic stage is the longest for Atlantic C. caretta which return to neritic life after 7 to 11.5 years at a size of 46 to 64 cm (curved carapace length (CCL))(Bjorndal et al., 2003). Chelonia mydas E. imbricata and L. kempi are thought to have a shorter ocean life stage, which is estimated to be 3 to 5 years for C. mydas (Reich et al., 2007). Size at recruitment into neritic foraging grounds is 20 to 35 cm for C. mydas and E. imbricata and 20 to 25 cm for

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22 L. kempi The third life history pattern or Type 3 pattern is exhibited by D. coriacea and East Pacific L. olivacea which undergo development comple tely within the oceanic zone. Life history is best char acterized for North Atlantic C. caretta which includes Floridas nesting population. Oceanic stage turtles are f ound in waters near the Azores, Canary Islands, and Madeira Islands where they arrive from na tal beaches via the Gulf Stream (Bolten, 2003b). Immature turtles are primarily epipelagic during the oceanic stage, but may become epibenthic or demersal near islands, seamounts, or other fo rmations (Bolten, 2003b). Following recruitment into the neritic zone, C. caretta become primarily epibenthic or demersal and utilize a variety of habitats as carnivore generalists. Pelagic feedin g aggregations and oceanic migration routes also have been identified for C. caretta originating from other rook eries (Musick and Limpus, 1997; Bowen and Karl, 2007). There are far fewer reports of oceanic C. mydas thus less is known about the life history of this spec ies. A key difference between the C. caretta and C. mydas is that neritic C. mydas primarily are herbivorous and feed on seagrasses and macroalgae. Differences in life history of sea turtle species result in differences in the size classes encountered in different regions, which is reflected in the strandi ng data. In Florida, peaks in size frequency distribution based on curv ed carapace length (CCL) of stranded C. caretta are between 60 to 80 cm and 90 to 105 cm CCL (Foley et al., 2007). In contrast, peaks for C. mydas are 30 to 40 cm and 100 to 105 cm; therefore, most stranded immature C. mydas are much smaller than the average immature C. caretta These comparisons do not include stranded posthatchlings, which are another majo r size class represented in the st randing data for both species. Spirorchiid Trematodes Family diagnosis The family Spirorchiidae Stunkard, 1921 is comprised of intr avascular trematode parasites of freshwater and marine turtles. These parasites primar ily are vascular generalists and

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23 various species inhabit arterial, venous, and ly mphatic systems (Smith, 1997a). Extravascular migration has been observed in some freshw ater forms (Smith, 1997a). The morphology of spirorchiids is diverse and ranges from li nguiform to filiform with both distomous and monostomous types. All known members are he rmaphroditic. Egg morphology also is highly variable in size and shape. On e or two polar filaments may be present and an operculum may be present or absent. Life cycles have only been discovered for fres hwater forms, which all utilize gastropod mollusks as a single intermediate host (Smith, 1972; Smith, 1997a). Freshwater spirorchiid cercariae develop through one or more sporocyst generations. Development of rediae has not been reported. Cercariae are brevifurcate and apharyngeate. Freshwater chelonian hosts are infected by penetration through the oral, nasal, conjuctival and cloaca integument and mucous membranes (Smith, 1997a). Taxonomy and phylogeny The first spirorchiid was described by Leared in 1862 as a parasite of the edible turtle or common turtle, which may have been Chelonia mydas (Smith, 1972). Almost 100 spirorchiid species within 19 genera have been described over the last nearly 150 years (Smith, 1997a; Platt, 2002). Reported host chelonian sp ecies represent all major families except Testudinidae (Platt, 1992). Record s are biased toward sea turtles and North American freshwater turtles. Many new spirorchiids undoubtedly will be discovered as it is estimated that only around 15% of chelonian species have been exam ined for these paras ites (Snyder, 2004). Spirorchiidae is within the superfamily Schistosomatoidea (Trematoda: Digenea: Strigeadida), which also includes Sanguinicolidae and Schistos omatidae. Sanguinicolidae are blood flukes of fish and Schistosomatidae incl udes blood flukes of mammals, birds, and crocodilians. Recent phylogenetic studies of Schistosomatidae and Spirorchiidae based on small and large subunit ribosomal DNA support that mari ne spirorchiids form a clade sister to the

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24 schistosomatids (Snyder, 2004). Spirorchiids of freshwater turtles are basal to marine spirorchiids, suggesting that marine forms are derived from a freshwater ancestor (Snyder, 2004). In addition, this study also demonstrated that Spirorchiidae is in fact paraphyletic, and thus taxonomically invalid (Snyder, 2004). This finding likely will result in revision of higher classification and thus additional complexity in the progression of spirorchiid taxonomy. The taxonomy of Spirorchiidae is confounded by divergent morphology, a limited specimen database, poorly preser ved/partial specimens, incomple tely described species, and inaccessible specimens (Platt, 2002). The literature is widely dispersed and replete with disputes over nomenclature and synonymy. There have been revisions and redescriptions of multiple taxa (Dailey et al., 1993; Platt, 1993; Pl att and Blair, 1998; Platt, 2 002). Even the spelling of the family name has been revisited, and appears in the literature as both Spirorchidae, as proposed by Stunkard (1921), and Spirorchiid ae, which was proposed by MacCallum later the same year. MacCallums spelling, although not etymologically correct, is preferred by the International Commission on Zoological Nomenclature (ICZN) (although Stunkhard is credited with the discovery) because many other digeneans with -orch is family names have an ii ending (Platt, 2002). An official ruling has not been rendered by the ICZN. Subdivision of Spirorchiidae into subfamilies has been proposed (Azimov,1970; Ya maguti, 1971; Smith, 1972), but these schemes are unsupported by phylogeneti c analysis (Snyder, 2004). Marine spirorchiids Ten genera of marine spirorhids are rec ognized in a recent compilation of keys for the family Spirorchiidae (Platt, 2002). However, four of these genera, Cheloneotrema (Simha & Chattopadhyaya, 1976), Neocaballerotrema (Simha, 1977), Satyanarayanotrema (Simha & Chattopadhyaya, 1980), and Shobanatrema (Simha & Chattopadhyaya, 1980), have only been described from the Indian subcontin ent and have not been available for subsequent review (Platt,

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25 2002). Platt noted that the descriptions of thes e parasites are insufficient to distinguish them from other previously described genera. In fact, he proposed that a fifth Indian genus, Squaroacetabulum (Simha & Chattopadhyaya, 1 970), is a junior synonym of Amphiorchis (Platt 2002). Table 1-1 includes reports of spirorchiids of sea turtles in the peer-reviewed literature. Excluded are abstracts and identifications beyond the gene ric level that were based solely on egg morphology, except where data were reasonably substantiated and contributed to either geographical or host range. Many of these re ferences were previously compiled by Smith (1997b). Modifications include recognized synonymies, reports since 1996, and additional data on parasite microhabitat, associated pathol ogy, and prevalence data. Many of these taxa probably should be further studied and re-eva luated for validity, as has been done for Hapalotrema (Cribb and Gordon, 1998; Platt and Blair, 1998) and Carettacola (Dailey et al., 1991). Reported sea turtle host species for spirorchiids are limited to C. mydas E. imbricata and C. caretta (Smith, 1997b). There are no published descriptions of spirorchiids of Lepidochelys, Natator, or Dermochelys Approximately fourteen spirorchiid species within eight genera have been described from C. mydas eight species within three genera from E. imbricata and six species within five genera from C. caretta (Table 1-2). Smith (1997) stated as many as 22 species within nine genera had been reported for C. mydas The lower number given here reflects reported synonymies, exclusion of those reports without specific identification, and two cases where definitive host identification wa s ambiguous or unsubstantiated (Leared, 1862; Mehrotra, 1973; Gupta and Mehrotra, 1981). Several marine spirorchiid species have been reported to parasitize multiple sea turtle species. As noted by Smith (Smith, 1972; Smith, 1997a), there is no evidence of strict

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26 phylogenetic host specificity at either the generic or specie s levels. Many freshwater spirorchiid species are known to parasitize multi ple chelonian host species, including chelonian species of different genera (Platt, 1993; Smith, 1997a). In contra st to this opinion, one study of digenean trematodes in Caribbean C. mydas categorized some spirorchiids as generalists, indicating parasitism of multiple host species, and others as specialists, indicating parasitism of a single host species (Santoro et al., 2006). This assumption of specificity may be premature given the limited information or lack of data for sea turtles in many regions. Perceived host specificity may reflect selective exposure to interm ediate hosts, as dictated by habitat utilization, rather than true specificity. Reports of several spirorchiid species suppor t a worldwide distribution for some taxa. Hapalotrema pambanensis Hapalotrema mistroides, Hapalotrema postorchis, Hapalotrema synorchis Learedius learedi, Montecellius indicum and Neospirorchis schistosomatoides have all been reported from distant regions (see Table 11). The spirorchiid literature is heavily biased toward C. mydas both in the number of reports and the num bers of individual turtles examined. In a 1972 review, Smith stated that: To undertake [a detailed description of world distribution] using known records would be premature and probably reflect only the pa st and present world di stribution of marine and freshwater research institutes, the attrac tiveness and/or accessibi lity of particular localities to research workers, and a lack of interest in the group. This statement is still valid as sea turtles in habiting many geographica l regions have yet to be studied or have received only limited study. The only marine spirorchiid species for which a restricted geographical distribution has been proposed is Carettacola hawaiiensis which has only been identified from C. mydas in Hawaii. Work et al. (2005) propose that C. hawaiiensis is an endemic parasite, whereas Hapalotrema species and Learedius were introduced. This hypothesis is based on limited reports of the parasite small parasite numbers in the turtle host,

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27 and minimal evident pathology. The latter were interpreted as evidence of a longer, more evolved host-parasite relationship as compared to other spirorchiid s. However, spirorchiid data from other areas of the Pacific, other than Aust ralia, are very limited and phylogenetic studies to support this hypothesis are lacking. Prevalence, microhabitat, and host pathology Most of the published reports of marine spir orchiids fall into the categories of primary parasitological descriptions/studi es and studies of associated host pathology. The former often lacks mention of associated pathology and the latter specific identification of parasites. Thus, the interpretation of significance of parasitism, in terms of health implications in the turtle host, frequently is disconnected from specific identification of the parasites. Also, most reports are limited to small numbers of turtles, thus prevalence cannot be inferred. Of those publications cited in Table 1-1, onl y eleven (excluding those cited by Smith that are unavailable) have included data from ten or more wild sea turtles (Looss, 1899; Looss, 1902; Fischthal and Acholonu, 1976; Glazebrook et al., 1981; Wolke et al., 1982; Rand and Wiles, 1985; Glazebrook et al., 1989; Glaze brook and Campbell, 1990; Dailey et al., 1991; Dailey et al., 1992; Gordon, et al., 1998; Work et al., 2005; Jacobson et al., 2006; Sa ntoro et al., 2006). Comparison of prevalence among most existing studies, as attempted by Glazebrook et al. (1990), is unsupported due to differences in methods of detection, inadequate descriptions of methodology, and examination of different age/size cl asses and species of turtles. For example, Wolke et al. (1982) reported that 14 of 43 acce ssions had spirorchiids; however, only sixteen complete carcasses were examined. Several studies report a combined prevalence of multiple spirorchiid genera and species (Glazebrook and Campbell. 1990; Gordon et al., 1998; Work et al., 2005), or a combined prevalence that includes multiple sea turtle species (Glazebrook et al., 1981; Glazebrook et al., 1989).

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28 Detection of spirorchiids and associated lesi ons requires a targeted ap proach that includes meticulous examination of vascular compartmen ts, dissection of the cardiovascular system, examination of all tissues (both grossly and hi stologically), and fecal examination. In those studies that apply targeted methodology, spirorchiid prevalence in many study populations is greater than 95% (Dailey et al ., 1992; Gordon et al., 1998; Work et al., 2005; Santoro et al., 2006). A study of digeneans in nesting female C. mydas in Tortuguero National Park, Costa Rica, by Santoro et al. (2006) is especially not able because this study population was comprised of nesting females killed by jaguar, and thus is most representative of a large group of otherwise healthy turtles. Many of the ot her studies primarily included dead or moribund stranded turtles or turtles with other health problems, such as fibropapillomatosis. Work et al. (2005) used enzymatic digestion of the spleen, followed by th e use of Flukefinders (a differential filtration technique) to recover spirorchiid ova as a means, although unva lidated, of estimating tissue ova numbers in C. mydas This method appears to be quite e ffective for detection given that all 99 C. mydas included in this study were found to be infected. Parasite microhabitat selection or host-org an/tissue fidelity has obvious implications on associated effects in the host and biology of th e parasite, e.g. successful propagation. In the marine and freshwater spirorchiid literature, there are examples suggesting varying degrees of host-organ fidelity, as well as reports that some species do not exhibit specific tropism. This aspect of spirorchiid biology is somewhat obscure d by vague descriptions of parasite locations within the host and lack of information on the parasite maturity in some reports. By far, the heart and major arteries, includi ng the pulmonary arteries and regions of the aorta, are the most commonly reported sites from which adult marine spirorchiids have been recovered (Table 1-1). It must be considered that there is at least some bias in the literature as

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29 these sites are easily assessed, and other organs and tissues, such as brain and smaller vessels, are more technically difficult to extr act and thoroughly examine, especially in large turtles. The heart and major arteries are consis tently mentioned in reports of Hapalotrema and Learedius species. Montecellius indicum also is described from the h eart in the two published studies. Multiple authors describe Carettacola species from hepatic and mesenteric vessels. Both Neospirorchis pricei and N. schistosomatoides have been described from the heart, and N. schistosomatoides also has been reported in meningeal vessels. Other genera and species are limited to reports in single animals, thus c onsensus observations on possible tropisms are not possible. Furthermore, in many other reports give n in Table 1-1, the locati on of parasites is very vague, such as visceral blood vessels, blood vessel, or circulatory sy stem, or location is not identified. There are reports of both host-organ specifi city and lack thereof in the freshwater spirorchiid literature. Wall (1941) reported a specific tropism of S. elephantis for gastroenteric arterioles. Another spirorchiid, Vasotrema robustum is described as undergoing early development in hepatic vessels followed by migr ation to the heart and major arteries (Wall, 1951). In contrast, multiple authors report a lack of host-organ specificity, as well as migration of adults outside of vessels, in species of the genus Spirorchis (Smith, 1972; Smith, 1997a). In two different studies of experimentally infected Chrysemys picta picta Spirorchis scripta and S. parvus were collected from a variety of anatom ic locations, including non-vascular sites (Holliman and Fisher, 1968; Holliman et al., 1971). In both studies, it was stated that adult and immature parasites were collected; however, th e maturity of the parasites from the various locations was not clearly indicated. In addition, Holliman et al. (1971) argue that their findings confirm a lack of host-organ specificity for adult S. parvus, yet they report that 217 of 247

PAGE 30

30 recovered parasites were removed from the cen tral nervous system. This finding arguably suggests a tropism for the central nervous system or its associated vasculat ure. In addition, some of the turtles in this study were infected with both S. parvus and S. scripta, and it appears that this dually infected group is included in two different publications (H olliman and Fisher, 1968; Holliman et al., 1971). Although the recovered spiror chiids were specifically identified, it is unclear if the locations from which they were recovered were combined because the reported numbers are inconsistent. It is reported that 217 S. scripta were recovered from the singly infected group and 23 from the dually infected group (240 total S. scripta ); however, anatomic locations are listed for 230 specimens (Holliman and Fisher, 1968). In considering this issue of host-organ fidelity in Spirorchis sp., Platt (1993) notes finding S. parvus in both the cranial and mesenteric circulation of naturally infected C. picta marginata Lastly, lack of host-organ specificity also is reported for S. elegans in C. picta picta although, again, maturity of parasites from various sites was unclear (Goodchild and Kirk, 1960). The host effects and pathological lesions asso ciated with spirorchiidiasis have been studied in both freshwater and marine turtles. The reported effects of sp irorchiid infection fall into the categories of vascular a nd tissue injury and inflammation associated with adult parasites and eggs and more general, nonspecific descri ptions of weight loss, lethargy, and chronic debilitation. The hypothesized a ssociation between fibropapillomatosis and spirorchiids will be discussed separately. Studies of the pathologic effect s of marine spirorchiids primarily have examined C. mydas (Looss, 1902; Glazebrook et al., 1981; Rand and Wiles, 1985; Glazebrook, et al., 1989; Glazebrook and Campbell, 1990; Gracz yk et al., 1995; Gordon et al., 1998; Raidal, et al., 1998; Cordero-Tapia et al ., 2004; Work et al., 2005), fewer C. caretta (Looss, 1902; Wolke et al., 1982; Glazebrook and Campbell, 1990; Jacobson et al., 2006), and a small number

PAGE 31

31 of E. imbricata (Glazebrook et al., 1989; Glazebrook and Campbell, 1990). In most of these studies, examined turtles were parasitized by multiple spirorchiid species and little or no effort is made to associate lesions with specific species. One of the earliest references to pathologica l lesions associated with spirorchiids was by Looss (1902), who noted changes in the vascular intima and endothelium associated with attached Hapalotrema in his studies of C. mydas and C. caretta in Egypt. Looss also noted a lack of correlation between the numbers of spir orchiid eggs observed and the number of adult flukes present. Wolke et al. (1982) reported spirorchiidiasis and associated lesions in 14 of 43 accessions, which included random tissues and 16 whole carcasses of C. caretta that stranded on the Atlantic coast of the Un ited States. Among these accessi ons, the authors describe an unspecified number of chronically debilitated, severely emaciate d turtles with heavy epibiota coverage and associated these cases with large nu mbers of spirorchiid ova in various tissues. It is stated that 90% of lesions were focal granulomata and two morphological forms of granulomas were described, one characterized by central necrosis and a second characterized by giant cells formation without necrosis. The au thors also note cases of bacterial enteritis secondary to spirorchiid egg de position, although the type of sp irorchiid is not indicated. Spirorchiid egg morphologies repr esenting the three main types ar e noted, but adults were not collected. Lastly, the finding of iron accumu lation in the liver, spleen, and kidneys was interpreted as evidence of hemolytic anemia, which is speculated to have contributed to the pathogenicity of spiror chiid infection. A report of pathological changes associated with spirorchiidiasis in a single moribund C. mydas moribund from Australia was described by Glazeb rook et al. (1981). The affected turtle

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32 was chronically ill and had large numbers of spir orchiids and mycotic pneumonia. Many (167) Hapalotrema sp. were recovered in the cardiac ventricl e and base of the right aortic arch and brachiocephalic artery, where there was localized endocarditis and endart eritis and thrombus formation. Spirorchiids also were noted in the br ain and other organs, and were presumed to be the same species, but were not identified. These au thors describe a very similar case or the same turtle in a second study eight years later and pres ent a photomicrograph of an egg mass and adult spirorchiid within the meninges that is identifiable as a Neospirorchis sp. (Glazebrook et al., 1989). The parasite is misidentified as Hapalotrema sp. in both papers. Large numbers of spirorchiid eggs were observed in multiple organs and were associated with granuloma formation. In the 1989 study, the authors reportedly examined 109 farmed turtles, 20 oceanarium turtles, and 39 wild turtles. Most presumably were C. mydas and an unspecified number of E. imbricata also were examined. The wild turtles ha d both the highest prev alence (77.2% (17/22)) and heaviest infections. Prevalence in farm ed and oceanarium turtles was 4.8% (5/104) and 33.3% (5/15), respectively. Although the ocean arium turtles are identified as oceanariumreared, it appears from a later publication that some of these turtles were captured as subadults or adults and may have been infected prio r to captivity (Glazebrook and Campbell, 1990). Clinical signs were observed in only 26% of turtle s with spirorchiidiasis and were described as nonspecific, including emaciati on. Many spirorchiids are recovered from the heart and major vessels (primarily the right aortic arch and brach iocephalic artery), and a dults are noted in other organs, including the central nervous system (likely Neospirorchis sp.), spleen, lungs, and skeletal muscle. The spirorchiids collected, Hapalotrema sp. from C. mydas and E. imbricata and Learedius were identified to genus only. Gross vasc ular lesions described as thickening and hardening of the arterial walls were observed in 5 of 17 infected wild turtles and three had

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33 associated thrombi. Two animals also had ch ronic pneumonia, although it is unclear if this lesion was associated with embolization of eggs. Proliferative endocarditis and endarteritis are described as is varying degrees of granulomat ous inflammation in multiple organs associated with egg embolization. The author s also described congestion and edema, which they associated with embolized eggs. Much of these data a ppear to have been published again by Glazebrook and Campbell (1990) in a subsequent survey of dis eases of oceanarium-reared and wild turtles. Rand and Wiles (1985) examined eleven moribund or dead C. mydas recovered in Bermuda and report identification of L. learedi and N. schistosomatoides in the group. Description of lesions is limited to granulomatous inflammation associated with spirorchiid eggs in tissues. Other findings are nonspecific and incl ude findings typical of chronically ill turtles, such as atrophy of fat and soft tissue edema. Significance of spirorchiidiasis in these cases was not specifically evaluated. A brief description of lesions associated with spirorchiid infection in a single C. mydas from Hawaii was given by Graczyk et al. (1995) in a study of spirorchiid exposure using an enzyme-linked immunosorbent assay (ELISA). Generalized lymphoplasmacytic endarteritis, thrombosis, and granuloma formation in multiple organs associated with spirorchiid eggs is described in an emaciated turtle with fibropapillomatosis. Para sites from this turtle included H. dorsopora (syn. pambanensis ), L. learedi and C. hawaiiensis which were used as source of antigen for the ELISA. One of the largest and most in-depth studies on the impact of spiror chiids in sea turtles was an examination of associated pathological lesi ons in stranded C. mydas in Queensland, Australia (Gordon et al., 1998). Th is was the first study to clearl y address some of the more important aspects of spirorchiidiasis, such as distinguishing incidental (to the cause of death)

PAGE 34

34 infections from significant a nd fatal pathological lesions. Other studies have mentioned incidental infections, but often failed to provide actual numbers or apparently did not have complete necropsy data on which to base confid ent interpretation. In the study by Gordon et al (1998), the criteria for categoriz ing significance, however, were not specifically stated. Spirorchiid infection was observe d in 97.9% (94/96) of examined green turtles, with 30.2% (29/96) of the turtles having one of multiple se vere problems due to spirorchiidiasis, and 10.4% (10/96) dying directly from spirorchiid infection. It a ppears that most of the turtles examined were chronically ill, as indi cated by emaciation, and all had ascites and anasarca, which the authors were unable to correlate with decrease d plasma protein. Pat hological lesions included mural endocarditis (33/96), loca lized near the atriove ntricular junctions, and endarteritis. Remarkably, arterial thrombosis is recorded in 81 turtles, and both mural thrombi and extensive occlusive thrombi are described. Aneurysm formation is reported as a common finding and the authors illustrate an unusual proc ess of exteriorization of thromb i from the vessel lumen. Septic thrombosis was a confounding problem in six turtles. The host response is as described by previous authors and includes granuloma formation accompanied by granulocytes and lymphocytes in most organs and tissues. Concentr ations of egg granulomas that were considered significant were observed in the lungs of six turtles, and in one case a more acute, i.e. granulocytic response is descri bed. Histologically, the cardi ac and vascular lesions were characterized by papillary prolifer ation and subintimal stromal proliferation, in addition to the inflammatory infiltrate and pigmented and mine ralized debris. Similar changes also were observed in splenic, pulmonary, and pancrea tic vessels. The authors could not confirm correlation of spirorchiids with circulatory disturbances, such as congestion, edema, or hemorrhage. Spirorchiid species are identified from a subset (n=8) of affected turtles, and

PAGE 35

35 include H. pambanensis (syn. mehrai ), H. postorchis and N. schistosomatoides. Most spirorchiids were recovered from the left aorta, and fewer from the right aorta and heart. In addition, the authors refer to flukes as macrosc opic and microscopic, with the latter referring to flukes that were only identified in tissue sectio n. It appears that many of these likely were Neospirorchis species, based on the authors description of a filiform body type. These parasites were observed in a variety of sections, includi ng brain (53/72), gastroin testinal tract (15/69), lungs (7/70), liver, thyroid gland, adrenal gland, a nd periarterial connective tissue. Associated meningeal lesions are described as considerably milder than in other organs, and were only considered significant in one turtle that had severe granuloma formation and hemorrhage. Serpiginous lesions are described in the blood vessels of the meninges and enteric submucosa that may have been Neospirorchis egg masses based on the descrip tion, but this is unclear. Lastly, the authors note a lack of correlation between the severity of lesions and the numbers of flukes recovered and state that this disparity in observations, concurre nt disease processes, chronic stage of disease in stranded turtles, and lack of knowledge regarding spirorchiid life cycles are major obstacles in characterizi ng and interpreting health implications of spirorchiidiasis. Raidal et al. (1998) report four cases of spirorchiidiasis associated with Gram-negative bacterial infections in immature green turtles th at stranded in western Au stralia. Parasitism by multiple species of spirorchiids is described and is associated with granulomatous vasculitis and, in two cases, thrombosis. Lesions supporting conc urrent bacterial infect ion only are presented for three of the four cases. Rec overed spirorchiid genera included Amphiorchis Hapalotrema and Carettacola which were not identified to species. In addition, a pho to of the brain from one

PAGE 36

36 infected turtle demonstrates a la rge number of spirorch iid eggs within the meninges, which likely was a Neospirorchis sp. This finding was observed in two turtles. Two reports of spirorchiid iasis in black turtles ( C. mydas agassizii ) describe L. learedi in turtles killed as by-catch in Magdalena Bay, Mexico (Corde ro-Tapia et al., 2004; Inohuye-Rivera et al., 2004). As in previous studies, adult parasites were remove d from the heart (three of four examined hearts in this report) and granulomas associated with spirorchiid eggs were found in every tissue examined. An interesting linear arrangement of L. learedi eggs in the pancreas is described and interpreted this as possible migration towards the en teric lumen (Cordero-Tapia et al., 2004). The prevalence of infecti on in these studeis is unclear. Work et al. (2005) used splenic spirorchiid egg numbers as an estimate of intensity of spirorchiid infection and compared these data to various parameters, including turtle size, body condition, fibropapilloma diagnosis and spirorchiid serological status in a study of Hawaiian C. mydas The use of splenic egg numbers as a surrogate for evaluation of the entire animal was not validated in this study. Spirorchiid eggs were detected in all of the examined turtles. Splenic egg burden was found to significan tly decrease with increasing st raight carapace length (SCL) and increasing body condition index (BCI). These re sults indicated that factors related to host age influence spirorchiidiasis, which also has b een suggested in studies of freshwater turtles (Holliman, 1971). A trend in turtles from different regions also was identified. Spirorchiids and serological studies Serological assays for the detection of expos ure to spirorchiid trematodes have been used in three different studies (Graczyk et al., 1995; Herbst et al., 1998; Work et al., 2005). Graczyk et al. (1995) developed an i ndirect ELISA using combined preparations of crude surface glycocalyx antigen from L. learedi H. dorsopora (syn. pambanensis ), and C. hawaiiensis In this study, plasma from 58 wild turtles (5 local ities, but 41 turtles from one locality) was tested

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37 and seroprevalence was 100% from all localit ies, except Ahu-O-Laka, Kaneohe Bay, Oahu. Herbst et al. (1998) used total crude antigen from L. learedi and an unidentified Hapalotrema sp. to develop an indirect ELISA fo r studying serological exposure of C. mydas with and without FP to herpesvirus and spirorchiids. Examined turtles included a group with experimentally produced FP, turtles from Indian River Lagoon w ith a high prevalence of FP, and turtles from reef habitat where FP is not obs erved. No significant associati on was found between spirorchiid seroprevalence and habitat. Interestingly, seropositive animals included the smallest turtle tested (SCL = 26.8 cm). Cross-reactivity was noted between Learedius and Hapalotrema as well as Neospirorchis and gastrointestinal digeneans, but was more specific when a higher cutoff value was applied. Using a higher cutoff limit, a lower seroprevalence was found in FP turtles as compared to FP-free turtles, which did not support an associated between spirorchiids and FP. Proposed explanations for these findings in cluded immunosuppression or immunomodulation in FP turtles, and that the lagoon may not support ac tive spirorchiid infection. As stated by the authors, all of these possibilities require significant additional stud ies. In a recent study, Work et al. (2005) developed ELISAs to detect antibodies against adult and e gg crude antigen using combined preparations from Learedius sp., Hapalotrema sp., and Carettacola hawaiiensis Animals in this study included wild hatchling, immature, and adult turtles from different localities, as well as captive-bred and reared tu rtles, some of which were exposed to coastal habitat. Captive turtles in filtered seawater had the lowest titers, followed by hatchlings, which were thought to have maternal antibody. Equiva lent exposure was noted in wild immature and adult turtles. Oceanic stages were not ex amined is any of these studies. Pathology associated with freshwater spirorchiids Many of the descriptions of host pathology associated with freshwater spirorchiids are reported in the context of experimental infections during life history studies. Irritation of the

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38 head region manifested by blinking, gaping, and vigorous clawi ng is described with exposure to infectious cercariae (Holliman and Fisher, 19 68; Holliman et al., 1971). Other clinical signs generally are nonspecific and some reported associated findings, such as osteopenia, are not confidently attributable to spirorchiidiasis (H olliman and Fisher, 1968). Reported clinical signs include listlessness, stupor, and ed ema of the head and thoracic limbs. There also are references to higher mortality in experime ntally infected turtles (Holli man and Fisher, 1968; Holliman et al., 1971). Much of the described associated host injury and inflammation is associated with eggs within various tissues (Wall, 1941; Goodchi ld et al., 1967; Johnson et al., 1998). Holliman (1971) made the interesting observa tion that hyperinfected turtles may contribute to the parasite life cycle by releasing eggs, including those trap ped within tissues withou t apparent alternative means for reaching the external environment. In addition, there are tw o reports that include neurological symptoms in freshwater turtles asso ciated with spirorchiids. Holliman et al. (1971) report one case of hemipl egia and circling in a Chrysemys picta picta due to a necrotic brain lesion associated with S. parvus. Similar neurological signs al so are reported by Johnson et al. (1998); however, there were no correl ative pathological findings presented to explain the clinical signs. Other associated pathology findings in freshwater turtles in clude proliferative endarteritis in captive Trachemys scripta elegans and Chrysemys picta (Johnson et al., 1998). Fibropapillomatosis and spirorchiids A review of the marine spirorchiid literatu re would be incomplete without mention of fibropapillomatosis. Considerable effort ha s been expended in investigating a possible association between spirorchiids and this disease. Spirorchiid eggs were first reported within FP tumors by Smith and Coates (1939). These author s ultimately concluded that the parasite ova probably were not the immediate cause of the disease based on further examination of two additional turtles with FP that did not have evidence of spirorchiid infection. An interest in FP

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39 re-emerged in the 1980s as a high prevalence of tumors was observed in C. mydas in Florida and Hawaii (Balazs, 1986; Erhart, 19 91). Spirorchiids were again re visited as causative factor in the pathogenesis of FP based on presence of eggs in some tumors and the precedence for fibrogenic host response, papillomatous lesion s, and malignancy associated with mammalian schistosomes (Smith and Coates, 1939; Harshba rger, 1984). Subsequent studies, however, did not support a causative association between spirorchiids and FP. There were two unsuccessful attempts to induce FP by injecting spirorchiid ma terial (Herbst, 1994; Dailey and Morris, 1995). In addition, two independent sero logical studies, one in Florid a and one in Hawaii, did not indicate any association between exposure to spirorchiids and FP (Graczyk et al., 1995; Herbst et al., 1998). Additional studies offered compelling ev idence that implicated a novel herpesvirus as the primary agent of concern (J acobson et al., 1991; Herbst and Klein, 1995; Herbst et al., 1998; Quackenbush et al., 1998). Studi es of FP have turned to further characterization of the associated herpesvirus, virus variants, and other possible contributing factor s, such as toxins and pollutants. Most, if not all i nvestigators have largely disregarded any significant role of spirorchiids in the pathogenesis of FP. Life cycles As previously stated, the life cycles of all marine spirorchiids are unknown, thus important aspects of epidemiology, disease ecology, and other cri tical features of spirorchiidiasis, e.g. time course of parasitism, ha ve yet to be studied. Inability to discover the life cycles of marine spirorch iids is a significant obstacle to better understanding of these parasites and their implications on sea turtle health (Gordon et al ., 1998). The life cycles of several freshwater spirorchiids, however, have be en identified, and may offer some insight into intermediate hosts of marine form s. In addition, other information relevant to marine life cycles includes evidence of disparity in sp irorchiid prevalence among different life stages of sea turtles,

PAGE 40

40 as well as documented infections in captive-hatched or cap tive-bred turtles at coastal facilities. These data can be used to deve lop hypotheses regarding the genera l habitats in which infections are acquired and studies of potential intermediate hosts. The life cycles have been characterized a nd experimentally reproduced for as many as seven species of freshwater spir orchiids (depending on resolution of the species inquirenda status of some Spirorchis species) (Smith, 1997a). There is a bias for temperate North American species in these studies. All spirorchiid life cycles include a si ngle gastropod mollusk intermediate host. The only examples of trem atodes within the superf amily Schistosomatoidea that utilize non-gastropo d intermediate hosts are some species of sanguinicolids, which parasitize lamellibranch mollusks (bivalves) and polychaete annelids (Smith, 1997a). In fact, sanguinicolids are only digeneans known to utili ze non-gastropods as primary intermediate hosts. Nonetheless, given the basal phylogenetic position of freshwater spirorchii ds relative to marine forms, and the consistent uti lization of gastropod mollusks by schistosomes, it reasons that marine spirorchiids most likely also utili ze gastropod mollusks as intermediate hosts. Gastropods that serve as intermediate hosts of freshwater spirorchiids include species within the genera Helisoma Physa Menetus Ferrissia and Indoplanorbis (Smith, 1997). Prevalence of spirorchiid infecti on in snail populations is low in many studies with evidence of seasonal variation. Holliman (1971) examined 3,502 Helisoma anceps and found that only 1.28% were infected with S. scripta and 0.11% with S. parvus. Holiman hypothesized that successful completion of the life cycles was more dependent on high cer carial productivity of the individual snail rather than on the number of snails infected. Turner and Corkum (1977) conducted a 16-month survey of digenean infec tions in three species of ancylid snail in Louisiana. Spirorchis scripta only was found in Ferrissia fragilis, with a peak prevalence in

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41 July (study period was April thr ough July) of only 1.3%. A remarkable seasonal prevalence and the highest prevalence recorded thus far for any sp irorchiid in its intermediate host was reported in snails inhabiting a reservoir in Kentucky (R osen et al., 1994). Infection of the snail H. anceps by S. scripta had peak prevalence of 18.2% in May to June that decreased into mid-summer and remained undetectable from November through April. Several freshwater spirorchiid life cycle studies have indicated that adult snails are less susceptible or resistant to infection (Wall, 1941; Holliman and Fisher, 1968; Holliman, 1971; Holliman et al., 1971; Rosen et al., 1994). Rosen et al. (1994) documented a higher prevalence of in fection in smaller snails, suggesting a rapid, parasite-associated mortality in the smaller cohor t. In contrast, Goodchild and Fried (1963) did not observe any age-related suscep tibility to spirorchiid infection in experimentally infected Menetus dilatatus buchanensis Spirorchiid cercariae develop through one or more generation s of sporocyst and, as with many aspects of spirorchiid development, the rapi dity of this process is temperature dependent (Wall, 1951; Goodchild and Kirk, 1960; Holliman and Fisher, 1968; Smith, 1972). Cercarial output varies with some reports of 100 or more in a single day (Pieper, 1953; Holliman, 1971). Infected snails can be quite l ong-lived with survival records as long as two to several months (Pieper, 1953; Goodchild and Fried, 1963). As w ith other blood flukes, emerged cercariae infect the turtle host by penetration. Experimentally exposed turtles ar e observed to exhibit irritation around the eyelids and proposed sites of penetration include the eyel ids, conjunctiva, nares, oral cavity and cloaca (Wall, 1941; Wall, 1951; Goodchild and Kirk, 1960; Holliman and Fisher, 1968; Holliman et al., 1971). Experimental reproduction of the life cycl es of freshwater spir orchiids has provided useful information on infection in turtle hosts. Th e longevity of spirorchii ds in turtle hosts is

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42 poorly defined. Holliman (1971) reported finding an adult spirorchiid in the mesentery 293 days after exposure to S. scripta cercariae. The prepatent periods reported by different studies are widely variable and range from six weeks to as lo ng as a year (Table 1-1) Possible explanations of this disparity include differen ces in parasite and host species or host interaction, temperature, and detection methods. Parasite maturation a nd oviposition are reporte d to be temperature dependent; however, this observation is based on a single study with a small sample size and exposure to different numbers of cercariae, thus additional work is necessary (Goodchild et al., 1967). Holliman (1971) also noted shorter matu ration times, as well as larger numbers of parasites, in younger turt les. In a study of S. parvus in C. picta investigators noted that eggs were never found in the feces, even from hosts infected as long as 332 days (Holliman et al., 1971). It was further noted that egg numbers were low in the en teric wall of some hosts, which may have limited detection in the feces. After e ggs are expelled or extracted from the turtle, hatching occurs in approximately 4 to 8 days and is temperature dependent. There are two unsuccessful attempts to discove r the life cycles of marine spirorchiids in the literature. Greine r et al. (1980) examined several ga stropod species found at the Cayman Turtle Farm, Limited (CTFL) where resident C. mydas are infected with L. learedi A second study is briefly mentioned by Dailey in a su rvey of digenean trematodes of Hawaiian C. mydas (Dailey et al., 1992). Although an intermediate host has yet to be id entified, there is evidence that at least some species infect turtles in th e neritic or inshore zone. Learedius learedi infects captive-bred C. mydas at the CTFL (Greiner et al., 1980). Ga stropods and other orga nisms are not commonly found in the rearing tanks at the CTFL, which suggests that in fectious cercariae are brought into the facility via seawater and th at the intermediate host lives in the adjoining marine habitat

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43 (Greiner et al., 1980). There are two additional examples indicating infection in the neritic zone. Captive-hatched C. mydas housed at a rehabilition facility in Marathon Key, Florida (The Turtle Hospital) were found to become infected by species of Neospirorchis (L. Herbst, personal communication). Lastly, Work et al. (2005) detected antibodies against spirorch iids in captive turtles that were transferred from captivity into coastal seawater ponds. It also is valuable to compare the prevalence of spirorchiids in differe nt life stages of sea turtles, as this may indicate habitats that suppor t spirorchiid transmission. These data are patchy at best because there are knowledge gaps for some life stages and investigators often neglect to include detailed measurements of the turtle hos t in spirorchiid studies. There are, however, several relevant references. Or os et al. (2005) did not observe any spirorchiids in 88 immature C. caretta recovered from the Canary islands, Sp ain. This study population was primarily comprised of oceanic stage turtles base d on carapace length, which suggests that C. caretta are infected following arrival in the neritic habitat. Very limited information is available on oceanic stage C. mydas and E. imbricata Looss (1902) noted that Hapalotrema sp. already was present in C. mydas at 20 to 30 cm (carapace length), which is the approximate size oceanic C. mydas recruit into the neritic zone (Musik and Limpus, 1997). Glazeb rook et al. (1989) noted that disease was not present in hatchling (possibl y referring to post-hatc hling) or juvenile C. mydas and E. imbricata and that all infected turtles from one examined locality we re greater than 28.5 cm (curved carapace length (CCL)). These author s do not indicate whether or not smaller turtles from other localities were infected or the numbe rs of examined turtles within different size classes. Work and Balazs (2002) did not find spirorchiids in two C. mydas captured as bycatch in the North Pacific. Both of these turtles were over 55 cm in straight carapace length. No serological studies have included oceanic stage turtles.

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44 A few parasitological surveys have not found spirorchiids in sea turtles despite methods that presumably would have detect ed infection and, in some cases, re latively large sample sizes. Spirorchiids are conspicuously absent from two parasitolo gical surveys of C. caretta (33 and 14 individuals) and C. mydas (seven individuals) in the Medite rranean region (Sey, 1977; Manfredi et al., 1998). Neither study gives the sizes of the examined turtles, which may have been oceanic stage animals. Lastly, no spirorchiids have been observed in D. coriacea or L. olivacea both of which are believed to exhibit a completely oceanic life history pattern (Manfredi et al., 1996; Work and Balazs, 2002; Oros et al., 2005). Molecular studies of digeneans and a pplications to spirorchiid research Parasite identification a nd taxonomy based solely on morphology has a number of limitations, many of which are highly relevant to the study of spirorchiid trematodes. These problems can be addressed by judicious use of molecular studies. Morphology-based classification is limited due to the small size of so me adult parasites, inab ility to specifically identify larval or developing life stages, subjective measures of descriptive characters, phenotypic plasticity, morphological similarities between closel y related species, and the lag time between speciation events and evolution of observable mo rphological differences (Nolan and Cribb, 2005). In addition, partial or poor quality specimens, inadequate descriptions, and sparse specimen database have been impli cated in the many taxonomic uncertainties of spirorchiids (Platt, 2002). A primary focus of this dissertational res earch was the use of molecular approaches to identify and detect spirorchiids, and assess for diversity. The following literature review will con centrate on application of ribosomal and mitochondrial genetic data for these purposes. Internal transcribed spacer regions of the ribosomal gene. Ribosomal DNA is present in the genome as a series of repeats and includes two internal transcri bed spacer regions (ITS 1

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45 & 2) separated by the relatively short 5.8s segmen t. The secondary structure of ribsomsomal sequence must be maintained to sustain function, thus the region stays re latively conserved. The ribosomal repeats evolve in a concerted ma nner via unbiased gene conversion and unequal crossing over, which is thought to rapidly homogenize and fix new variants within reproductive units. Thus, individuals are believed to be representative of th e interbreeding group (Morgan and Blair, 1995; Nolan and Cribb, 2005) However, Vilas et al. ( 2005) point out that concerted evolution may be slower than assumed in platyhel minths, and resultant hybridization, incomplete lineage sorting, and retention of ancestral polym orphism could confound studies. Nonetheless, the ITS are commonly used markers for species st udies and studies of cr yptic diversity, and are useful for these purposes in digeneans when a ppropriately applied (Nolan and Cribb, 2005). Some studies report greater variability in the ITS1 as compared to the ITS2; however, this finding appears to be incons istent (Vilas et al., 2005). Th e ITS1 includes a variable 5 region and more conservative 3 region (von der Schulenberg et al ., 1999). Tandem repeats have been identified within the 5 region in multiple digenean families, including Schistosomatidae (Nolan and Cribb, 2005). Mitochondrial cytochrome c oxidase I. The mitochondrial cytochrome c oxidase I (MCOI) gene is another common choice for inve stigation of species delimitation and cryptic diversity. In an examination of ITS and mitochondrial sequen ce data for use in molecular prospecting studies in platyhelm inths, Vilas et al. (2005) ad vocate the use of mitochondrial sequence based on higher rate of evolution, sm aller effective population size, and clearer demarcation between inter-specific and intra-specif ic variation as compared to the ITS. The potential limitations of mitochond rial sequence, including the MCOI for these studies is intraspecific and intra-individual variation; however many of the same potential mechanisms for

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46 generating such confounding results must be co nsidered in both ITS and mitochondrial DNA (Vilas et al., 2005). Furthermore, MCOI sequen ce can be translated to detect pseudogenes, which is not possible for ITS data. Review of ITS and MCOI in species diversity studies. Ideally, a genetic marker for species identification or assessmen t of species diversity should have a high level of divergence between closely related species and low intrasp ecific variation (Vilas et al., 2005). In addition, when examining poorly studied groups, a comparative data set across a broad array of different taxa, digenean trematodes in this case, is help ful for data interpretation and confidence in conclusions. The ITS is a suitable marker for species identification based on evidence for clear interspecific differences between closely related species and low intraspecific variation (Nolan and Cribb, 2005). The MCOI exhibits greater variability than the ITS and is useful for both identifying diagnostic characters and detecti ng cryptic diversity (V ilas et al., 2005). In a recent review, Nolan and Cribb (2005) re port the use of ITS data in 63 studies involving 19 different families of digeneans. Of these studies, only three reported an absence of differences in ITS sequences between species with strong biological and/or morphological evidence supporting classification as different species (Despres et al., 1992; Blair et al., 1998; Niewiadomska and Laskowski, 2002). A review of sequence data from these studies and additional sequence made available since original publication indicate that the results from two of these studies are suspect (N olan and Cribb, 2005). Thus, in most instances the ITS regions appear to correlate well with species di fferences, the only convincing exception being Paragonimus miyazakii and P. skrjabini (Blair et al., 1998). Howeve r, this study only examined the ITS2, which is generally regarded as more c onserved than the ITS1. In fact, a minority of studies report the use of the enti re ITS, with most using only th e shorter ITS2 (Nolan and Cribb,

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47 2005). It is proposed that good stud y design with regard to the ITS should include the entire ITS in order to increase the ability to discriminate closely related species (N olan and Cribb, 2005). The second essential feature of a genetic mark er to identify species is low intraspecific variation. Intraspecific variati on results in uncertainty regarding which gene variant to use for comparison. Erroneous reports of intraspecific variation are blamed on failure to adequately confirm sequences (sequencing or PCR errors) a nd failure to recognize multiple species (Nolan and Cribb, 2005). Actual intraspecific varia tion may occur due to ge ographic variation or variability in the number of tandem repeats that comprise part of the ITS1 (Nolan and Cribb, 2005). The later is one of the common examples of intraspecific variat ion observed within the ITS, and is a feature of the ITS1 in some digeneans (Vilas et al., 2005), and may have been overlooked in others. In these inst ances it is critical to examine the different variants and include other sequences, such as the ITS2 and mitochondrial genes, in any analysis. Comparison of ITS sequence is a useful me thod for discriminating digenean trematode species in most instances and has produced compelling evidence for merging synonymous species or more clearly delineating different or cryptic species (Nolan and Cribb, 2005). In some examples, however, intraspecific variation or lack of divergence in ITS sequence between obviously biologically different species has required the use of altern ative, more variable genes. These cases serve to illustrate important consid erations that must be addressed in studies involving the ITS regions. First, many of th e proposed exceptions that argue against the reliability of the ITS involve studies where only the ITS1 or the ITS2 were sequenced. As previously stated, the compara tive evidence available across dige nean taxa support that both ITS regions should be examined. Second, is the importance of a large sample size and adequate replication to confirm sequence data. These fact ors lend confidence to results and significance to

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48 minor differences that may otherwise be overlook ed or regarded as intraspecific variation. Similarly, supportive data in terms of geographi c locality, host species, and other morphological or biological informati on may be essential. There are examples that sugge st that the ITS may not be th e most sensitive indicator of species diversity, at least for some taxa (Nolan and Cribb, 2005). Overlap between observed interspecific and intraspecific di stances has been reported in clos ely related species (Vilas et al., 2005). Mitochondrial genes with greater vari ability, such as NAD H dehydrogenase-1 and cytochrome c oxidase I, may be used as additiona l supportive data to detect differences in the taxa of interest (Villas et al ., 2005). Large-scale studies are needed to better understand and resolve debate over intraspecific variation in the ITS. In the aforementioned study comparing the use of ITS and mitochondrial sequence for molecular prospecting, Vilas et al. (2005) co mpared two mitochondrial sequences, the MCOI and NADH dehydrogenase-1 between multiple congene ric species. The major limitation of this study is that it relied heavily on sequences obt ained from GenBank, which requires great caution, especially when interpreting small nucleotid e differences. Greater divergence between congeneric species was found as compared to ITS, and the authors commented that mitochondrial sequence differing by greater than 5% warrants furt her investigation of possible species differences. There are many key differences between mitochondrial DNA and nuclear DNA that have implications on the use of mito chondrial sequence for examining sp ecies, i.e. cryptic diversity, and inferring phylogeny. These differences include variability in the degree of recombination, effective population size, mutation rate, and introgression (Ballard and Whitlock, 2004). First, mitochondrial DNA typically does not undergo recomb ination, except in fungi and plants, thus

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49 the mitochrondrial genome is phys ically linked (Birky, 2001). As a result, inferences about the history of a population cannot be replicated using other mitochondrial genes. Second, mitochondrial genomes have a smaller effectiv e population size (due to a haploid genome), whereby new alleles are fixed fast er than for nuclear DNA. This feature is advantageous for phylogenic studies because it provides faster re solution of coalescence (Ballard and Whitlock, 2004). A third feature of mitochondrial DNA is hi gher mutation rate, which is dependent in the population size. Small populations tend to accumu late deleterious mitochrondrial alleles, whereas there is greater negative selection in larg e populations. Actual mutation rates relative to nuclear genes, however, is dependent on the biology of the taxa (Ballard and Whitlock, 2004). Lastly, introgression may be more likely in mitochondrial DNA than nuclear DNA. Introgression is an important consideration in closely relate d, sympatric taxa due to the probability of hydridization. This process may resu lt in discrepancies in interspecific differences and may be confused with ancestral polymorphi sm and incomplete lineage sorting in the assessment of gene geniologies (Nielsen a nd Wakeley, 2001; Ballard and Whitlock, 2004). There are many examples of mitochondrial intr ogression without nuclear introgression or morphological changes (Ballard and Whitlock, 2004).

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50Table 1-1. Reported marine spirorchiid trematodes by genus, host species, geographic location, an atomic location, associated pathology, reported prevalence, and citation. Spirorchid species Host species Geographic location Anatomic location (adults) Associated Pathology Prevalence Referencea Genus Amphiorchis Amphiorchis amphiorchis C. mydas Captive Visceral blood vessels Not described N/A Price, 1934 E. imbricata Puerto Rico Blood vessels of large intestine Not described N/A Fischthal and Acholonu, 1976 Amphiorchis caborojoensis E. imbricata Puerto Rico Pulmonary vessels Not described N/A Fischthal and Acholonu, 1976 E. imbricata Puerto Rico Not given Not described N/A Dyer et al. 1995 Amphiorchis indicum E. imbricata Gulf of Manar, India Mesenteric [sic] capillaries of the intestine Simha and Chattopadhyaya, 1980a Amphiorchis indicus E. imbricata(?) Gulf of Manar, India Heart Not described 1/3 Gupta and Mehrotra, 1981 Amphiorchis lateralis E. imbricata Palao Islands Oguro, 1938a Amphiorchis (Squaroacetabulum) solus* C. mydas Gulf of Manar, India Heart Not described N/A Simha and Chattopadhyaya, 1970 C. mydas Tortuguero, Costa Rica Intestine Not described 1/40 Santoro et al., 2006 Amphiorchis sp. C. mydas Australia (western) Mesenteric arteries Granulomatous vasculitis, thrombosis, microabscess formation associated with eggs (multiple spirorchid spp. found) N/A Raidal et al., 1998 Genus Carettacola ( Haemoxenicon ) Carettacola bipora C. caretta Florida, USA Coelomic blood vessel Not described N/A Manter and Larson, 1950 Carettacola hawaiiensis C. mydas Hawaii Hepatic vessels Simila r findings to other studies 3/10 Dailey et al., 1991; Dailey et al., 1992 C. mydas Hawaii Not givem Describe eggs in fibropapillomas N/A Dailey and Morris, 1995 C. mydas Hawaii Hepatic vessels Lymphoplasmacytic endarteritis w/ thrombosis; multi-organ granulomatous inflammation(multiple spirorchid spp. found) N/A Graczyk et al., 1995 C. mydas Hawaii Not given Not described 99/99d Work et al., 2005 Carettacola stunkardi (C. chelonenecon) C. mydas Baja California Mesenteric veins Not described N/A Martin and Bamberger, 1952

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51Table 1-1. Continued Spirorchid species Host species Geographic location Anatomic location (adults) Associated Pathology Prevalence Referencea Carettacola stunkardi (C. chelonenecon) (cont.) C. mydas Panama, Central America Caballero et al., 1955a Carettacola sp. C. caretta Atlantic coast, USA Not given Some emaciated; multi-organ granulomatous inflammation (multiple spirorchiid spp. Found) 14/43d Wolke et al., 1982 C. mydas Australia (western) Mesenteric veins, liver, aorta Granulomatous vasculitis, thrombosis, microabscess formation associated with eggs (multiple spirorchid spp. found) N/A Raidal et al., 1998 Genus Cheloneotremab Cheloneotrema testicaudata C. mydas Gulf of Manar, India Mesenteric capillaries of intestine Simha and Chattopadhyaya, 1980a Genus Hapalotremac ( Mesogonimus) Hapalotrema pambanensis ( H. mehrai, H. dorsopora) C. mydas Gulf of Manar, India Heart Not described N/A Rao, 1976 C. mydas Gulf of Manar, India Heart Not described 2/2 Gupta and Mehrotra, 1981 C. mydas Hawaii Heart Not described 8/10 Dailey et al., 1993 C. mydas Hawaii Not given Described eggs in fibropapillomas N/A Dailey and Morris, 1995 C. mydas Hawaii Heart, major vessels Lymphoplasmacytic endarteritis w/ thrombosis; multi-organ granulomatous inflammation(multiple spirorchid spp. found) N/A Graczyk et al., 1995 C. mydas Queensland, Australia Heart, pulmonary a. Not described N/A Cribb and Gordon, 1998 C. mydas Queensland, Australia Heart, pulmonary a. Endocarditis/e ndarteritis; aneurysm formation; thrombosis 91/96d Gordon et al., 1998 C.mydas Queensland, Australia Heart Not described N/A Platt and Blair, 1998 E. imbricata Queensland, Australia Heart Not described N/A Platt and Blair, 1998 Hapalotrema mistroides (H. constrictum, H. loossi) C. caretta Italy Aorta (A. celomica) (stated in Looss 1899) Monticelli, 1896a C. caretta Egypt Heart, major vessels Vaguely describes endothelial irregularities and vascular thickening 20/21 Looss, 1899; Looss, 1902 C. caretta Nile Valley Gohar, 1934; Gohar, 1935a C. caretta Western Australia Heart, liver Not described N/A Platt and Blair, 1998 C. mydas Egypt Heart, major vessels Vaguely describes endothelial irregularities and vascular thickening N/A Looss, 1902

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52Table 1-1. Continued Spirorchid species Host species Geographic location Anatomic location (adults) Associated Pathology Prevalence Referencea Hapalotrema mistroides (H. constrictum, H. loossi) (cont.) C. mydas Nile Valley Gohar, 1934; Gohar, 1935a Hapalotrema postorchis C. mydas Gulf of Manar, India Heart Not described N/A Rao, 1976 C. mydas Hawaii Heart Not described 3/10 Dailey et al., 1993 Hapalotrema postorchis (cont.) C. mydas Torres Strait & Queensland, Australia Heart, major arteries Not described N/A Platt and Blair, 1998 C. mydas Queensland, Australia Left & right aortas Endarteritis; aneurysm formation; thrombosis 91/96d Gordon et al., 1998 C. mydas Queensland, Australia Left & right aortas Not described N/A Cribb and Gordon, 1998 C. mydas Tortuguero, Costa Rica Heart, great vessels Not desc ribed 8/40 Santoro et al., 2006 Hapalotrema synorchis (H. orientalis ) C. caretta Florida, USA Heart Not described 1/3 Luhman, 1935 C. caretta Western Australia & Queensland Heart Not described N/A Platt and Blair, 1998 E. imbricata Okinawa, Japan Takeuti, 1942a E. imbricata Puerto Rico Heart Not described N/A Fischthal and Acholonu, 1976 Hapalotrema sp. C. caretta Atlantic coast, USA Not given Some emaciated; multi-organ granulomatous inflammation (multiple spirorchiid spp. Found) 14/43d Wolke et al., 1982 C. mydas Australia (northern) Heart, major arteries Endcarditis ; endarteritis w/ thrombosis; Multiorgan granulomatous inflammation 17/22 (wild only) d Glazebrook et al., 1981 and 1989 C. mydas Australia (northern) Heart [T]hickening and hardening of arterial walls with thrombus formation; generalized emaciation; chronic pneumonia 16/21 (wild only)d,e Glazebrook and Campbell,1990 C. mydas Hawaii Heart 8/10 d Dailey et al., 1992 C. mydas Queensland, Australia Barker and Blair, 1996 C. mydas Queensland, Australia Liver/lung wash Not described N/A Platt and Blair, 1998 C. mydas Australia (western) Aortas Granulomatous vasculitis, thrombosis, microabscess formation associated with eggs (multiple spirorchid spp. Found) N/A Raidal et al., 1998 C. mydas Hawaii Not given Not described 99/99 d Work et al., 2005 E. imbricata Australia (northern) Endcarditis; endarteritis w/ thrombosis; Multiorgan granulomatous inflammation 17/22 (wild only) d Glazebrook et al., 1989 E. imbricata Australia (northern) Not specified [T]hickening and hardening of arterial walls with thrombus formation; generalized emaciation; chronic pneumonia 1/1 (wild only) d,e Glazebrook and Campbell, 1990

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53 Table 1-1. Continued Spirorchid species Host species Geographic location Anatomic location (adults) Associated Pathology Prevalence Referencea Genus Learedius Learedius learedi C. mydas Captive Circulatory system Not described N/A Price, 1934 C. mydas Panama Caballero, et al., 1955a C. mydas Gulf of Manar, India Gulf of Manar, India Gupta and Mehrotra 1975a C. mydas Australia Heart Australia Blair, 1979 (quoted by Glazebrook et al., 1989) C. mydas Grand Cayman Island Heart, major arteries Greiner et al., 1980 C. mydas Bermuda Heart Multi-organ gr anulomatous inflammation 11/11 Rand and Wiles, 1985 C. mydas Puerto Rico Heart Not described N/A Dyer et al., 1991 C. mydas Puerto Rico Williams et al,. 1994 C. mydas Hawaii Heart Not described 4/10 Dailey et al., 1992 and 1993 C. mydas Hawaii Not given Describe eggs in fibropapillomas N/A Dailey and Morris, 1995 C. mydas Hawaii Heart, major vessels Endarter itis, thrombi formation, multi-organ granulomatous inflammation (multiple spirorchid spp. found) N/A Graczyk et al., 1995 C. mydas Tortugeuro, Costa Rica Heart, major vessels, other sites Not described 39/40 Santoro et al., 2006 C. agassizii Baja California Heart Multi-orga n granulomatous inflammation 11/11 Cordero-Tapia et al., 2004 E. imbricata Puerto Rico Heart Not described N/A Dyer et al., 1995 Learedius loochooensis C. mydas Japan Takeuti, 1942a Learedius orientalis (possibily syn. w/ L. learedi ) C. mydas Arabian Sea Heart Arabian Sea Mehra ,1939a Learedius orientalis (possibily syn. w/ L. learedi ) (cont.) C. mydas Gulf of Manar, India Heart Not described 2/2 Gupta and Mehrotra, 1975a C. mydas Puerto Rico Heart, aorta Not described 1/4 Dyer et al., 1995 E. imbricata Puerto Rico Heart Not described 3/14 Fischthal and Acholonu, 1976 Learedius similis C. mydas Captive Circulatory system Not described N/A Price, 1934

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54 Table 1-1. Continued Spirorchid species Host species Geographic location Anatomic location (adults) Associated Pathology Prevalence Referencea Learedius sp. C. mydas Australia (northern) Heart, right aortic arch, brachiocephalic artery [T]hickening and hardening of arterial walls with thrombus formation; generalized emaciation; chronic pneumonia 16/21 (wild only) d Glazebrook and Campbell, 1990 C. mydas Australia (northern) Heart, major arteries Endcarditis ; endarteritis w/ thrombosis; Multiorgan granulomatous inflammation 17/22 (wild ) d Glazebrook et al., 1989 Learedius sp. (cont.) C. mydas Hawaii Not given Not described 99/99 d Work et al,. 2005 E. imbricata Australia (northern) Heart, major arteries Endocarditis; endarteritis w/ thrombosis; Multiorgan granulomatous inflammation 17/22 (wild only) d Glazebrook et al., 1989 Genus Monticellius Monticellius indicum C. mydas Arabian Sea Heart Mehra, 1939a C. mydas Tortuguero, Costa Rica Heart Not described 5/40 Santuro et al., 2006 Genus Neospirorchis Neospirorchis pricei C. caretta Florida, USA Heart Not described N/A Manter and Larson, 1950 Neospirorchis schistosomatoides C. mydas Captive Visceral blood vessels Not described N/A Price, 1934 C. mydas Bermuda Heart Multi-organ gr anulomatous inflammation 2/11 (6/11 had Neosp eggs) Rand and Wiles, 1985 C. mydas Queensland, Australia Meningeal venules Granulomatous inflammation 91/96d Gordon et al., 1998 Neospirorchis sp. C. caretta Atlantic coast, USA Not given Some emaciated; multi-organ granulomatous inflammation (multiple spirorchiid spp. found) 14/43d Wolke et al., 1982 C. caretta Florida, USA Meningeal venules Granulom atous inflammation Jacobson et al., 2007 Genus Neocaballerotrema Neocaballerotrema caballeroi C. caretta Gulf of Manar, India Enteric blood vessel Simha, 1977a Genus Satyanayanotremaa Satyanayanotrema satyanarayani C. mydas Gulf of Manar, India Blood vessel Simha and Chattopadhyaya, 1980a Genus Shobanatrema Shobanatrema shobanae C. caretta Gulf of Manar, India Mesenteric [sic] capillaries of large intestine Simha and Chattopadhyaya, 1980a aReference unavailable, cited in Smith 1997; bMultiple spellings; cBased redescriptions of species in the genus Hapalotrema by Platt & Blair (1998), Cribb & Gordon (1998), and Platt (2002). Junior synonyms are given in parentheses; dPravelence data includes multiple spirorchiid genera or species; ePrevalence data includes multiple sea turtle species; *Pla tt 2002 placed as junior synonym of Amphiorchis

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55Table 1-2. Marine spirorchii d trematodes by turtle definitive host specie s with locality and reference citation. Host species Spirorchid species Locality Reference C. mydas Amphiorchis amphiorchis Captive Price ,1934 Amphiorchis sp. Australia (western) Raidal et al.., 1998 Carettacola hawaiiensis Hawaii Dailey et al., 1991 and 1993; Dailey and Moris 1995; Graczyk et al., 1995; Work et al., 2005 Carettacola stunkardi Baja California Martin and Bamberger, 1952; Cabellero et al., 1955 Carettacola sp. Australia (western) Raidal et al., 1998 Cheloneotrema testicaudata India Simha and Chattopadhyaya, 1980 Hapalotrema pambanensis India Rao 1976; Gupta and Mehrotra 1981 Hawaii Dailey et al., 1993; Dailey and Mo rris, 1995; Garczyk et al., 1995 Queensland, Australia Cribb and Gordon, 1998; Gordon et al., 1998; Platt and Blair, 1998 Hapalotrema mistroides Egypt Looss, 1902 Hapalotrema postorchis Egypt Rao, 1976 Hawaii Dailey et al., 1993 Australia Platt and Blair, 1998, Gordon et al.. 1998; Cribb and Gordon, 1998 Hapalotrema sp. Australia Glazebrook et al., 1981 and1989; Glazebrook and Campell, 1990; Ba rker and Blair, 1996; Platt and Blair, 1998; Raidal et al., 1998 Hawaii Dailey et al., 1992; Work et al., 2005 Learedius learedi Captive Price, 1934 Panama Caballero et al., 1955 India Gupta and Mehrotra, 1975 Australia Blair ,1979 Caribbean Greiner et al., 1980, Dyer et al., 1991; Williams et al., 1994 Bermuda Rand and Wiles, 1985 Hawaii Dailey et al., 1992 and 1993: Dailey and Morris, 1995; Graczyk et al., 1995; Baja, California Cordero-Tapia et al., 2004 Learedius loochooensis Japan Takeuti, 1942 Learedius orientalis Arabian Sea Mehra, 1939 India Gupta and Mehrotra, 1976 Puerto Rico Dyer et al., 1995 Learedius similis Captive Price, 1934 Learedius sp. Australia Glazebrook & Campbell 1990; Campel et al., 1989 Hawaii Wolk et al. 2005 Montecellius indicum Arabian Sea Mehra 1939 Neospirorchis schistosomatoides Captive Price 1934 Bermuda Rand and Wiles, 1985 Australia Gordon et al., 1998

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56Table 1-2. Continued Host species Spirorchid species Locality Reference C. mydas (cont.) Satyanayanotrema satyanarayani India Simha and Chattopadhyaya ,1980 Squaroacetabulum solus India Simha and Chattopadhyaya ,1970 C. caretta Carettacola bipora Florida Manter and Larson, 1950 Carettacola sp. Atlantic coast, USA Wolke et al., 1982 Hapalotrema mistroides Italy Monticelli, 1896 Egypt Looss, 1899 and 1902; Gohar, 1934 and 1935 Australia Platt and Blair, 1998 Hapalotrema synorchis Florida, USA Luhman, 1935 Australia Platt and Blair, 1998 Hapalotrema sp. Atlantic coast, USA Wolke et al., 1982 Neospirorchis pricei Florida, USA Manter and Larson, 1950 Neospirorchis sp. Atlantic coast, FL Wolke et al., 1982 Florida, USA Jacobson et al., 2007 Neocaballerotrema caballeroi India Simha, 1977 Shobanatrema shobanae India Simha and Chattopadhyaya, 1970 E. imbricata Amphiochis amphiorchis Puerto Rico Fischthal and Acholonu, 1976 Amphiorchis caborojoensis Puerto Rico Fischthal and Acholonu, 1976; Dyer et al., 1995 Amphiorchis indicum India Simha and Chattopadhyaya, 1980 Amphiorchis indicus* Palao Islands Oguro, 1938 Hapalotrema pambanensis Australia Platt and Blair, 1998 Hapalotrema synorchis Japan Takeuti, 1942 Puerto Rico Fischthal and Acholonu, 1976 Hapalotrema sp. Australia Glazebrook et al., 1989; Gl azebrook and Campbell, 1990 Learedius learedi Puerto Rico Dyer et al., 1995 Learedius orientalis Puerto Rico Fischthal and Acholonu, 1976 Learedius sp. Australia Glazebrook et al., 1989

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57 CHAPTER 2 SPIRORCHIID TREMATODES OF STRA NDED SEA TURTLES IN FLORIDA AND ASSOCIATED PATHOLOGY Introduction Florida and its coastal waters are critical foraging and nesting hab itat for the Atlantic loggerhead turtles (Caretta caretta ) and Caribbean-Atlantic green turtles ( Chelonia mydas ). Approximately 1,200 to 1,500 strandings are docu mented annually in Florida, which far surpasses other Atlantic and Gu lf regions monitored under the S ea Turtle Stranding and Salvage Network (STSSN). Health-related studies in Florida sea turtles ha ve included investigations of fibropapillomatosis, brevetoxico sis, hypothermic (cold)-stunni ng, and other unusual mortality events (Witherington and Erhart, 1989; Herbst et al., 1994; Foley et al ., 2005; Jacobson et al., 2006). These studies have provided insight into important health i ssues, but also have revealed fundamental gaps in our knowledge of sea turtle di seases. Spirorchiid trematode infections in particular have been a recurring health issue of concern in Florida sea turtles largely due to observations of heavily infected individuals in the stranding populat ion and an uncertain role in at least one unusual mortality event (Wolke et al., 1982; Jacobson et al., 2006). Sea turtles serve as definitive hosts for approximately twenty-four different described species of spirorchiid trematode s representing ten genera (Smit h, 1997b). These numbers almost certainly are an under representation of actual parasite diversity as turtles inhabiting many geographical regions and some species have yet to be thoroughly examined. Studies of marine spirorchiids tend to be polarized as formal morp hological descripti ons and surveys of parasites, or descriptions of associated host pathology. Ve ry few investigators have effectively combined specific identification of sp irorchiid species, characteriz ation of host pathology, and interpretation of significance in the context of complete necropsies. Furthermore, previous studies have been heavily biased towards chronically ill turtles, which often are diagnostically

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58 complex and require heavily qualified or conser vative interpretation (Wol ke et al., 1982, Gordon et al., 1998). As a result, the picture of spirorchiids and their impact on sea turtle health is somewhat distorted and very incomplete. Basic information, such as prevalence of spirorchiids and associated pathological lesions in stranded turtles, is sparse or completely lacking in many regions, including Florida. The aim of these studies was to assess the sign ificance of spirorchiidi asis and characterize associated disease in stranded C. caretta and C. mydas by examining the following factors: impact of spirorchiids as a cau se of death or contributing cau se of death; diversity and prevalence of spirorchiids, microhabitat utilization in the turtle host, and associated pathological lesions; and relationships between parasitism and duration of illness and host age/size class. These various facets were selected to address cr itical unknown information relevant to Floridas stranded sea turtles, as well as health implica tions in sea turtles worldwide. The resulting findings provide the first comprehensive assessment of spirorchiidiasis in Florida turtles and new insight into host-parasite-d isease relationships. Materials and Methods Necropsies and Parasite Collection Necropsies were performed on stranded C. caretta and C. mydas recovered in Florida by the Sea Turtle Stranding and Salvage Networ k from November 2004 through October 2007. Two stranded turtles from Georgia (Glynn and Ch atham counties) also were examined. The study was limited to wild turtles that were found dead, died soon af ter discovery, or that were in rehabilitation centers for 10 days or less. Four categories of postmortem examination were performed depending on circumstances, postmor tem condition, and whether or not the carcass had been previously frozen. Category 1 n ecropsies included gross and histopathological examination of all organs and tissues, and complete parasitological methods for detection of

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59 spirorchiid trematodes. Category 2 necrops ies included gross assessment, complete parasitological methods for detection of spiror chiid trematodes, and, in some cases, limited histopathology of major lesions. Category 1 and 2 necropsies were performed at the University of Florida, College of Veterina ry Medicine. Category 3 necrops ies consisted of gross necropsy and evaluation of target organs and sites for spirorchiid tremat odes (see parasitological methods) and associated lesions. These necropsies prim arily were performed at the Florida Fish and Wildlife Conservation Commission (FWCC) Ma rine Mammal Pathol ogy Laboratory in St. Petersburg, Florida. Category 4 examinations were limited to evaluation of the brain and meninges for the presence of spirorchiid tremat odes. Categories 2 through 4 generally were previously frozen and/or autolyzed carcasses. Parasitological Methods The primary objective of the parasitologica l methods performed during necropsy was to detect and recover spirorchiid trematodes. These methods included a combination of gross examination, examination target organs using a dissecting microscope, screening of body fluids and organ washes, and histopathology. During the course of the necropsy, the liver, body cavity, heart (and base of aortas and pulmo nary arteries), aorta (left, ri ght and dorsal), and mesenteric arteries were examined and vascular compartments were thoroughly washed and filtered using a #45 sieve. The filtrate was examined for the pr esence of spirorchiids. In addition, the brain, heart, any vascular lesions, t hyroid gland, thymus, adrenal glands urinary bladder, and sections of stomach and intestine were examined using a dissecting microscope for the presence of spirorchiid eggs and adults. For category 3 n ecropsies, the complete aorta was evaluated for areas of endarteritis and the brain, thyroid gl and, adrenal glands, and thymus were examined under a dissecting microscope. Category 4 necropsi es were limited to examination of the brain using a dissecting microscope. Identification of spirorchiids was based on examination of adults

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60 whenever possible. When only eggs were dete cted, morphology alone was used to identify the presence of the genera Neospirorchis and Carettacola ; however, egg morphology was not used to distinguish Hapalotrema and Learedius egg types in green turtles. In such cases, the presence of eggs with bipolar processes (Type I) was noted but neither generic nor specific identification was made. Lastly, observed sp irorchiid eggs, especially Neospirorchis sp., in tissues were classified as primary site ovipos ition verses embolization to dist ant locations. Primary sites of oviposition were characterized by the presence of large and/or discrete egg masses (typically greater than 100 eggs) and often we re observed in the presence of adult spirorchiid s. Embolized eggs most commonly were diffuse ly distributed throughout a tissu e or organ and were either individual eggs or small cl usters of less than ten. Laboratory Methods Samples for histological examination were fixed in 10% neutral phosphate buffered formalin and processed by routine methods into paraffin blocks, wh ich were cut into 5 m-thick sections and stained with hematoxylin and eosin. Spirorchiid trematodes were fixed in alcoholformalin-acetic acid (AFA) if intact or nearly complete and representative specimens (including adults and eggs) were fro zen at -80 Celsius. Categorization of Duration of Illness Necropsied turtles were placed into three ca tegories according to the estimated duration of illness. The subjective criteria for classi fication included both nutritional condition and necropsy findings (Table 2-1). Nutritional cond ition was the primary determining factor and was assessed by evaluation of skeletal muscle, adipose tissue, skeletal mineraliz ation, and presence or absence of a generalized catabolic state. The s econdary factor is the apparent duration of any identifiable fatal or contributor y pathological lesion. Assessment of lesion chronicity was based on standard gross and histopathol ogical interpretation, e.g. primar ily granulocytic inflammation

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61 and fibrinous exudation indicate acute processe s and predominantly mononuclear inflammation, granulation tissue formation and fibrosis are indi cators of chronicity. The actual time scale of these categories is a crude estimate as the progr ession of weight loss in sea turtles has not been studied and many factors may influe nce the progression of disease, such as severity of insult, degree of debilitation, and envir onmental parameters. Acute insu lts include immediate fatality and animals that were estimated to be ill on the order of days to weeks. Turtles with intermediate insults were estimated to have been ill for weeks to months. Those animals categorized under chronic insult likely have been ill on the order of months or longer. Primary Diagnosis / Cause of Death Designations Necropsied turtles also were categorized by the primary diagnosis or cause of death. Categories included traumatic injury, brevetoxicosis, hypothermic (cold-stunning), mass mortality events (MME) of unknow n etiology, drowning/aspiration, in fectious disease, enteric impaction, fibropapillomatosis-associated, emaci ation, multifactorial, and undetermined. These designations were given to represent the primary health problem, if determinable. For example, a turtle that died of secondary bacterial infection of a boat pr opeller wound was categorized as a traumatic injury. Similarly, animals that ul timately drown from underlying disease were categorized by the nature of the underlying disease, if identified. Prim ary drowning/aspiration cases were diagnoses by exclusion, often were accompanied by circumstantial evidence (e.g. recovered from trawler net), and were not associated with rec ognized MME's. Brevetoxicosis, unsolved MME's, and hypothermic deaths were supported by epidemiological stranding data, environmental data, and supportive diagnostics from the investigati ons of these events, rather than specific pathological fi ndings. Emaciation was listed as the primary diagnosis if poor nutritional condition was the only necropsy finding of significance. Deaths were ruled multifactorial if multiple processes were present and were interpreted to have contributed to

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62 death. In every use, this designation reflected multiple health problems in chronically ill, emaciated turtles. Spirorchiid Impact Rating Spirorchiid trematode infection and associat ed pathological lesions was evaluated based on intensity of spirorchiid infec tion, severity of lesions, proportion of organ involvement, and the organ or tissue affected. All data were asse ssed in the context of other necropsy findings and cause of death, if determined, and scored into one of five categories. The criteria for scoring are given in Table 2-2. Intensity of parasitism classically is measured by quantifying adult parasites and or eggs, e.g. fecal egg counts. With regard to spirorchiid trematodes, the numbers of adult parasites often do not appear to correlate wi th severity of pathological lesions and the intravascular location and small size of adults makes confident quantification extremely difficult or impossible (Gordon et al., 1998). Also, the va riety of tissues in which eggs selectively accumulate makes any objective measure that would be comparable between cases very difficult. Therefore, the intensity of spirorchiidiasis in this study is based on a relative assessment of absent, rare, small, moderate, or large numbers, reflecting both adult and egg numbers within various organs and tissues as observed at the gr oss and subgross levels. Photographs of cases and postmortem data were then retrospectively compared to support that abundance ratings were consistent within categories. In addition, spec ific grading criteria were created for vascular, neurological, thyroid/thymic, a nd gastrointestinal lesions (Tab le 2-3). A spirorchiid impact rating was only applied to category 1 and 2 necropsies. Body Condition Calculations for Immature C. mydas Error in body weight measurements due to variation between equipment, epibiota accumulation, and severe traumatic injuries pr evented confident assessment of body condition indices in the general study population. A group of immature C. mydas that died from

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63 hypothermic-stunning did not have these conf ounding problems, thus additional body condition data were collected. Two body condition indices were calculated, a simple mass to length ratio (Body mass [BM] [kg]/Straight carapace length [SCL][cm]) and estimated volume ratio (BM/SCL3), which has been used in previous se a turtle studies (Bjorndal et al., 2000; McMichael, 2005; Work et al., 2005;). Statistical Methods Comparisons were made between various aspects of spirorchiidiasis, including presence/absence, pathological lesions, and grade, in turtles categorized by duration of illness (see previous criteria) and size. Three size classes used were based on straight carapace and included SCL < 65 cm, >65-85 cm, and >85cm. Some factors were combined to support statistical analysis when appropriate. Paramete rs were compared by Pearson's chi square or Fisher exact test (95% confidence interval), depending on the expected frequencies, using Analyse-it version 2.11. Also examined we re differences in body condition indices in immature C. mydas with and without moderate or large numbers of embolized spirorchiid eggs and intra-thyroid Neospirorchis species using the Mann-Whitney test. Results Caretta caretta Study population demographics, duration of illness categories A total of eighty-nine C. caretta were necropsied. The number of C. caretta in each necropsy data category by geographical region is shown in Figure 2-1. The frequency distribution of turtle sizes had a bimodal distribution with peak frequencies in th e 60-75 cm SCL and 85-95 cm SCL size ranges (Figure 2-2). All turtles >85 cm SCL were confirmed to be sexually mature. The female to male sex ratio wa s strongly biased at 3.94 :1 (71/18). Category 1 and 2 data included 54 turtles, 20 in the acute insult category, 14 in the intermediate duration

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64 category, and 20 in the chronic insult category (Tab le 2-4). The size class compositions of the duration of illness categories were compared. The only statistical difference was that there was a higher proportion of turtle s (category 1 & 2 data) gr eater than 85 cm SCL in the acutely affected group as compared to the those wi th chronic insults (p<0.05). Th e same bias was observed when all necropsy data categorie s (1-4) were analyzed (p<0.005). The primary reason for this bias was traumatic deaths of adults dur ing breeding and nesting season. Primary diagnosis or cause of death by duration of illness Acute insult. The primary diagnoses or causes of death of C. caretta that died from acute insults included traumatic injury (6/20), brevetoxi cosis (7/20), and deaths associated with a mass mortality event of unknown etiology (4/20) (Table 2-4). Traumatic injuries were due to watercraft collisions (2/6), injuries resulting from a fall (from a sea wall) (1/6), shark attack (1/6), and a probable hooki ng injury (1/6). One turtle died of a unilateral coracoid fracture of unknown cause that resulted in second ary perforation of the left aort a. All brevetoxicosis cases were associated with a Karenia brevis bloom in southwestern Flor ida (Zone 2) in 2005. The turtles that died associated with a mass morta lity event of unknown etiology were all from the same event, which was documented in northeastern Florida and Georgia co asts (Zone 3) in 2006. Some type of acute toxicosis was suspected based on the lack of any evidence of primary infectious disease, traumatic injuries, circumstanti al environmental data, or other apparent cause. Testing for brevetoxin, ciguatoxi n, saxitoxin, palytoxin, and domoic acid were al l negative (data not shown). Of the remaining three turtles in the acute insult category, one drown in a trawler fishing net (1/20), one died of aspiration pneumonia due to unknown circumstances (1/20), and the cause of death of a third case could not be determined (1/20). Insult of intermediate duration. Necropsy cases categorized in the intermediate duration category included deaths associated with th e aforementioned mass mo rtality event of unknown

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65 etiology in 2006 (6/14), inflammatory/infectious disease (5/14), traumatic injuries (2/14), and one turtle that died from an en teric impaction associated with seve re colitis (1/14) (Table 2-4). Deaths associated with infecti ous disease or inflammatory cond itions were due to a variety of causes and affected systems. Cases included sept ic cholecystitis (1/5), fungal tracheobronchitis (1/5), chronic pneumonia of unknown etiology (1/5), necrotizing dermatitis (1 /5), and septicemia with thromboembolic disease (1/5). The case of thromboembolic disease had atrial thrombi that formed in association with areas of intense endocardial spirorchiid egg deposition, which was interpreted as predisposing or confounding condition. The two deaths resulting from traumatic injuries were both cases with ch ronic propeller wounds that entere d the spinal canal and resulted in severe ascending bacterial meningitis (2/14). Chronic insults. Most postmortem findings in chronically ill C. caretta were regarded as either nonspecific lesions associated with chroni c illness and advanced poor nutritional status or opportunistic infections. Interpretation of some pathological fi ndings as opportunistic (secondary) infections was based on the relatively acute nature of the inflammatory response (predominantly granulocytic) as compared to advanced poor nutri tional status/chronic illness. Some chronic conditions also we re identified, but only in a sing le case (discussed below) could these findings be argued to be an inciting or primary cause of general illne ss. Nonetheless, these processes may have ultimately contributed to death in many cases or exacerbated decline in clinical status. Most of the necropsied C. caretta categorized under chronic disease had multiple disease processes of significance and cause of death was classified as multifactorial (13/20) (Table 2-4). No underlying primary cause of disease could be c onfidently identified in these cases, nor was there any apparent temporal or other association with known mass mortality events. In addition

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66 to being profoundly emaciated, common diagnoses in terpreted to be contributory to death in these animals included ulcerative gastritis (8/ 13), ulcerative enteritis and/or colitis (7/13), dermatitis (most often associated with accumulati on of epibiota) (8/13), granulomatous hepatitis (4/13), and cutaneous and/or musc uloskeletal trauma associated with advanced catabolic state (2/13). In fewer cases, emaciation alone wa s the primary significant finding (3/20). In 4/20 cases, a single or predominant proces s was evident as an underlying condition or proximate cause of death. One turtle had se vere chronic cardiovascul ar and gastroenteric spirorchiid-associated lesions that were interpreted as the cause of death. Tw o turtles had severe chronic colon impactions with associated ulcerat ive colitis. Lastly, one turtle had chronic, obstructive inflammation of all major bile ducts (choledochitis) and drowned after entering a coastal power plant facility. Prevalence by genus and identifiable species (category 1 & 2 data). Spirorchiids detected in the study populat ion were species within the genera Neospirorchis, Hapalotrema and Carettacola Period prevalence of Neospirorchis sp. was 96.3% (52/54), prevalence of Hapalotrema sp. was 77.8% (42/54), and prevalence of Carettacola sp. was 22.2% (12/54). All adult Carettacola were identified as C. bipora Mixed infections by all three genera were observed in 11 /54 infected turtles, two genera in 32/54 turtles, and single species infections were detected in only 9/54 turtles. Three species of Hapalotrema were found in C. caretta These included H. mistroides H. pambanensis and a novel species (Figure 2-3). Iden tification of species was performed in 20/25 cases in which adults were collected. Hapalotrema mistroides was identified in 17/20 cases, H. pambanensis in 5/20 cases, and the novel Hapalotrema sp. in 2/20 cases. Specific species identification by morphology wa s not possible for most of the recovered Neospirorchis species because intact specimens suitable for identification were extremely

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67 difficult to collect. A diagnosis of Neospirorchis was based on egg morphology, elongate body type, and examination of anterior and posterior ends of some fragmented specimens. The only exception was N. pricei which was easily recovered intact from the heart and major arteries. Prevalence of this species was 11.1% (6/54). Site predilection and associated pathological lesions Hapalotrema species. Species within the genus Hapalotrema were observed in the heart, aortas, mesenteric arteries and hepatic vessels, an d pathological lesions in major arteries were seen in 37.2% (29/78) of C. caretta in category 1-3 necropsies (Tab le 2-5). Of those parasites identified to the species level, the sites from wh ich adult spirorchiids were recovered are given in Table 2-6. When categorized by duration of illness (category 1 & 2 necropsies), there were no detectable differences in numbers of infected turtles. There were, however, differences when status of infection was compared by size class. There were more infected turtles in the >85 cm SCL group than the < 65 cm SCL group ( p <0.05). Lesions of major arteries, including the aort as and major branches were associated with Hapalotrema species based on the presence of adults attached within areas of arteritis in representative cases. Only one case had endarteritis with no evidence of Hapalotrema infection. Lesions in this case were minimal and chronic, wh ich may reflect a resolved or mild infection. The discovery of multiple Hapalotrema species in C. caretta somewhat complicates association of a specific species with pathologic lesi ons because four of 20 turtles in which Hapalotrema species were identified had mixed infections including H. mistroides and H. pambanensis However, only two turtles with mixed inf ections had major arterial disease, and H. mistroides was the only species identified in the remainder of cases from which adults were recovered. Also, neither of the two cases infected with the novel Hapalotrema species had arterial disease affecting larger arteries.

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68 In category 1 and 2 necropsies, the most common site of endarteritis/arteritis was the left aorta (n=25), followed by the superior mesenteric ar tery (n=11), right aorta (n=4), gastric artery (n=3), coeliac artery (n=1), and dorsal aorta (n=1 ) (Figure 2-4). Multiple vessels were affected in 9/29 cases. Gross lesions ranged from single sm all plaques of endarteri tis to larger areas of arteritis with associated generali zed thickening of the artery wall (3 to 4 mm) involving the entire left aorta and, in some cases, the superior mesenteric, coeliac, and gastric arteries. Mural thrombi were observed in 10 cases and all were non-occlusive. Aneurysm formation was present in a single turtle at the proximal superior mesenteric artery. Major arterial lesions were histologically examined in category one necrops ies (n=24). Mild lesions (Grade 1) were characterized by mild subintim al fibrosis and a predominantly mononuclear inflammatory infiltrate. More severe lesions included variably intense pleocellular infiltrate, fibromuscular proliferation (often villus) of the intima (resulting in severe thickening of the artery wall), and degeneration and necrosis of the tunica media (Figure 2-5). The latter was a distinctive lesion characterized by formation of well-demarcated necrotic areas of smooth muscle surrounded and infiltrated by heterophils (Figure 2-6). This lesion was observed in seven cases. Inflammation extended transmurally in fifteen cases and ofte n formed patchy or diffuse cuffs of intense pleocellular infiltrate within the adventitia (F igure 2-5). Granuloma formation within lesions was common and most often included intralesio nal spirorchiid eggs, necrotic debris, and pigmented material. No significant differences were detected in the presence of arteritis or grade of arteritis when turtles were categorized by duration of illness. There were, however, significant differences based on turtle size. More large turtles (>85 cm SCL) ha d endarteritis than either the >65-85 cm or < 65 cm SCL categories (p<0.005 and p< 0.0001, respectively). There were no

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69 detectable significant differences in grade of arteritis among infected turtles of the different size groups. Pathological lesions affecting small arte ries were histologically observed in gastrointestinal tract and associated other organs in eleven turtles, all of which were infected by Hapalotrema species. Observed lesions includ ed endarteritis, medial hypertrophy, microvascular proliferation, and pe riarteritis (Figure 27). Organs/sites with affected vessels included the gastrointestinal tract (n=9), pancreas (n=2), kidney (n=1), and adrenal gland (n=1). All but two turtles had concurrent endarteritis of major vessels and 6/9 had grade 3 lesions. Some turtles had grossly visible Hapalotrema egg masses within subserosal vessels. Two distinct gross appearances of these egg masses were observed, the most common of which was small, raised, brown masses two to three millimeters in diameter (Figure 2-8). Most (6/7) of these turtles with observable emboli had grade 2 or 3 lesions in major arteries and were infected with H. mistroides or had mixed (H. mistroides and H. pambanensis ) infections. The second gross lesion type was represente d by a single case with large tran smural egg granulomas visible from the mucosal and serosal surfaces (Figure 2-9) This turtle did not have major arterial disease and was in fected by the novel Hapalotrema species. Adults and egg emboli were limited to the smaller arteries. Six C. caretta also had vasculitis of hepatic portal ve ins. All cases were infected with Hapalotrema sp., but no adults were recovered from the hepatic vasculature in any case. Two turtles also were infected with C. bipora, and two adults were removed from the liver vasculature in one of these animals, thus it is possible that C. bipora is a cause of this lesion in some cases. Additional lesions associ ated with infection by Hapalotrema sp. included two cases with papillary lesions associated with submucosal granulomatous inflammation in response to

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70 spirorchiid eggs. The first was the formation of colonic polyps in a turtle infected by H. mistroides (Figure 2-10). The second case had severe granulomatous cholecystitis with papillary mucosal hyperplasia asso ciated with the novel Hapalotrema species (Figure 2-11). Neospirorchis species. Spirorchiid eggs consistent in morphology with the genus Neospirorchis were widely embolized throughout the body. Dense concentrations of eggs, often associated with adult parasites, were consiste ntly found in several anatomic locations including the vasculature of the leptomeninges, endocrine organs, thymus, and submucosa of the alimentary tract. The period prevalence of Neospirorchis species observed in these locations for all necropsy categories is given in Table 2-7. Neospirorchis sp.: central nervous system. Adult Neospirorchis sp. and/or egg masses were observed in the leptomeninges of almost half (44/89) of the necropsied stranded C. caretta (all categories included) (Table 2-8) (Figure 2-12). The major ity (29/45) of these cases were grade 1 infections, although, more than ten percent were classified as grade 3. There were no significant differences detected in either the propor tions of infected turtle s or grade of infection when the study population was categorized by duration of illness. There were, however, differences when turtles were compared by size. A significantly higher proportion of turtles >85 cm SCL were infected as compar ed to turtles between >6585 cm SCL (p < 0.01) and turtles < 65 cm SCL (p <0.0001). There were no demonstr able significant diffe rences in the grade among infected turtles between the size categories. However, very few infected turtles were < 65 cm SCL (n=5), thus more confident assessm ent would require a larger sample size. Adult trematodes and egg masses were most commonly within venules of the leptomeninges and surface vessels of the brain an d spinal cord. Individual eggs and egg masses were both intravascular and perivascular. Th ere was minimal or no inflammatory response

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71 associated with adult trematodes. Egg masse s often were surrounded by granuloma formation, including intravascular granuloma formation, and a predominantly mononuclear infiltrate, resulting in meningitis and choroiditis. The seve rity of inflammation, with regard to distribution and intensity, corresponded to the number and di stribution of eggs present in most cases. A fibrin thrombus was observed in a single case with grade 3 infection. No asso ciated ischemic or hemorrhagic lesions affecting the brain or spinal cord were obse rved in any infected turtles. Neospirorchis sp.: endocrine organs. The endocrine organ in which spirorchiid infection was most commonly observed was the thyroid gl and (Figure 2-13). Of category 1, 2, and 3 turtles, 54.6% (41/75) had adult Neospirorchis sp. and/or eggs within the thyroid gland and 22.0% (9/41) of those infected were classified as grade 3 (Table 2-9). Of the category 1 and 2 necropsies, 44.4% (24/54) had a dult spirorchiids observed with in the thyroid gland. When categorized by duration of illness, significantly more chronically ill turtles were infected as compared to animals in the acute (p<0.005) and intermediate duration categories (p <0.05). Also, proportionately more chronica lly ill turtles had grade 3 infec tions as compared to acutely affected turtles (p<0.05); however no significan t difference was observed when analysis by grade was limited to turtles with thyroid spirorchiids only (negative animals not included). No significant relationships between in fection or grade of infection and turtle size were detected. Severity of associated pat hological lesions correlated with numbers of distribution of eggs. In grade 3 infections, the thyroid gland often appeared black on section due to the numbers of spirorchiid eggs, which formed large egg ma sses within the thyroid and surrounding cervical vasculature. Histologically, these lesions were characteri zed by an intense mononuclear infiltrate, granulomatous inflammation, thrombosis and loss of thyroid fo llicles. Adults and

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72 vascular lesions, including medi al hypertrophy and vasculitis, were observed in venules and arterioles. Adult spirorchiids also were frequently obs erved in venules of the adrenal glands. Among category 1 and 2 turtles, adult Neospirorchis sp. were observed in 32.0% (16/50). No egg masses were observed in this location, but individual embolized sp irorchiid eggs were common (not included in period prev alence). Adrenalitis was minimal or mild in all cases and consisted of mild, mononuclear peri vasculitis and egg granulomas. Other endocrine organs in which Neospirorchis species also were observed included the pineal gland (adults [n=7] and eggs), parathyroid gland (eggs only), pancreas (eggs only), and pituitary gland (eggs only). These observations were not included in any analyses due to small sample sizes. Neospirorchis sp.: thymus. Another common site in whic h spirorchiids were observed was the thymus, which was infected in 48.7% (37/76) of category 1, 2, and 3 tu rtles (Table 2-10). The most commonly observed gross finding was egg masses within thymic vessels (Figure 2-14). All but one turtle with intrathymic spirorchiid eggs and/or adults also had thyroid involvement. Of the thymic infections observed, 27.0% (10/37 ) were grade 3. When categorized by duration of illness, chronically ill turtles included a significantly higher proportion of thymic infections than acutely ill turtles (p <0.0001). Significant diffe rences in proportion of infection turtles were detected when turtles were categorized by size. More turtles in the < 65 cm and >65-85 categories were infected than turtles >85 cm (p <0.05). No significant relationships between grade of infection and duration of illness or size were detected. Neospirorchis sp.: gastrointestinal tract. The period prevalence data reported in this section include Neospirorchis species in which adults were found within the enterocolic

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73 submucosa and produced large black, serpiginous e gg masses (typically 2.0 to 5.0 cm in length) that were visible from the mucosal surface (Figure 2-15). The enterocolic Neospirorchis sp. were found in 62.0% (31/50) of the study populati on (Table 2-11). There were proportionately more infected chronically ill turtles than acu tely ill animals (p <0.005). No significant differences in grade were detectable between dur ation of illness categorie s. When categorized by size, there was a higher proportio n of infected turtles in the >65-85 SCL si ze group than in the >85 cm size group (p <0.01). No differences in grade of infection were observed among the different size groups. Submucosal egg masses produced ulceration an d secondary bacterial infection as eggs migrated through and injured the mucosa (Figure 216). Associated ulcerative enteritis was most severe in grade 3 infections in which large area s of the mucosal surface were affected. Lesions were most common in the small intestine, where egg mass numbers and density were the greatest. There were observable differences in the morphology of adult Neospirorchis when specimens were examined under a dissecting micr oscope (Figure 2-17). Some individuals appeared longer, robust, and yello w as compared to others that were narrower in diameter and white. The eggs associated the more robust type also were larger (average of 59 x 47 m) as compared to those of the more petite forms, which averaged 43 x 36 m (Figure 2-17). In addition, both forms lacked the prominent pigmented cecae that were easily observed in adults recovered from the nervous system a nd endocrine organs (Figure 2-17). Additional egg masses were observed during examination of the alimentary mucosa and urinary bladder mucosa using a dissecting micr oscope (Figure 2-18). These egg masses were smaller (<1.0 2.0 mm diameter) and lighter brown than the more commonly observed

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74 enterocolic forms and the eggs were characterize d by distinct fine surface projections (Figure 219). These small egg masses were found in ten C. caretta and locations included the stomach (n=1), M2 (n=2), M3 (n=2), cloaca (n=5), and ur inary bladder (n=5). No adult spirorchiids were observed in association with these egg masses in any case. Neospirorchis sp.: heart and major arteries. The one identifiable species of Neospirorchis, N. pricei was found in six C. caretta When infected turtles were categorized by duration of illness, three were chronically ill and three were in the intermediate category. All infected turtles were >65 cm SCL, three were >65-85 cm SCL and three were >85 cm SCL. Most adults were removed from the heart in all bu t one case. Small numbers of adults also were collected from the mesenteric vessels (n=3), aortas (n=1), and carotid artery (n=1). It was not possible to confidently quantify the numbers of adults collected because individuals frequently were fragmented and often were extensively inte rtwined in the muscular ventricular trabeculae and were extremely difficult to flush out of the heart. Total numbers of complete and fragmented individuals from each case were 85, 2 4, 20, 9, 8, and 1. One turtle infected with N. pricei died of septicemia and thromboembolic diseas e and had dense aggregates of eggs within the atria over which mural thro mbi had formed (Figure 2-20). Other findings related to Neospirorchis sp. Adult Neospirorchis species were observed in several addition sites either by organ lavage, subgross examination, or histological examination. The observations were limited to a few individual turtles. The most extensive injury was observed in the testes of three C. caretta in which adults were present and abundant eggs were seen associated the granulomatous in flammation (Figure 2-21). One of these cases had a severe associated orchitis and moderate se verity was observed in th e other cases. Several specimens of Neospirorchis species were flushed from th e hepatic vasculature of one C. caretta

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75 and microscopic trematodes, consistent in morphology with Neospirorchis sp. were observed in the sinusoids of three additional turtles. No a ssociated inflammatory response in the liver was observed. Additional locations were limited to single observations and rare or individual parasites. Sites included the lungs, nasal submuc osa, arterial periadve ntitia, and bile duct periadventitia. Spirorchiid impact rating Spirorchiid infection in most of the turtles ne cropsied was categorized as either incidental (27/54) or undetermined (18/54), followed by cont ributory (6/54), uninfect ed (2/54), and fatal (1/54) (Table 2-12). Turtles were categorized by duration of illness and placed into two groups: an uninfected and incidental infection verses those turtles with undetermined, contributory, or fatal impact ratings. There were significantly more acutely ill turtles in the uninfected and incidental group as compared to the chronically ill turtles (p<0.005). No significant differences were detected when the same comparis ons were made among size classes. Criteria for categorization of spirorchiid significance in contributory, fatal, and undetermined categories reflect common arrays of findings. Within the contributory category, 5/6 turtles were emaciated and had multi-organ lesions. The key determining factor for assigning a spirorchiid impact rating of contributory, as stated in the scoring criteria, was the presence of significant multisystemic lesions associated with eggs or adult parasites. Of these cases, one turtle had extensive ulceration of the pylorus as a primary necropsy finding that was associated submucosal embolization of Hapalotrema eggs. Severe arteritis ( Hapalotrema mistroides ) was observed in four turtles, thyroiditis ( Neospirorchis sp.) in two cases (one of which also had thymitis), and two cases had abundant (grade 3) meningeal spirorchiids ( Neospirorchis sp.). Considerable embolizat ion of eggs was interprete d as contributory in three cases in which abundant eggs were found in th e gastroenteric submucosa and lungs. One case

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76 also had abundant enteric Neospirorchis egg masses with associated enteritis and another had severe orchitis ( Neospirorchis sp.). The only non-emaciated turt le in the contributory category was an underweight animal with septicemia and th romboembolic disease. In this case, atrial thrombi formed over areas of intense endocardial Neospirorchis egg deposition. The only case in which spirorchiidiasis could be confidently confir med as the cause of death was a loggerhead with massive egg granulom as in the gastrointest inal tract, liver, gall bladder, and other organs associated with a novel Hapalotrema sp. The extent of spirorchiid egg granulomas in this case resulted in secondary ischemia of the enteri c mucosa, ulceration, and probable septicemia. Of those cases in which the impact of spir orchiidiasis was undeter minable, half (8/18) were chronically ill and half (10/ 18) were in the acutely ill or in termediate duration categories. As stated in the scoring criteria, all had identi fiable causes of death or multiple findings that could not be confidently associated with spir orchiidiasis based on le sion location and disease process or severity. For example, the chroni cally ill turtles were emaciated and had findings identifiable as a proximate cause of death or contributing health problems, but did not have the multisystemic lesions comparable to those given a contributory rating. The most common spirorchiid associated lesions in animals in th e undeterminable category was grade 3 arteritis (12/18), neurospirorchiidiasis (6/18), and thyroid itis (6/18). Of these animals, 11/18 had lesions involving more than one organ or system, typically ar teritis and neurospirchiid iasis or thyroiditis. Findings represented by individual cases included one turtle with large numbers of N. pricei in the heart, one animal with severe thymitis (concu rrent with thyroiditis), and one case with large numbers of eggs embolized in the enteric mucosa.

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77 Chelonia mydas Study population demographics and duration of illness categories Fifty-nine C. mydas were examined. The total number of C. mydas in each necropsy data category by region of stranding is shown in Figur e 2-22. Frequency distribution of turtles by SCL is shown in Figure 2-23. The study popula tion was heavily biased towards smaller, immature animals less than 45 cm SCL. The female to male ratio was 2:1 (38/19) (data unavailable for 3 turtles). Category 1 and 2 da ta included 50 turtles, 37 in the acute insult category, 8 in the intermediate duration category, and 5 in the chronic insult category (Table 213). The large number of turtles in the acute insult category reflected 30 animals that stranded during a cold-stunning event in St Joseph Bay in January 2008. In addition, all tu rtles greater than 65cm SCL fell within the ac ute insult category, thus the inte rmediate duration and chronic insult categories were comprised completely of turtles less than 65 cm SCL. As in C. caretta a primary reason for this bias was acute trauma tic deaths of adults during breeding season. Primary diagnosis or cause of death by duration of illness Acute insult. Most of the C. mydas cases in the acute insult category were deaths associated with hypothermic stunning (cold-stunning) (30/37) and traumatic injuries (7/37). The hypothermia cases all originated from a single event in Zone 1 in January 2008. Most of the traumatic injuries (6/7) were watercraft-related. A seventh turtle was a nesting female that died from suffocation after a dune collapse d on her while nesting (1/6). Insult of intermediate duration. Cases classified as insults of intermediate duration included turtles with traumatic injuries (3/8), infectious diseas es/inflammatory conditions (3/8), and fibropapillomatosis (2/8). Two turtles with tr aumatic injuries had watercraft-related injuries with secondary bacterial infec tion (2/3) and one had a chroni c, strangulating entanglement wound on its neck (1/3). Infectious diseases and inflammatory conditions included fungal

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78 pneumonia (1/3), fungal tracheitis (1 /3), and probable septicemia (1/3). Of the two turtles in which fibropapillomatosis was the primary finding, one had been euthanized due to the presence of visceral tumors, and one had a severe marine leech infestation ( Ozobranchus sp.) and anemia resulting in secondary drowning after entering a coasta l power plant. Chronic insults. Health problems observe d in chronically ill C. mydas were varied. Primary diagnoses included bacterial meningoence phalitis (1/5), fibropapillomatosis (1/5), and a chronic entanglement wound (1/5). Emaciation was the only significant finding in one animal (1/5) and the death of another was interpreted as multifactorial (1/5). Prevalence by genus and identifiable species (category 1 & 2 data). Three genera of spirorchiid trematodes we re detected in the study population, including Neospirorchis, Learedius and Hapalotrema Period prevalence of Neospirorchis was 92.0% (46/50), Learedius was 8.0% (4/50), and Hapalotrema was 2.0% (1/50). All Learedius specimens were identified as L. learedi and the only example of a Hapalotrema infection was identified as H. postorchis Type I eggs (Hapalotrema sp. or Learedius sp.) were observed in two additional turtles from which no a dults were recovered. Among infected C. mydas the majority 45/50 were infected by one genus and five were infected by two different genera. As in C. caretta specific species identification of Neospirorchis specimens was not possible due to the inability to ob tain intact parasites suitable fo r identification. Classification of adult and egg specimens as Neospirorchis was based on egg morphology and elongate body type. Site predilection and associated pathological lesions Learedius learedi. Sites from which L. learedi were recovered included the heart and hepatic vessels. Three of the four infected turtles were adults and one was an immature animal with a SCL of 25.6 cm. Thus, out of 46 immature turtles examined (category 1 & 2 data), only 1

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79 had evidence of L. learedi infection. The three a dult turtles died from tr aumatic injuries (acute insult category) and the juven ile was underweight and had fungal pneumonia (intermediate duration category). The latter animal is estimated to have had at most 1-2 adults, which were observed on histological examinati on and did not lavage from the heart during parasite recovery. The adult turtles had intensities of 21, 35, and 36 adult parasites pe r host. Two of these turtles had associated distinct focally ex tensive endocarditis located suprav alvular to the atrioventricular valves (Figure 2-24). These lesions were obser ved grossly as well-demarcated, raised, tan pink areas with an irregular surface and histologically were characterized by a mixed mononuclear and granulocytic infiltrate, fibrosis, and surf ace villous proliferation. Milder, more diffuse endocarditis also was present in both turtles. In addition, both animals with endocardial lesions also had severe thickening of the aortic system due to marked subintimal proliferation and mild pleocellular endarteritis. The extent of these lesions in one case was more extensive and involved the pulmonary, brachiocepha lic, carotid, mesenteric, and asso ciated smaller arteries, as well as prominent generalized thickening/hype rtrophy of the myocardium (Figure 2-25). Hapalotrema postorchis. Only one C. mydas was confirmed to be infected by a species of Hapalotrema which was identified as H. postorchis This parasite has not been previously reported in Florida. The infected turtle was an a dult male that died as th e result of head trauma sustained during entrainment in a coastal power f acility (acute insult category). Twenty-one adult H. postorchis were collected and fragments of an additional four to ten individuals were observed. The aortic system was prominently thic kened, which reflected chronic arteritis similar to that observed in turtles infected with L. learedi (Figure 2-26). The left aorta was most severely affected and had multifocal, grossly visi ble plaques of endarteritis containing several

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80 embedded H. postorchis Lesions in small arteries, including the mesenteric, renal, adrenal, and carotid arteries, also were observed. Neospirorchis sp. As in C. caretta spirorchiid eggs consis tent in morphology with Neospirorchis sp. were observed embolized throughout th e body and were most readily visible in the lung and submucosa of the gastrointestinal trac t. In addition, dense concentrations of eggs and/or adult parasites were found in some locations similar to that observed in C. caretta specifically the central nervous system, endocrine organs, an d rarely within the gastric submucosa. The period prevalence of Neospirorchis species found in these sites for the four necropsy categories is shown in Table 2-14. Neospirorchis sp.: central nervous system. Adult spirorchiids and/or egg masses identified as Neospirorchis sp. were found in leptomen inges of 11.9% of necropsied C. mydas (7/59). These findings in C. mydas must be qualified with regard to grade and size class considerations. Foremost, all egg masses tended to be very small (often 1 mm or less), thus the criteria used to quantify/grade C. caretta infections were scaled by abundance of eggs, rather than egg mass size to represen t equivalent abundance in C. mydas Second, adult parasites only were observed in subadult or mature C. mydas which included 5/7 infected turtles. Four were adults with SCL of 95 cm or greater that died from acute traumatic events during nesting season. The fifth turtle was submitted as a head only and based on its size was estimated to be a large subadult or adult. Thus, all of the mature gr eens examined had neurolog ical infections. Two cases were classified as grade 1 and three as grade 2. The rema ining two turtles were immature, one of unknown size and another wi th a SCL of 43.1 cm. In both cases, no adults were observed and egg masses consisted of very small numbers of eggs (both grade 1), which may reflect embolization rather than oviposit ion within meningeal vessels, as is apparent when adult

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81 parasites are present. Nevertheless, these cases were included because similar patterns of egg embolization (from distant sites) were not obs erved in the other 52 turtles examined. Neospirorchis sp.: endocrine organs. Spirorchiid parasitism of the thyroid gland was observed in 33.3% (17/51) of necropsied C. mydas (Table 2-14). Of the infected turtles, adult parasites were observed in 58.8% (1 0/17) and 5.9% (3/51) were classified as grade 3 infections (Figure 2-27). Comparisons with other factors were limited given the biases of the study population, and limited number of ch ronically ill, mature, and larg e immature turtles examined. Associated pathological lesi ons were as described for C. caretta Adult parasites were recovered from the pineal gland in two turtles and eggs were focally concentrated at this site in a third cas e. One turtle was found to have adult Neospirorchis sp. in the parathyroid gland. Findings in other endocrine organs were limited to embolization of eggs. No adult parasites were obser ved in the adrenal glands. Neospirorchis sp.: gastrointestinal tract. The only spirorchiid-related necropsy finding in the alimentary tract that exhibited a distinct pattern, other than wide spread or regionally intense egg embolization, was the finding of sm all egg masses within the gastric submucosa (Figure 2-28). Egg deposition at this location was observed in 10.4% (5/48) of examined C. mydas which were all immature and represented all duration of illness categories. This finding typically only was visible by examination of th e gastric mucosa under a dissecting microscope and associated inflammatory lesions were minimal or mild. No adult spirorchiids were observed grossly, but were seen histological ly in two cases. In an add itional case, similar eggs and a single adult spirorchiid were observed in the sma ll intestine by histologi cal examination. Other than this example, no discrete enteric Neospirorchis egg masses, as observed in C. caretta were seen in C. mydas

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82 Other findings related to Neospirorchis sp. In addition to finding ad ult spirorchiids and egg masses in the above anatomical locations, widespread egg embolization in relatively high densities were observed in seven cases, all but on e of which were immature turtles in the acute insult category. Eggs were most readily observed in the lungs and gastro intestinal mucosa of four cases, primarily the lungs in two cases, and primarily the gastrointest inal tract in one case (Figure 2-29. Adult parasites a nd/or egg masses in these cases was limited to the thyroid gland, which was parasitized in four turtles. Spirorchiid impact rating Most spirorchiid infections in C. mydas were in the incidental category (35/50), followed by undetermined (10/50), uninfected (4/50), and cont ributory (1/50) (Table 2-16). Spirorchiid infection was not identified as the cause of death in any C. mydas The size class and duration of illness bias in the study population prevented co mparison of rating within these categories. Three findings of concern were observed in C. mydas with infections that were categorized as undeterminable impact: ca rdiovascular disease associated with L. learedi and H. postorchis in adult turtles (n=3), severe (grade 3) parasitism of the thyroid gland in immature turtles ( Neospirorchis sp.) (n=3), and intense egg em bolization in the lungs and/or gastrointestinal tract of immature turtles ( Neospirorchis sp.) (n=4). Three of the four latter cases also had grade 2 thyroid parasiti sm. The three adult turtles were all males that died of acute traumatic injuries. Six of the immature turtles died from hypothermic stunning. The tenth case was an emaciated immature turtle with severe fibropapillomatosis and presumptive septicemia. The single case in which spirorchiidiasis wa s interpreted as contri butory to death had massive numbers of Neospirorchis sp. within the pulmonary vasculature and severe fibropapillomatosis. The lungs in this case were discolored due to the large numbers of spirorchiid eggs.

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83 Body condition indices in hypothermic-stunned immature C. mydas Body condition indices were calculated for the thirty C. mydas that died of hypothermic stunning (Table 2-17). One case was discarded as an outlier because abnormal conformation of the carapace (old trauma) affected BCI calculations. No significant differ ences were observed in body condition indices, using eith er calculation, when turtles were compared based on the presence/absence of Neospirorchis in the thyroid gland or pres ence of relatively large numbers of Neospirorchis eggs concentrated in one or more or gan systems. The latter group primarily included turtles with large numbers of intra-t hyroidal eggs and moderate to large numbers embolized to the lungs and alimentary submucosa. Discussion The study populations of C. caretta and C. mydas reflect the predominant size classes of each species represented in the historical strandin g data for Florida (Foley et al., 2007). There are, however, factors that must be considered in any extrapolation of findings of this study to the general stranding population. The goal of necropsy efforts was not to mimic actual demographic proportions of the total stranding population, but to include represen tation of major size classes to gain a more complete understanding of the hos t-parasite relationship. With the exception of a few studies, correlation of spiror chiid data with size classes fre quently has been overlooked yet is critical to understanding hostparasite relationships in specie s with a complex life history and broad geographical range. In a ddition, turtles with acute insults were sought whenever possible to avoid the bias and confounding h ealth issues that limit interpre tation of findings in chronically ill animals, which comprise a large proportion of stranded sea turtles. Thus, the design and execution of this study tempered examination of a representative subset of animals with pursuit of fundamental questions regarding spir orchiidiasis in Florida sea turtles.

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84 The study population include d 148 stranded turtles, 89 C. caretta and 59 C. mydas Included in the category 1 and 2 data were 57 turtles identified that died of relatively acute insults, the most common examples of which included hypothermic stunning (C. mydas n=30), traumatic injury (both speci es, n=13), brevetoxicosis (C. caretta n=7), and an unsolved MME ( C. caretta n=4). Twenty-two turtles of both species died from illnesses classified as intermediate duration, which included animals th at stranded in thin nut ritional condition during the unsolved MME ( C. caretta n=6), chronic traumatic inju ries (both species, n=5), miscellaneous infectious diseases (both species, n=8), fibropapillomatosis ( C. mydas n=2), and enteric impaction ( C. caretta n=1). The chronic insult category included twenty-five turtles, the deaths of more than half of which were classified as multifactorial (n=14) and reflected a combination of advanced poor nutritional conditi on and opportunistic disease processes. Three major features of the study population and categorization of da ta require discussion with regard to rationale and implications on data interpretation. First, most C. mydas died during single hypothermic stunning event, which provided 30/59 C. mydas examined and 30/37 of those classified with acute insults. Although inclusion of these turtles biased the total study population, the event provided an opportunity to examine a relatively large number of seemingly robust C. mydas with a known cause of death and was in sightful in interpreting the significance of spirorchiidiasis. However, as outlined in the following discussion, this bias must be considered when comparing these data to other size classes, as well as turtles from other regions. Second, the use of an intermediate duration of insult category arguably was a very subjective classification because it included turtles ranging fr om mildly underweight to visibly thin, but not in sufficiently poor condition to be classified as emaciated, chronically ill turtles. The necropsy and parasitological data, however support this categorization. Most turtle s included in the

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85 intermediate duration category ha d singular identifiable causes of death, as did the acute insult category, in contrast to those tur tles with advanced chronic disease. Furthermore, in the various aspects of spirorchiidiasis examined, data for turtles in the intermediate duration category often fell between statistically significant differen ces observed between the acute and chronic categories, as expected in a continuum of dis ease relationships. Lastly, the bias produced by proportionately more adult C. caretta in the acute insult category may have confounded some of the statistical analyses, specifically the differe nces noted among size classes of turtles with thymic and enteric spirorchiidias is. In both instances, proportionate ly more turtles in the smaller size categories were infected as compared to adult turtles; however, there also were more chronically ill turtles infected as compared to animals that died of acute problems. Thus, the differences noted in the size class of infected turtles actually may have reflected the greater proportion of smaller turtles in the chronically ill group. Assessment of the significance of spirorchiidi asis in the study populat ion was one of the primary objectives of this study. The spirorchiid impact rating was devised as conservative means for estimating significance and distinguishi ng pathological lesions of concern (but of unknown significance) from those in which a cau sal role in death and stranding could be reasonably inferred based on complete necropsy. In C. caretta spirorchiidiasis was interpreted as contributory to death or causal in 13.0% (7/5 4) of examined animals, and was a finding of concern, but of unknown significance in an additional 33.3% (18/54) Of the latter group, it is notable that over half (10/18) of those animal s were in the acute or intermediate illness categories. Thus, relatively robus t turtles with unrelate d causes of death had pathological lesions of concern based on severity. In C. mydas spirorchiidiasis was found to be contributory to death in only one case (2.7%, 1/37) and no examples of fatal spirorchiidiasis were observed. The

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86 proportion of C. mydas in the undermined category, however, was comparable to C. caretta and most (9/10) C. mydas in this category died of acute in sults. These finding s warrant comparison with the only published large study of spirorchiid-associated patholog ical lesions in sea turtles, which examined C. mydas stranded in Moreton Bay, Queensland, Australia (Gordon et al., 1998). Several notable similariti es and differences between this study and the current study will be cited throughout this discussion. Gordon et al. reported spirorch iidiasis as the cause of death or a contributory cause in 10.4% (10/96) and 30.2 % (29/96) of stranded tu rtles, respectively, which, at first glance, appears much greater than was observed in Florid a turtles, especially C. mydas The disparity in these findings may be due to demographical and geographical differences in the study populations, the criteria for interp reting significance, and health status at the time of stranding. Unfortunately, the size classes of examined animals were not given in the Australian study, which omits a critical component of the host-parasite-d isease relationship. In addition, the criteria for determini ng spirorchiidiasis as the cause of death were not stated, which also severely limits comparisons among studies. The authors certainly describe significant pathological lesions in select cases, such as occlusive aortic thrombi, septic thrombosis, and intracranial hemorrhage; however, the precedence for vaguely concluding cause of death relationships in the spirorchiid disease literature prevents confident comparison of interpretative opinion. The other significant diffe rence between the Moreton Bay C. mydas study and the present study is that many of the Australian turtles appear to have been chronically ill, whereas a significant number (71/106) of Fl orida turtles died of acute insults. If examination had been limited to chronically ill C. caretta ( C. mydas were underrepresented), the proportion of causal (5.0%, 1/20) and contributory (25.0 %, 5/20) relationships actually are relatively comparable to the Australian C. mydas study. In recognition of the caveats considered when examining

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87 chronically ill animals, Gordon et al. (1998) stated that further ex amination of tur tles with acute illness is necessary to better define incidental or harmless, i.e. background, infections and lesions. Another previous study that bears comparis on is a relatively recent investigation of epizootiology of spirorchiidiasis in Hawaiian C. mydas that examined splenic egg counts (Work et al., 2005). This study found th at splenic egg density was inversely related to both SCL and BCI. Regional relationships also were identified. These trends contrast with some of the findings of the present study in that some of the parameters measured, e.g. neurospirorchidiasis, were greater in larger turtles. There are severa l factors that must be considered in comparing these studies. First, as noted by investigator s in the Hawaiian study, splenic egg density likely reflects chronic accumulation and may not correlate with intensity of adul t parasite infection, whereas the Florida study documented active inf ections with lesions associated with adult spirorchiids. Second, there appear to be differe nces between the spirorchiid fauna of Hawaiian C. mydas and Florida turtles, the most significant of which is the apparent absence of Neospirorchis species in Hawaii. Neospirorchis was the most commonly detected genus in both C. caretta and C. mydas in Florida and lesions associated with members of this genus were a major finding in the present study. Splenic egg de nsity may not accurately reflect the selective accumulation of Neospirorchis eggs in various anatomic sites. Minimally, this technique would be difficult to validate for use in Florida turtles given the spectra of lesions and sites parasitized by Neospirorchis in both C. caretta and C. mydas Several pathological lesions of concern associated with spirorchiids were documented in Florida turtles. These observ ations provided further informa tion on recognized problems, such as neurospirorchiidiasis, and id entified new or poorly documented health issues, such as major

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88 arterial disease and spirorchiid-associated thyroid itis. It is not prudent to disregard these findings as insignificant or merely incidental, despite the fact that many examples were documented in turtles that died of unrelated acute problems. Rather the lesi on severity and potential for physiological effects or sequelae suggest a nega tive impact on host fitness. Furthermore, infection and severe associated lesions in relati vely healthy turtles suppo rt that spirorchiids can act as primary pathogens, i.e. they have the ab ility to produce injury in otherwise healthy individuals, as opposed to a more limited role as secondary, opportunistic agents in debilitated animals. Conversely, these otherwise robust and, in many cases, actively foraging infected turtles with pathological lesions provide an important context in which to interpret similar findings in chronically ill anim als with other confounding health issues. Thus, based on the findings of this study, spirorchiid s are regarded as potential primary pathogens and a health concern in free-ranging turtles, but interpretation of significance in individual cases requires caution, especially with regard to co ncluding proximate cause of death. The high prevalence of major arterial disease primarily associated with H. mistroides in C. caretta was notable and underscores th e importance of complete examination of the aortic system and larger arteries when performing n ecropsies. There were no significant differences among duration of insult categories, a nd severe lesions were observed in turtles that died of acute traumatic injuries and brevetoxicosis. Significantly more adult turtles were infected by Hapalotrema sp. and more had associated arteritis, whic h suggests that age-related factors, such as exposure over time or differences in exposure intermediate host(s) are present. Furthermore, the occurrence and popul ation significance of Hapalotrema -associated arteritis may be underestimated as adult C. caretta comprise a small proportion of the stranding population.

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89 Although different spirorchiid species were involved, a similar picture emerged in C. mydas of this study. Three of four adult C. mydas examined had severe arterial disease associated with L. learedi and H. postorchis In contrast, infection by L. learedi was only observed in one of 46 examined immature C. mydas and very few parasites and no significant lesions were present in this single case. Thus, from this sampling, Learedius and Hapalotrema appear to be more of a health issue in adult C. mydas in Florida waters. This observation is consistent with a parasitological survey of ne sting females in Tortuguero, Costa Rica, in which L. learedi was found in 39 of 40 turtles (Santoro et al., 2006). There were some notable differences in the ar terial lesions observed in the present study and those described by Gordon et al. (1998). Both studies observed lesi ons in the heart and major arteries in significant num bers of stranded turtles. Alth ough different spirorchiid species were identified, the left aorta was the most common site of both lesions an d localization of adult spirorchiids. Gordon et al. desc ribed arterial thrombus forma tion, mural and occlusive, in 81 turtles, as well as an interesting pattern of externalization of thro mbi. In contrast, in category 1 necropsies, mural thrombus formation was only obs erved in nearly half of affected turtles (10/24) and only one of these had a partiall y occluding luminal thrombus. Part of the discrepancy may lie in use of the term throm bus. Gordon et al. include descriptions of spirorchiid eggs and pigmented debris, some of which appeared to be involved in the exteriorization process, in their definition of thrombi. In the current study, the use of the term thrombus was limited to lesions characterized by th e formation of fibrin clots on the endothelial surface or within the vessel lumen. The exterior ization process, as described by Gordon et al., was not observed, although the inflammation wa s transmural in 15 or 24 cases examined histologically and there were granulomas cont aining spirorchiid eggs or pigmented debris

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90 observed within these lesions. In a few cases, the impression of an exteriorization process could be claimed. In addition to semantics, some of the variation in desribed lesions could reflect differences in lesions associated with different species of spir orchiids. Specific identification only was performed for a small subset of turtles in the Australian study, and identified species in the heart and majo r vessels included H. pambanensis and H. postorchis There was only a single example of arteritis associated with H. postorchis infection in the current study population and severe lesions were more often associated with H. mistroides infection in C. caretta Although the large pigmented th rombi as described by Gordon et al. (1998) were not observed, an interesting pattern of degeneration and necrosis of the tunica media was seen in 7 of 24 category 1 cases. It was remarkable that sequelae, such as thrombosis, aneurysm formation, or rupture were not observed more frequently given the severity of some of these lesions. This pattern of medial necrosis has not been previously described in spirorchiid-associated lesions. Multiple spirorchiid species, including N. pricei H. pambanensis H. mistroides, and L. learedi were recovered from the hearts of Florida turtles. Patchy areas of mural endocarditis were observed in many of these cases, but distinctiv e lesions also were noted in turtles infected with N. pricei and L. learedi One turtle infected with N. pricei had intense areas of egg accumulation within the atria with associated at rial thrombi and thrombolic disease resulting from septicemia. Intense egg accumulation in the atria was limited to this one of six turtles infected with N. pricei Only one C. mydas was found to have an adul t spirorchiid in the heart that was consistent in morphology with a Neospirorchis species. Presumptive identification was based on histological examination because apparent very few (possibly one) adult parasites were present and did not flush from the ventricle during parasite collec tion procedures. Two Neospirorchis species have been previously repor ted from hearts of sea turtles, N. pricei from C.

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91 caretta and N. schistosomatoides from C. mydas Another example of a distinct cardiac lesion was observed in two C. mydas infected with L. learedi Both turtles had well-demarcated areas of papillary endocarditis a ssociated with attached L. learedi located supravalvular to the atrioventricular valves. Similar lesions have been observed in captive turtles at the Cayman Turtle Farm, Limited associated with L. learedi infection, as well as in nesting females from Tortugeuro National Park, Costa Rica (unpublished data). Also, Go rdon et al. (1998) describe a highly similar lesion at th is location in Australian C. mydas but did not report L. learedi as one of the identified spirorchiid species. Perhaps infection by L. learedi was overlooked in the 1998 study (only a small subset of parasites were iden tified) or the supravalvular location is a common attachment site for both Learedius and Hapalotrema species. Neurospirorchiidiasis (Neospirorchis sp.) has been a concern in C. caretta in Florida since being implicated in a mass mortality event in south Florida in 2000 and 2001 (Jacobson et al., 2006). The findings of this study have identified several features of neurological spirorchiid infection in C. caretta that are critical to future disease st udies and better understanding of the host-parasite relationship. First, neurospirorchi idiasis was observed in nearly half of the study population (44/89), of which 64.4% ( 29/45) were classified as inci dental infections. Over 11.1% (5/45) of cases, however, were intense infections classified as grade 3. The major question regarding these parasites is their clinical or h ealth significance. There are anecdotal reports of improvement of vague neurological symptoms following antihelminthic therapy; however, correlative clinical studies with confirmation of infection are l acking, primarily due to lack of methods for specific antemortem diagnosis. Un fortunately, the findings of this study do not answer these concerns. Of the five turtles with se vere (grade 3) neurospiro rchiidiasis, all either were found dead or died soon after discovery an d did not receive a neurological examination.

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92 Three of these animals were severely emaciated with multiple disease processes, including spirorchiid-associated lesions in other organs Of the remaining two cases, one died of brevetoxicosis, and thus would have exhibi ted confounding neurological symptoms, and the other died of a gastrointestinal perforation and secondary coelomitis. Therefore, of these five cases, it can only be stated for a single turtle that abundant meningeal spirorchiids were observed, but were unrelated to cause of d eath based on good nutritional condition and known cause of death (gastrointes tinal perforation). Of th e 44 brains examined with neurospirorchiidiasis, no associated hemorrhagic or ischemic events were identified. Another notable finding was the significant relations hip between neurospirorchiidiasis and adult C. caretta As with Hapalotrema infections, this observation may reflect increasing probability of exposure over time, selective exposu re, or other age-related factors. An agedependent effect is relevant to prev ious investigation of the 2000 and 2001 C. caretta mortality event in south Florida. At the time of the summ arization and analysis of this event, estimated intensity of neurospirorchiidiasi s was compared with existing unrel ated cases in lieu of actual prevalence data. The control cases primarily we re stranded turtles from northern Florida with a median SCL of 61.5 cm. In retrospect, this comparison was inappropriate given the higher prevalence of neurospirorchiidiasis in la rge turtles. Thus, any association of neurospirorchiidiasis and the 2000/2001 event can not be concluded based on this comparison. Neurological Neospirorchis infections were not limited to C. caretta but also were observed in C. mydas Similar to C. caretta there appears to be a si ze relationship in stranded Florida C. mydas as well, as all of the infected turtles with adult parasites were adult or large subadult turtles. Neurospirorch iidiasis was not found in any of th e smaller turtles, with only two possible exceptions in which eggs, but no adul t parasites were observed. No infected C. mydas

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93 had high intensity neurological infections. Neurospirorchiidiasis has been previously documented or depicted in photographs of C. mydas in at least three previous studies, although the genus was correctly identified in only one report (Glazebrook et al., 1989; Gordon et al., 1998; Raidal et al., 1998). Gordon et al. desc ribed microscopic fluke s, consistent with Neospirorchis sp., in the meninges of 73.6% (53/72) of examined C. mydas (1998). The species N. schistosomatoides was identified in two of these cases. Another pathological finding of concern in bo th species was parasitism of the thyroid gland, which resulted in extensive glandular injury in severe cases The thyroid gland was one of the most common sites inhabited by spirorchiids in both C. caretta and C. mydas Comparable proportions of the different age/ size classes were infected. There were significantly more infections and more intense in fections in chronically ill C. caretta The same comparisons were not possible for C. mydas; however, two cases of grade 3 lesi ons were observed in relatively robust C. mydas that died of hypothermic stunning. Futhermore, there was no evidence of any significant effect on BCI in the immature C. mydas that died of hypothermic stunning that were observed to have intra-thyroidal Neospirorchis The significance of thyr oid injury associated with Neospirorchis infection requires further study. There were several interesting differences in parasitism of endocrine organs by Neospirorchis sp. in the C. caretta and C. mydas Infection of the thyroid gland was identified in both species; however, concurrent parasitism a nd egg embolism within the thymus, which was frequently observed in C. caretta was not seen in C. mydas nor has involvement of this site been described in the litera ture. Another notable difference was occurrence of adult Neospirorchis in the vasculature of the adrenal gla nds, which was observed in 32.0% (16/50) of category 1 and 2 C. caretta Although microscopic flukes, presumably Neospirorchis sp.,

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94 occasionally were observed in adrenal glands of C. mydas in a previous study, none were seen in the 50 examined Florida C. mydas (Gordon et al., 1998). These differences could reflect differences in spirorchiid species involved or variation in host-parasite interaction. The organ system most commonly involved in spirorchiidiasis in terms of egg embolization and occurrence of adult spirorch iids was the gastrointestinal tract. Again, differences were observed between C. caretta and C. mydas Adult Neospirorchis sp. within the enteric submucosa and localized egg deposition characterized by large black, serpiginous egg masses were observed in over 65.8% (50/76) of stranded C. caretta Proportionately more chronically ill turtles were infect ed than turtles in the acute insu lt category, and all severe (grade 3) infections were in ch ronically ill animals. In contrast to spirorchiid infectio ns in other organ systems, proportionately more infected turtles with enteric involvement were large immature turtles (>65-85 cm SCL group), as compared to mature turtles, and most of the grade 2 and higher infections were animals in this size class. This possible relationship with size class must be cautiously interpreted given the aforementi oned bias in the study population. Wolke et al described bacterial enteritis asso ciated with spirorchiid eggs, as was observed in the present study, in stranded Atlantic C. caretta, but did not identify the spirorchiid type. In addition, similar serpiginous Neospirorchis egg masses in the enterocolic submucosa were not observed in Florida C. mydas in the present study, although very sim ilar egg masses have been described in C. mydas in Australia (Gordon et al., 1998). Smaller submucosal Neospirorchis egg masses, visibly distinct from the larger serpiginous masses or diffuse embolization, were observed in the stomach of over 10.4% (5/48) of examined C. mydas and were associated with adult parasites in histological sections in some cases. A

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95 sixth C. mydas had similar egg masses in the intestine. These eggs were observed to have fine surface projections as was seen in eggs obser ved in the colon, cloaca, and stomach of C. caretta The other gastrointestinal lesions obs erved were those associated with Hapalotrema species in C. caretta which ranged from incidental diffusely embolized eggs and embolized egg masses within subserosal and submucosal vessel s to more substantial, transmural egg masses with associated ischemic lesions and ulceration of the stomach and intestine. One of these cases was the only confirmed example of fatal spirorchiidia sis. There may be differences in the enteric lesions associated with different Hapalotrema species. The egg masses in the fatal case, which was infected with a novel species of Hapalotrema were much more extensive than was observed in turtles infected with H. mistroides and H. pambanensis A second case with similar pathological lesions was observed in a C. caretta infected the novel Hapalotrema species, but was excluded from the study due to a partial data se t. Additional lesions in the digestive tract associated with Hapalotrema eggs included enteric polyp form ation, similar to schistosomal polyposis of humans (Mesquita et al., 2003), and papillary lesions in th e gall bladder secondary to granulomatous cholecystitis. The testes were another organ in which signi ficant pathological lesi ons were associated with parasitism by Neospirorchis species. Although only observed in three turtle s, these cases comprised 25.0% (3/12) of examined adult male C. caretta There may have been persistent, negative effects on reproduction had these turtles recovered or been rehabilitated. This finding was not observed in C. mydas nor was parasitism of the ovary or other aspects of the female reproductive system identified in either species. In addition to pathological lesions associated with adult para sites and/or concentrated egg deposition in specific sites, diffuse embolizat ion of eggs, especia lly to the lungs and

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96 gastrointestinal tract, was severe in a number of cases. With the exception of the aforementioned specific examples, most of the eggs were Neospirorchis species in both C. caretta and C. mydas Multi-organ accumulation of eggs was a primary finding in 4/7 cases in which spirorchiidiasis was interpreted as contributory to the cause of death and was a lesion of concern in several turtles in the undeterminable significance cate gory. Interpretation of significance must be approached very cautiously, as noted in the group of C. mydas that died from hypothermic stunning. Six of these turtles were observed to have moderate or large numbers of Neospirorchis eggs embolized to the lungs and/or gastrointestinal tract, most of which also had adult Neospirorchis and eggs in the thyroid gland. The significance of egg accumulation in these cases is unknown, which was reflected in the unde terminable impact classification. There was no observable effect on nutritiona l condition as assessed by body we ight, abundance of internal fat, and body condition indices. Effects may ma nifest, however, when other confounding health problems, such as concurrent disease proce sses or environmental stressors, are present. Spirorchiid infection (all species include d) was observed in 96.4% (54/56) of C. caretta and 92.0% (46/50) of C. mydas examined in category 1 and 2 necropsies. These numbers were comparable to previous studies that also used targeted me thodology to detect spirorchiid infection (Work et al., 2005; Santuro et al., 2006; Gordon et al ., 1996; Dailey et al., 1992). Infection by multiple genera was frequently observed in C. caretta and all adult C. mydas but rarely in immature C. mydas The diversity of spirorchiid species identified in this study increase the number of species do cumented in the West Atlantic region. Species reported in the reviewed literature prior to 2004, included C. bipora H. synorchis N. pricei, and N schistosomatoides. The current study adds to this list H. mistroides and a novel Hapalotrema in C. caretta and H. postorchis and L. learedi in C. mydas The only spirorchiid species that was

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97 previously documented in Florida waters th at was not observed in the study population is H. synorchis In addition to these species, there is po tential for great unrecognized diversity in the genus Neospirorchis that could not be investigated by morphological examination due to the inability to collect intact specimens. There were obvious site predilections for adult Neospirorchis, including the leptomeninges, heart and major vessels, endocrine organs, thymus, and submucosa of the alimentary tract. Although intact adults could not be examined, there were distinct differences in size and general morphological features among a dult parasites and eggs from different anatomic locati ons that require further study. Notably, identifiable spirorchiid species were limited to either C. caretta or C. mydas These records contribute to existing data on host range. Several marine spirorchiids are known to parasitize multiple sea turtles species and it ha s been generally stated that there is no evidence of any host specificity at the ge neric or species levels (Smith, 1972; Smith, 1997a). At least one study, however, has offered evidence of potential host restriction in freshwater spirorchiids (Byrd, 1939, cited in Goodman, 1987). It must be c onsidered that any apparent host restriction in published reports could reflect bias as most studies have only examined C. mydas Only few studies have examined both C. caretta and C. mydas inhabiting the same general region and all examined either an unspecified number of or ve ry few turtles (Simha and Chattopadhyaya, 1980; Platt and Blair, 1998; Loss, 1902). Thus far, L. learedi has only been described in C. mydas and E. imbricata and reports of H. postorchis are limited to C. mydas (Smith, 1972; Smith, 1997b). Neither of these species was observed in Florida C. caretta included in the cu rrent study, thus infection may be rare or absent in this species, at least in this region. Another Hapalotrema species, H. pambanensis has only been reported in C. mydas and E. imbricata (Smith, 1997b). Based on the current study, C. caretta is now added to definitive hosts for this species. None of

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98 the specimens of H. mistroides or the novel Hapalotrema were collected from C. mydas ; however, examination of additional larger turtle s is necessary given the apparent rarity of Hapalotrema species in smaller immature C. mydas in Florida waters. Lastly, the single species of Carettacola C. bipora was limited to C. caretta which was the identified host in the only report and original descripti on of this species (Manter and Larson, 1950). Evidence of specialization or host restriction of marine spirorchiids requires further investigation. Results from the present study of Florida turtles support that there are at leas t large differences in regional prevalence among C. caretta and C. mydas Differences in parasite fauna would not be surprising given the differences in diet and habi tat utilization between the two turtle species. The occurrence of adult parasites and egg de position/accumulation in specific sites may reflect some degree of microhabita t selectivity. There are report s of both host-organ specificity, as well as lack of specific tropisms in the fres hwater spirorchiid literature (Wall, 1951; Holliman et al., 1971; Platt, 1993). It is reasonable to hypothesize that specialization woul d evolve, in at least some circumstances, as it has in mamma lian and avian schistosom es (Marquardt et al., 2000). Although this issue will requi re additional techniques, e.g. molecular characterization, to further study Neospirorchis species, there is evidence of site tropism in the more easily studied linguiform genera. All reports of Carettacola species in which anatomic location is specified have collected adult parasites from hepatic bloo d vessels (Dailey et al., 1991; Dailey et al., 1992; Graczyk et al., 1995). Similarly, all adult C. bipora in the present study were collected from either hepatic vessels or mesent eric vessels, the latter of which may have reflected presence of parasites in extrahepatic portal circulation. Similarly, the three Hapalotrema species identified were observed primarily in the heart and major ar teries in this study a nd in previous reports (Smith, 1997b). Both H. mistroides and L. learedi also have been reported in hepatic circulation

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99 (Smith, 1997b; Santoro et al., 2006), as obs erved in the present study. Although adult spirorchiids have been collected from these various vessels, there appears to be a predilection for the left aorta in some Hapalotrema species, as observed by Gordon et al. (1998) and for H. mistroides and H. postorchis in Florida turtles. The left aort a is the major conduit to the superior mesenteric artery, and thus provides access to sma ller enteric vessels where eggs may migrate to the mucosa and reach the external environment. Microhabitat utilization is releva nt to both health effects in th e turtle host and life cycle of the parasite. Sites where eggs accumulate or where adult parasites, e.g. Hapalotrema species, cause inflammation and tissue injury may affect or gan function or result in pathologic sequelae, such as enteric ulceration or arterial thrombosis. In addition, in sites where external access is seemingly remote, such as the brain or thyroi d gland, the strategy for propagation of the life cycle is unclear. Similar observations have been made in freshwater turtles. Holliman (1971) suggested that the death of hype rinfected turtles may contribute to the parasite life cycle by releasing eggs entrapped within internal tissues. In dead stranded sea tu rtles examined in the present study, live miracidia were observed in spirorchiid eggs following postmortem intervals of 48 to 72 hours, which is ample time for disper sal of a carcass by marine scavengers. Another scenario that has been anecdotally discussed is the possibility that a given sea turtle species is an aberrant host in some instances, such as in cases of neurospirorchiidiasis in C. caretta However, the high prevalence and apparent in cidental nature of most infec tions, as observed in this study, would be an unusual presentation of an aberrant host. Alternatively, the numbers of eggs that reach the lungs and/or gastrointestinal tract over time may be sufficient for shedding.

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100 Conclusions Spirorchiid trematodes and their associated cardiovascular lesions are among the oldest disease-related observations in the sea turtle literature (Looss, 1902). Over the last hundred years, significant progress has been made in the ch aracterization of these pa rasites. There are, however, fundamental gaps in our understanding of their impact on sea turtle health, as our ability to study diseases is limited by difficultly in obtaining fresh carcasses, the investment required for thorough postmortem examination, and the advanced ch ronic state of disease that typifies many stranded turtles. This study is the product of a colla borative effort to investigate the basic aspects of the host-parasite relationship and the health implications of spirorchiidiasis in Florida turtles. Prevalence of spirorchiid in fection was high in examined C. caretta and C. mydas and was comparable to that observed by other inve stigators using similar methodology. The number of turtles of both species, especi ally adults, with spirorchiid-asso ciated cardiovascular lesions of concern was notable. Several examples were obs erved in robust turtle s with acute causes of death, which support that these para sites have the ability to infect and produce severe lesions in seemingly healthy turtles. It was surprising that more exampl es of fatal sequelae were not observed, as described in previous studies, gi ven the extent of some lesions and apparent diminished integrity of affected arteries. Many turtles that develop fatal complications may die at sea and may not be well-repres ented in the stranding data. The findings of this study contribute to both the diversity of spirorchiids known to parasitize Florida sea turtles a nd the spectrum of organs infect ed and associated pathological lesions. Many questions regardi ng the health implications of spirorchiidiasis remain. The effects of neurological in fections and parasitism of endocrine organs, especially the thyroid

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101 gland, require further investiga tion. The prevalence data and si ze class relationships identified herein hopefully will be used to gui de and facilitate such studies. Any comparisons between the findings of this study and previous reports require caution, as does extrapolation of these data to different sea turtles species, tu rtles of different size classes, or populations inhabiting different regions. The differences observed between host turtle species and among different size cl asses expand upon a very small nu mber of studies that have considered such variables. The effort to unde rstand spirorchiids and their implications on sea turtle health would greatly benefit from bette r definition of methodology, inclusion of biological host data (such as size class), and specific id entification of parasites whenever possible to promote comparability among studies. At present, many generalizations are premature and much of the complexity of the host-parasite relationship re mains to be revealed.

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102 Figure 2-1. Examined C. caretta by geographical region and category of necropsy data. Light gray areas are counties in which n ecropsied turtles were found stranded. Category 1 Category 2 Category 3 Category 4 Total Zone 1 2 2 7 4 15 Zone 2 11 1 1 1 14 Zone 3 14 4 8 6 32 Zone 4 20 0 8 0 28 Total 47 7 24 11 89

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103 0 2 4 6 8 10 12 14 16 18<50>50-55>55-60>60-65>65-70>70-75>75-80>80-85>85-90>90-95>95100 >100Straight carapace length (cm) (notch-tip)No. of turtle s Frozen necropsy Fresh necropsy Figure 2-2. Histogram of size cl ass frequencies of necropsied Caretta caretta Fresh necropsies are category 1 turtles and frozen necr opsies include all other categories.

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104 Figure 2-3. Photomicrographs of three Hapalotrema species collected from C. caretta

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105 Figure 2-4. Arteritis associated with Hapalotrema infection in C. caretta The top image depicts extensive thickening of the left aorta (LA) celiac artery (CA), superior mesenteric artery (SMA) and a congested, irregular endothelial surface (chronic arteritis). Multifocal lesions also are apparent within the right aorta (RA). In the lower left image, chronic arteritis of the left aorta (L A) is contrasted with a normal right aorta (RA). A cluster of Hapalotrema mistroides (white arrowhead) are attached within an area of endarteritis in the lower right image.

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106 Figure 2-5. Photomicrographs of arteritis associated with Hapalotrema infection in C. caretta The upper left image shows marked, diffuse thickening of the artery wall due to proliferation of the intima and a severe inflammatory infiltrate. A spirorchiid ( Hapalotrema mistroides) is visible within the lumen (black arrowhead). At higher magnification (upper right image), an intens e heterophilic infiltrate surrounds the acetabulum (black arrowhead) of a H. mistroides More chronic lesions are shown in the lower images, which illustrate fibromus cular intimal proliferation and chronic granuloma formation with pigmented debris (left lower image), and a transmural infiltrate with intralesional spirorchiid egg granulomas (white arrowhead) and a cuff of mononuclear cells within the ad ventitia (lower right image).

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107 Figure 2-6. Necrosis of the tunica medi a in arteritis lesions associated with Hapalotrema infection in C. caretta In the left image, there is ex tensive, well-demarcated necrosis of the tunica media (black arrowhead) underlyi ng an area of endart eritis (H&E). In the right image, disruption of the reticulin fibers is visible at the margin of the necrotic segment (black arrowheads) (reticulin stain).

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108 Figure 2-7. Histopathological lesions of small arteries (gastrointestinal tract) in C. caretta with spirorchiidiasis. The upper left image shows a small thrombosed artery with a mononuclear and granulocytic inflammatory in filtrate. The artery in the upper right image is similarly inflamed and has a small cluster of spirorchiid eggs ( Hapalotrema sp.) within the lumen, as well as perivasc ular multinucleated giant cell formation. The lower left image demonstrates an area of microvascular proliferation in an animal that had other lesions, including arteritis and medial hypertrophy. The lower right image shows severe medial hype rtophy of two small arteries.

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109 Figure 2-8. Masses of spirorchiid eggs ( Hapalotrema sp.) within subserosal vessels of a C. caretta This lesion is the typical appearance of subserosal egg masses observed in turtles infected with Hapalotrema mistroides or H. pambanensis Figure 2-9. Extensive enteri c lesions associated with Hapalotrema infection in a C. caretta There are large transmural egg masses and th rombosis with ischemic necrosis of the enteric mucosa (right image) in this C. caretta infected with a novel species of Hapalotrema These lesions are more extensive that those shown in Figure 2-8.

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110 Figure 2-10. Colonic polyp in a C. caretta associated with granulomatous inflammation and intralesional Hapalotrema eggs. Figure 2-11. Granulomatous cholecystitis wi th papillary mucosal proliferation in a C. caretta associated with Hapalotrema eggs ( Hapalotrema nov. sp.)

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111 A B C Figure 2-12. Comparison of grades of neurospirorchiidiasis ( Neospirorchis sp.) in C. caretta A) Only two small egg masses, both less th an 3 mm in diameter. B) Approximately eight to ten distinct egg ma sses, all of which are less than 3 mm in diameter. C) Greater than 10 egg masses, including seve ral large, coalescing masses that distend meningeal vessels.

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112 Figure 2-13. Severe infec tion of the thyroid gland by Neospirorchis sp. in C. caretta Large black masses of spirorchiid egges efface th e gland and distend blood vessels within the surrounding cervical tissues. Figure 2-14. Occlusion of th ymic blood vessels by eggs of Neospirorchis sp. in C. caretta Large black spirorchiid egg masses are seen within thymic vessels and egg emboli are demonstrated within the higher magnification inset.

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113 Figure 2-15. Abundant submucosal egg masses ( Neospirorchis sp) in the small intestine of a C. caretta Numerous black, serpiginous egg mass es are visible within the submucosa. Figure 2-16. Enteric ulceration s econdary to spir orchiidiasis ( Neospirorchis sp.) in C. caretta In this image, an ulcer is forming along the path of a large egg mass (EM). The filiform adult spirorchiid can be seen migr ating through a submucosal vessel and the anterior portion has been freed from the tissue and is laying on the mucosal surface.

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114 Figure 2-17. Images illustrating variati on in adult and egg morphology among observed specimens of Neospirorchis from C. caretta The upper (A) and middle (C) images show Neospirorchis specimens adjacent to egg masses within the enteric submucosa. Example A is more petite and white as compar ed to the larger and yellow example C. The eggs in the adjacent images (B a nd F) correspond to the adult morphological types and demonstrate the smaller egg size for the a dult in A (both bars = 50 m). In addition, the Neospirorchis in the leptomeninges (E) have prominent pigmented cecae, which are not observed in the two gastrointestinal examples. Figure F demonstrates variation in Neospirorchis egg morphology in a squash preparation from the lung of an infected C. caretta (bar = 100 m). Two Hapalotrema eggs with bipolar processes also are present.

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115 Figure 2-18. Spirorchiid egg masses ( Neospirorchis sp.) within cloacal su bmucosal vessels of a C. caretta Each cluster (black arrowheads) measures approximately 1.0 mm in diameter. Figure 2-19. Cytology and scanning electron microscopy (SEM) of cloacal Neospirorchis egg masses from a C. caretta Cytological impressions (left image) of the egg masses from Figure 2-18 reveal fine surface proj ections on the shell surface (bar = 25 m). These projections are readily observed by scanning electron microscopy (lower right)

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116 Figure 2-20. Atrial thrombosis associated with Neospirorchis pricei infection in a C. caretta The left atrium has been opened and a throm bus (white arrowheads) is adhered to the endocardium. The higher magnificantion in set shows the dens e aggregates of Neospirorchis eggs underlying the thrombus. Figure 2-21. Severe verminous orchitis in a C. caretta infected with Neospirorchis sp.. Numerous Neospirorchis eggs and adults (not visible) are distributed throughout the testis. Shown are the testis in situ (top ) in which eggs are observed within adjacent vessels. Individual eggs are more easily observed in the closer image (bottom).

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117 Figure 2-22. Examined C. mydas by geographical region and category of necropsy data. Light gray areas are counties in which n ecropsied turtles were found stranded. Category 1 Category 2 Category 3 Category 4 Total Zone 1 1 31 0 1 33 Zone 2 1 0 1 4 6 Zone 3 6 1 0 1 8 Zone 4 10 0 0 2 12 Total 18 32 1 8 59

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118 0 2 4 6 8 10 12 14 16 18 2020-25 >25-30 >30-35 >35-40 >40-45 >45-50 >50-55 >55-60 >60-65 >65-70 >70-75 >75-80 >80-85 >85-90 >90-95 >95-100 >100Straight carapace length (cm) (notch-tip)No. of turtle s Frozen necropsy Fresh necropsy Figure 2-23. Histogram of size class frequencies of necropsied C. mydas Fresh necropsies are category 1 turtles and frozen necropsies include all other categories.

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119 Figure 2-24. Endocarditis in C. mydas associated with L. learedi infection. The atria have been opened in both photographs exposing the s upravalvular endocardium. Both cases have well-demarcated, raised areas of e ndocarditis (white arrows). An adult spirorchiid ( Learedius learedi) (black arrowhead) can be seen attached to the lesion in closer image (inset).

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120 Figure 2-25. Severe arteritis in a C. mydas associated with L. learedi infection. There is severe thickening of the left brachiocephalic ar tery (BCA), left aorta (LA), and left pulmonary artery (PA). The lumina are seve rely reduced, especially in the left aorta and brachiocephalic artery (inset), due to in timal proliferation and chronic arteritis. Figure 2-26. Arteritis in a C. mydas associated with H. postorchis infection. The artery wall is severely thickened and th ree adult spirorchiids ( Hapalotrema postorchis ) are seen protruding from the vessel lumen.

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121 Figure 2-27. Thyroiditis in C. mydas associated with infection by Neospirorchis sp: Numerous spirorchiid eggs ( Neospirorchis sp.) and associated gra nulomatous inflammation are distributed throughout the thyroid gland. Figure 2-28. Eggs of Neospirorchis species within the ga stric submucosa of a C. mydas Two morphologies of Neospirorchis eggs are observed. The la rger, dark brown eggs are embolized deep within the submucosa. Th e smaller, golden eggs are dispersed into loosely organized clusters within the superficial submucosa.

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122 Figure 2-29. Numerous embolized Neospirorchis eggs in the lungs and gastric mucosa of a C. mydas Numerous Neospirorchis eggs are embolized th roughout the lungs (A), stomach (B), and other organs in these C. mydas that died during a hypothermic stunning event. Note the multifocal mu cosal erosion and inflammation (white arrowheads) in the lower image.

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123 Table 2-1. Criteria for categorization of the duration of illness. Category Criteria for classification Acute insult 1. Carcass in good nutritional condition based on musculature (no atrophy) and adipose stores (minimal or no atrophy present). 2. Any identifiable fatal pathological lesion is characterized by an acute inflammatory response or other cellular injury. Intermediate insult 1. Atrophy of skeletal muscle is present or ab sent, adipose tissue is atrophied, bone density is within normal limits, no catabolism of connective tissues. 2. Any identifiable fatal pathological lesion is characterized by a chronic inflammatory response or other cellular injury and fibrosis. Chronic insult 1. Carcass exhibits advanced atrophy of skeletal muscle and adipose tissue (emaciation). Osteopenia and catab olism of connective tissues present. 2. Any identifiable fatal pathological lesion is characterized by a chronic inflammatory response or other cellular injury and fibrosis. Table 2-2. Criteria for determining sp irorchiid trematode impact score. Score Definition and criteria 1 None detected. 2 Incidental infection. Numbers of adult spirorch iids, if detected, were low (4 or less) and egg deposition in tissues consisted either of rare individual embolized eggs and/or isolated egg masses. Associated tissue/organ injury or inflammation is mild and focal in distribution. 3 Contributory. Large numbers of adult spirorchiids and/or eggs are associated with significant organ injury. Included in this category are tur tles in which severe, multisystemic spirorchiid egg embolization is one of multiple significant pathological findings. Also included are turtles in which injury/inflammation associated with spir orchiids has resulted in or exacerbated other conditions, including thrombosis an d secondary bacteria infection. 4 Fatal. The severity and extent of organ/tissue in jury or inflammation associated with spirorchiid adults or eggs supports spirorch iidiasis is the cause of death. 5 Unknown. Large numbers of adult spirorchiids and/or eggs are present and/or there is significant associated organ injury, but significance cannot be confidently determined from necropsy data. Included in this group are turtles with a known cause of death in which association, if any, with spirorchiidiasis is unknown.

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124 Table 2-3. Criteria for gradi ng spirorchiid infection and as sociated pathological lesions. I. Arteritis of large ar teries associated with Hapalotrema infection Grade Criteria for grading 0 No gross lesions Histological correlate: Within normal limits 1 Focal gross lesion (< 1.5 cm diameter) Histological correlate: inimal or mild subint imal inflammatory infiltrate and/or fibrosis 2 Focally extensive (1.5 cm to < 4.0 cm in greatest dimension) OR multifocal: maximum of 3 lesions (any single lesion < 2.0 cm diameter or < 4.0 cm in greatest dimension) Histological correlate: Moderate changes including inflammation and intimal proliferation 3 Regionally extensive (single or multiple) (>4.0 cm in any dimension) OR Diffuse lesions involving one or more vessels Histological correlate: Changes as under 2 but severe and/or any one of the following: thrombus formation, aneurysm formation, necrosis of tunica media II. Neurospirorchiidiasis ( Neospirorchis species) Grade Criteria for grading 0 No egg masses or adults / individual embolized eggs only 1 Small numbers: typically 1-5 egg masses measuring less than 3 mm in greatest dimension and/or small numbers (1-3) adults 2 Moderate numbers: typically 5 -10 egg masses with most measuring less than 3 mm in greatest dimension and/or 1-2 larger coalescing egg masses distending meningeal vessels 3 Large numbers: >10 egg masses with regionally intense or diffuse distribution and/or >2 larger coalescing egg masses distending meningeal vessels III. Parasitism of the thyroid gland and thymus ( Neospirorchis species) Grade Criteria for grading 0 No eggs OR rare to small nu mbers of embolized eggs only 1 Small numbers of adults and/or egg masses 2 Moderate numbers (includes focally extensive aggregates and diffuse lesions) and/or adults 3 Large numbers (large areas of gland/thymus effaced by eggs and associated inflammation) IV. Enteric spirorchiidiasis ( Neospirorchis species) Grade Criteria for grading 0 No egg masses 1 1 10 distinct egg mass 2 >10 50 distinct egg masses 3 >50 distinct egg masses

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125 Table 2-4. Causes of death in C. caretta categorized by duration of illness (category 1 & 2 data). Primary Diagnosis/ Cause of Death Acute Intermediate Chronic Total Traumatic injury 6 2 8 Brevetoxicosis 7 7 Unusual mortality event of unknown etiology 4 6 10 Drowning / aspiration 2 2 Infectious disease 5 2 7 Enteric impaction 1 2 3 Emaciation 3 3 Multi-factorial 13 13 Undetermined 1 1 Total 20 14 20 54

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126 Tabe 2-5. Prevalence of Hapaltrema-associated large vessel arteritis in C. caretta by grade, necropsy data category, duration of illness, and size class. Necropsy data category Category 1 Category 2 Category 3 Total Grade 0 51.1% (24/47) 85.7% (6/7) 79.2% (19/24) 62.8% (49/78) Grade 1 8.5% (4/47) 0% (0/7) 4.2% (1/24) 6.4% (5/78) Grade 2 12.8% (6/47) 14.3% (1/7) 12.5% (3/24) 12.8% (10/78) Grade 3 27.7% (13/47) 0% (0/7) 4.2% (1/24) 17.9% (14/78) Duration of illness Acute Intermediate Chronic Grade 0 64.0% (16/25) 66.7% (12/18) 60.0% (21/35) Grade 1 12.0% (3/25) 5.6% (1/18) 2.9% (1/35) Grade 2 16.0% (4/25) 5.6% (1/18) 14.3% (5/35) Grade 3 8.0% (2/25) 22.2% (4/18) 22.9% (8/35) Size class (Straight carapace length) < 65 cm >65-85 cm >85 cm Grade 0 66.7% (14/21) 77.1% (27/35) 14.3% (2/14) Grade 1 9.5% (2/21) 0% (0/35) 14.3% (2/14) Grade 2 9.5% (2/21) 11.4% (4/35) 21.4% (3/14) Grade 3 14.3% (3/21) 11.4% (4/35) 50.0% (7/14)

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127 Table 2-6. Anatomic locations from which adult specimens of Hapalotrema species were collected from C. caretta Species Heart Aortas Mesenteric a. Hepatic vessels H. mistroides 4 11 8 1 H. pambanensis 3 2 0 1 Hapalotrema. sp. (novel) 0 0 2 1 Each cell indicates the number of individual turtles from which a given species was recovered from a specific site, not the number of parasites. Table 2-7. Prevalence of Neospirorchis sp. by anatomic location (adults and/or localized egg deposition) and necropsy data category. Location Category 1 Category 2 Category 3 Category 4 Total Meninges 51.1% (24/47) 42.9% (3/7) 54.2% (13/24) 36.4% (4/11) 49.4% (44/89) Thyroid gland 57.4% (27/47) 0% (0/7) 85.7% (18/21) 60.0% (45/75) Thymus 46.8% (22/47) 28.6% (2/7) 59.1% (13/22) 48.7% (37/76) Gastrointestinal 66.0% (31/47) 71.4% (5/7) 63.6% (14/22) 65.8% (50/76) Heart/major arteries 12.8% (6/47) 0% (0/7) 11.1% (6/54) Adrenal glands 31.9% (15/47) 33.3% (1/3) 32.0% (16/50) Testes 25.0% (3/12)a 25.0% (3/12) aOnly males included in prevalence.

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128 Table 2-8. Prevalence of neurospirorchiidiasis (Neospirorchis sp.) in C. caretta by grade, necropsy data category, duration of illness, and size class. Necropsy data category Category 1 Category 2 Category 3 Category 4 Total Grade 0 45.7% (21/46) 57.1% (4/7) 45.8% (11/24) 63.6% (7/11) 48.9% (43/88) Grade 1 34.8% (16/46) 42.9% (3/7) 33.3% (8/24) 18.2% (2/11) 32.9% (29/88) Grade 2 13.0% (6/46) 0% (0/7) 20.8% (5/24) 0% (0) 12.5% (11/88) Grade 3 6.5% (3/46) 0% (0/0) 0% (0) 18.2% (2/11) 5.7% (5/88) Duration of illness Acute Intermediate Chronic Grade 0 50.9% 16/32) 55.6% (10/18) 44.7% (17/38) Grade 1 40.6% (13/32) 22.2% (4/18) 31.6% (12/38) Grade 2 3.1% (1/32) 22.2% (4/18) 15.8% (6/38) Grade 3 6.3% (2/32) 0% (0/18) 7.9% (3/38) Size class (Straight carapace length) < 65 cm >65-85 cm >85 cm Grade 0 80.0% (20/25) 41.0% (16/39) 11.8% (2/17) Grade 1 16.0% (4/25) 33.3% (13/39) 52.9% (9/17) Grade 2 4.0% (1/25) 17.9% (7/39) 23.5% (4/17) Grade 3 0% (0/25) 7.7% (3/39) 11.8% (2/17)

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129 Table 2-9. Prevalence of t hyroid gland parasitism (Neospirorchis sp.) in C. caretta by grade, necropsy data category, duration of illness, and size class. Necropsy data category Category 1 Category 2 Category 3 Total Grade 0 42.6% (20/47) 100% (7/7) 33.3% (7/21) 45.3% (34/75) Grade 1 23.4% (11/47) 0% (0/7) 19.0% (4/21) 20.0% (15/75) Grade 2 23.4% (11/47) 0% (0/7) 28.6% (6/21) 22.7% (17/75) Grade 3 10.6% (5/47) 0% (0/7) 19.0% (4/21) 12.0% (9/75) Duration of illness Acute Intermediate Chronic Grade 0 68.0% (17/25) 50.0% (9/16) 26.5% (9/34) Grade 1 16.0% (4/25) 31.3% (5/16) 17.6% (6/34) Grade 2 16.0% (4/25) 12.5% (2/16) 32.4% (11/34) Grade 3 0% (0/25) 6.2% (1/16) 23.5% (8/34) Size class (Straight carapace length) < 65 cm >65-85 cm >85 cm Grade 0 45.0% (9/20) 30.3% (10/33) 57.1% (8/14) Grade 1 15.0% (3/20) 24.2% (8/33) 21.4% (3/14) Grade 2 25% (5/20) 27.3% (9/33) 21.4% (3/14) Grade 3 15.0% (3/20) 18.2% (6/33) 0% (0/14)

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130 Table 2-10. Prevalen ce of parasitism (Neospirorchis sp.) of the thymus in C. caretta by grade, necropsy data category, duration of illness, and size class. Necropsy data category Category 1 Category 2 Category 3 Total Grade 0 53.2% (25/47) 71.4% (5/7) 40.1% (9/22) 51.3% (39/76) Grade 1 23.4% (11/47) 28.6% (2/7) 36.4% (8/22) 27.6% (21/76) Grade 2 8.5% (4/47) 0% (0/7) 9.1% (2/22) 7.9% (6/76) Grade 3 14.9% (7/47) 0% (0/7) 13.6% (3/22) 13.2% (10/76) Duration of illness Acute Intermediate Chronic Grade 0 84.0% (21/25) 56.3% (9/16) 25.7% (9/35) Grade 1 16.0% (4/25) 31.3% (5/16) 34.3% (12/35) Grade 2 0% (0/25) 6.3% (1/16) 14.3% (5/35) Grade 3 0% (0/25) 6.3% (1/16) 25.7% (9/35) Size class (Straight carapace length) < 65 cm >65-85 cm >85 cm Grade 0 40.0% (8/20) 44.1% (15/34) 78.6% (11/14) Grade 1 25.0% (5/20) 26.5% (9/34) 21.4% (3/14) Grade 2 15.0% (3/20) 11.8% (4/34) 0% (0/14) Grade 3 20.0% (4/20) 17.6% (6/34) 0% (0/14)

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131 Table 2-11. Prevalence of enteric Neospirorchis sp. in C. caretta by grade, necropsy data category, duration of illness, and size class. Necropsy data category Category 1 Category 2 Total Grade 0 38.6% (17/44) 33.3% (2/6) 38.0% (19/50) Grade 1 31.8% (14/44) 66.7% (4/6) 36.0% (18/50) Grade 2 20.5% (9/44) 0% (0/6) 18.0% (9/50) Grade 3 11.4% (5/44) 0% (0/6) 10.0% (5/50) Duration of illness Acute Intermediate Chronic Grade 0 66.7% (12/18) 35.7% (5/14) 11.1% (2/18) Grade 1 22.2% (4/18) 42.9% (6/14) 44.4% (8/18) Grade 2 11.1% (2/18) 21.4% (3/14) 22.2% (4/18) Grade 3 0% (0/18) 0% (0/14) 22.2% (4/18) Size class (Straight carapace length) < 65 cm >65-85 cm >85 cm Grade 0 46.2% (6/13) 20.0% (3/15) 71.4% (10/14) Grade 1 46.2% (6/13) 20.0% (3/15) 21.4% (3/14) Grade 2 7.7% (1/13) 40.0% (6/15) 7.1% (1/14) Grade 3 0% (0/13) 20.0% (3/15) 0% (0/14)

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132 Table 2-12. Spirorchiid impact ratings (SIR) in C. caretta categorized by duration of illness. SIR 1 SIR 2 SIR 3 SIR 4 SIR 5 Acute 1 15 0 0 4 Intermediate 1 6 1 0 6 Chronic 0 6 5 1 8 Total 2 27 6 1 18 Table 2-13. Causes of death in necropsied C. mydas by duration of insult. Primary Diagnosis/ Cause of Death Acute Intermediate Chronic Total Hypothermic stunning 30 30 Traumatic injury 7 3 1 11 Fibropapillomatosis 2 1 3 Infectious disease 3 1 4 Emaciation 1 1 Multifactorial 1 1 Total 37 8 5 50 Table 2-14. Prevalence of Neospirorchis sp. in C. mydas by anatomic location (adults and/or localized egg deposition) and necropsy data category. Location Category 1 Category 2 Category 3 Category 4 Total Meningeal 22.2% (4/18) 0% (0/32) 0% (0/1) 37.5% (3/8) 11.9% (7/59) Thyroid gland 22.2% (4/18) 40.6% (13/32) 0% (0/1) 33.3% (17/51) Gastric 22.2% (4/18) 3.3% (1/30)a 10.4% (5/48) aSubgross examination not performed in omitted animals.

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133 Table 2-15. Prevalence of t hyroid gland parasitism (Neospirorchis sp.) in C. mydas by grade, necropsy data category, duration of illness, and size class. Necropsy data category Category 1 Category 2 Category 3 Total Grade 0 77.8% (14/18) 59.4% (19/32) 100% (1/1) 66.7% (34/51) Grade 1 11.1% (2/18) 18.8% (6/32) 0% (0/1) 15.7% (8/51) Grade 2 5.6% (1/18) 15.6% (5/32) 0% (0/1) 11.8% (6/51) Grade 3 5.6% (1/18) 6.3% (2/32) 0% (0/1) 5.9% (3/51) Duration of illness Acute Intermediate Chronic Grade 0 57.9% (22/38) 100% (8/8) 80.0% (4/5) Grade 1 21.0% (8/38) 0% (0/8) 0% (0/5) Grade 2 15.8% (6/38) 0% (0/8) 0% 0/5) Grade 3 5.3% (2/38) 0% (0/8) 20.0% (1/5) Size class (Straight carapace length) < 65 >65-85 >85 Grade 0 63.8% (30/47) 100% (1/1) 75% (3/4) Grade 1 17.0% (8/47) 48.5% (0/1) 0% (0/0) Grade 2 10.6% (5/47) 27.3% (0/1) 25.0% (1/4) Grade 3 8.5% (4/47) 18.2% (0/1) 0% (0/0)

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134 Table 2-16. Spirorchiid impact ratings (SIR) in C. mydas categorized by duration of illness. SIR 1 SIR 2 SIR 3 SIR 4 SIR 5 Acute 4 24 0 0 9 Intermediate 0 7 1 0 0 Chronic 0 4 0 0 1 Total 4 35 1 0 10

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135 Table 2-17. Body condition indices (BCIs) and Neospirorchis-related findings in hypothermic-stunned immature C. mydas St. Joseph Bay, Florida. Necropsy No. BCI (g/cm3) BCI (g/cm) Spirorchiid-associated findings GST0801 1.33 0.116 None present GST0802 1.16 0.112 Moderate numbers embolized Neospirorchis eggs in lungs GST0803 1.27 0.145 Adult Neospirorchis in thyroid gland GST0804 1.25 0.106 None present GST0805 1.17 0.104 Moderate numbers Neospirorchis eggs in thyroid; moderate embolization to lungs and stomach GST0806 1.16 0.126 Small numbers diffusely embolized Neospirorchis eggs GST0807 1.17 0.105 Small numbers diffusely embolized Neospirorchis eggs GST0808 1.31 0.110 Small numbers diffusely embolized Neospirorchis eggs GST0809 1.26 0.092 None present GST0810 1.31 0.120 Small numbers diffusely embolized Neospirorchis eggs GST0811 1.12 0.093 Small numbers diffusely embolized Neospirorchis eggs GST0812 1.14 0.117 Small numbers diffusely embolized Neospirorchis eggs GST0813 1.29 0.061 Small numbers diffusely embolized Neospirorchis eggs GST0814 1.10 0.101 Adult Neospirorchis in thyroid gland GST0815 1.15 0.128 Neospirorchis egg masses in thyroid gland GST0816 1.17 0.107 Moderate numbers Neospirorchis eggs in thyroid; moderate embolization to embolized to lungs, stomach, intestine GST0817 1.22 0.102 Small numbers diffusely embolized Neospirorchis eggs GST0818 1.19 0.069 None present GST0820 1.30 0.142 Moderate to large numbers Neospirorchis eggs and adults in thyroid gland; moderate embolization to lungs, stomach, intestine GST0821 1.27 0.135 Adult Neospirorchis in thyroid gland GST0822 1.12 0.088 Small numbers diffusely embolized Neospirorchis eggs; Neospirorchis egg masses in gastric submucosa GST0823 1.18 0.121 Small numbers diffusely embolized Neospirorchis eggs GST0824 1.17 0.058 Moderate numbers Neospirorchis eggs and adults in thyroid; moderate embolization to lungs, stomach, intestine GST0825 1.13 0.112 Adult Neospirorchis in thyroid gland GST0826 1.11 0.098 Small numbers diffusely embolized Neospirorchis eggs GST0827 1.34 0.154 Neospirorchis egg masses in thyroid gland GST0828 1.13 0.076 Large numbers Neospirorchis eggs in thyroid; m oderate embolization to stomach/intestine GST0830 1.30 0.072 Small numbers diffusely embolized Neospirorchis eggs GST0831 1.13 0.094 Adult Neospirorchis in thyroid gland

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136 CHAPTER 3 GENETIC DIVERSITY OF MARINE SPI RORCHIIDS AND CORRELATION WITH MORPHOLOGY, MICROHABITAT USAGE, AND HOST SPECIES Introduction Spirorchiids trematodes have long been recogn ized as parasites and causes of disease in sea turtles. Studies of the spir orchiids have been conducted for nearly 150 years and the list of described species and their turtle hosts has become extensive (Smith, 1997b). Taxonomy of these parasites, however, has been complicated by divergent morphology, inadequate specimen database, incompletely described species, and lack of accessibility to some type specimens (Platt, 2002). Furthermore, identification is problematic or impossible in many instances due the difficulty in obtaining suitabl e specimens. The genus Neospirorchis has been exceptionally difficult to study in Florida sea turtles because of its small size, elongate (filiform) body type, and tendency for adult parasites to inhabit small vessels. Also, the relatively delicate tegument of spirorchiids as compared to the connective tiss ue elements of host tissues limits the utility of enzymatic digestion techniques in harvesting parasites. Collected specimens often are fragmented, incomplete, or otherwise unsuita ble for confident morphological examination. Inability to accurately identify spirorchiids not only limits our understanding of the taxonomy and biology of these parasites, bu t also their relationship, including associated disease, in their sea turtle hosts. Molecular approaches as an adjunct to morphological and other methods of parasitological studies can be used to addre ss problems created by incomplete or suboptimal specimens, phenotypic plasticity, questionable valid ity of some morphologi cal features, and the potential time-lag between speci ation and evolution of disti nguishing morphological features (Nolan and Cribb, 2005). Most molecular studies of Digenea have examined ribosomal and/or

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137 mitochondrial DNA, and of these targets the inte rnal transcribed spacer (ITS) regions of the ribosomal gene and the mitochondrial cytochrome oxidase I (MCOI) gene have been the most extensively examined (Nolan and Cribb, 2005; Vilas et al., 2005). The ITS and MCOI have several desirable features for this purpose in th at both are relatively easy to characterize, both exhibit a rapid evolutionary rate, and in many in stances, both provide a clear indication of the presence or absence of genetic diversity that may be used in assessment of biological species diversity. In the present study, a combination of molecu lar and classical parasitological approaches was used to prospect for and i nvestigate the diversity of spirorchiids in sea turtles. Study objectives included: assessment of genetic diversity in some rec ognized species of spirorchiids, including specimens from Atlan tic, Caribbean, and Pacific regions ; genetic characterization of the two described species of Neospirorchis and comparison with Neospirorchis specimens collected from stranded sea turtles in Florida; and correlation of genetic data from Neospirorchis with microhabitat utilization, de finitive host species, and avai lable morphological features. Actual species delimitation was not feasible due to significant knowledge gaps in parasite life cycle, morphology, data from other geographical regions, and host range. The findings of this study, however, support that the di versity of marine spirorchiids far exceeds what has been previously recognized, especially for parasite s tentatively classifi ed within the genus Neospirorchis. Furthermore, the correlation of gene tic findings with limited biological and morphological data has implications on both taxonomy and infection in the sea turtle host. Methods and Materials Parasite Specimens Most of the parasites examin ed were recovered from dead stranded sea turtles in Florida from November 2004 through August 2008 that were recovered by the Sea Turtle Stranding and

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138 Salvage Network (STSSN). Parasites were collected during complete n ecropsies using targeting collection techniques that included a combination of gross examination, examination of organs using a dissecting microscope, and examination of filtrates of blood, body fluids, washes of organs using a #45 sieve. For each parasite specimen, the host species, host identification number, and anatomic location were noted. Sample s of eggs were placed into two categories, diffuse embolization or localized deposition. Localized deposition was recognized as the occurrence of distinct egg masses, which often displaced or effaced host tissue and frequently were associated with adult parasites, whereas diffusely embolized e ggs were observed as individual eggs without any appa rent association with nearby a dult spirorchiids. Spirorchiids with a filiform body type and/or ovo id, operculated eggs that lack ed processes were tentatively identified as Neospirorchis species. Intact or nearly in tact parasites were collected as morphological voucher specimens and were fixed in alcohol-formalin-acetic acid (AFA) and stained by routine methods. Representative specim ens (including adults and eggs) were frozen at -80 Celsius. In addition, permission was granted to extract DNA from original voucher specimens of N. schistosomatoides submitted to the U.S. National Parasite Collection (Beltsville, Maryland) by TG Rand (accession 77971) (Rand and Wiles, 1985). Other parasite specimens included limited numbers of Learedius learedi and Hapalotrema postrochis collected from adult female Chelonia mydas in Tortugeuro National Park, Costa Rica by M. Santoro, L. learedi and H. postorchis collected by Hawaiian C. mydas by T. Work, and L. learedi obtained from captive C. mydas at Cayman Turtle Farm, Limited on Grand Cayman Island. PCR and Sequencing DNA was extracted from frozen or ethanol-f ixed samples using the DNeasy Kit (Qiagen, Valencia, CA). Primer sequences for PCR and sequencing of the ITS2 were designed using comparative alignments from mammalian and av ian schistosomes and two additional primers

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139 described by Dvo k et al. (2002) were used to amplify the ITS1 (Figure 3-1). The MCOI was amplified use the forward primer JB3 (5-TTTTTTGGGCATCCTGA GGTTTAT-3) and the reverse primer COI-R (5-CACCAAATCATGAT GCAAAAGG-3) (Miura et al, 2005). The reverse primer JB4.5 (5 TAAAGAAAGAACAT AATGAAAATG-3) also was used (Bowles et al, 1995). From the initial sequence s, the custom forward primer MCOIF (5TGGGCATCCTGAGGTTTATG-3) and the cu stom reverse primer MCOIR (5TAAAGAAAGAACATAATGAAAATGA-3) were de signed to avoid amplification of C. caretta MCOI. Standard PCR was performed usi ng the Taqman PCR kit (Qiagen) in a 20 l reaction volume according to standard protocol. Th e mixtures were amplified in a thermal cycler (PCR Sprint, Thermo Hybaid, Franklin, MA). Reaction conditions for ITS amplification included initial denaturation at 95C for 5 min, then 45 cycles of denaturation at 95C for 60 s; annealing at 50C for 45 s, and DNA extension at 72C for 120 s, followed by a final extension step at 72C for 10 min. Similar conditions were used for the MCOI primers, except for the annealing temperature, which was set at 45C. The PCR products were resolved in 1% agarose gels. The bands were excised and purified usi ng the QIAquick gel extraction kit (Qiagen). Direct sequencing was performed using the Big-Dye Terminator Ki t (Perkin-Elmer, Branchburg, NJ) and analyzed on ABI 377 automated DNA sequencer s at the University of Florida Center for Mammalian Genetics DNA Sequencing Facilities. All sequences were bidirectional with a minimum of approximately 75% overlap or were repeated to obtain consensus sequence. All chromatograms were manually reviewed using FinchTV 1.4.0 and any ambiguous or questionable data were discarded or repeated for confirmation. Nu cleotide variation represented by single specimens was consistently re-evaluated by repeated PCR and sequencing and, in some instances, repeated DNA extraction from additional samples.

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140 Sequence Alignments and Comparisons All sequences were aligned and examined us ing Mega 4.0 (Kumar et al., 2008). Parasites initially were categorized by ITS2 sequence base d on the findings of studies of other Digenea that support minimal or no intraspecific variatio n in the ITS. Comparisons of ITS1, MCOI, and biological data, when available, were then ex amined within and between genotypes for further evidence of similarity or diversity. Pairwise distances were calcula ted using Mega 4.0 based ClustalW alignments and default parameters with complete deletion of gaps and missing data. The predicted translation of the MCOI sequenc e was determined using the Virtual Ribosome (Wernersson, 2006) with the trematode mitochondrial translation. Where appropriate, differences in pairwise distances were tested for significance and evaluated graphically using the statistical program Analyze-it Version 2.11. Phylogenetic Analyses Phylogenetic analyses were performed on ITS2 and MCOI sequences obtained from specimens of Neospirorchis. Homologous ITS2 sequences were aligned using ClustalW (Thompson et al., 1994), T-Coff ee (Notredame et al., 2000), and MUSCLE (Edgar, 2004). No gaps were present in the predic ted 142 amino acid sequence of th e MCOI, thus alignments were performed using only ClustalW. Bayesian analyses were conducted on each alignment using Mr.Bayes 3.1 (Huelsenbeck and Ronquist, 2001) using gamma dist ributed rate variation and a pr oportion of invariant sites. In addition, mixed amino acid substitution models were included in the analyses of MCOI sequences. The first ten percent of 1,000,000 iterations were discarded from the analyses as a burn in. Maximum likelihood (ML) analys es of each alignment were performed using PHYLIP (Phylogeny Inference Package, Version 3.66) (Felse nstein, 1989). Each alignment was run using

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141 DNA Maximum Likelihood (DNAML) program fo r the ITS2 and the Protein Maximum Likelihood (ProML) program for the MCOI. Fo r analysis of MCOI, three amino acid substitution models were compared, includi ng JTT (Jones, Taylor et al. 1992), PMB (Veerassamy et al., 2003), and PAM (Kosiol and Goldman, 2005). Other set parameters included global rearrangements, five replicat ions of random input or der, and gamma plus invariant rate distributions. A ll analyses were unrooted. The values for the alpha of the gamma distributions and proportion of invariant sites were taken from the Bayesian analyses. Members of two other spirorchiid genera, Hapalotrema mistroides and Learedius learedi, were used as an outgroup. Data subsets were then generated from the ITS2 alignment that produced the most likely tree according to both Bayesian and maxi mum likelihood results and were analyzed using bootstrap analysis to evaluate th e strength of the tree topology (200 re-samplings) (Felsenstein, 1985). For the MCOI, these analyses were perfor med on the most likely tree resulting from the three amino acid substitution models and bootst rap analysis was based on 100 re-samplings (greater than 100 re-samplings produced a fatal computer error). Results ITS2 and MCOI Sequencing Hapalotrema species Specimens from which sequence data were collec ted are listed in Table 3-1. Twenty four adult Hapalotrema specimens representing four different species were examined. Sequence data were collected from eggs from three additional specimens, but were not included in the genetic analysis. Pairwise distances between ITS2 and MCOI sequences obtained from Hapalotrema species, L. learedi and C. bipora are given in Tables 3-2 and 3-3. The ITS2 was 100% homologous for all thirteen adult H. mistroides examined (Figure 32). Identical ITS2 sequence also was obtained from Hapalotrema eggs from two cases, one case

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142 associated with polyp formation in the colon and another with egg masses in subserosal vessels. Slight variation in the MCOI nucleic acid sequence was observed (Figure 3-3). Five variants were identified and differed by only one or two nucleotides over 367 positions (>99% homology). Of the two turtles from which multiple H. mistroides were examined, both had two different variants represented. These minor differences in the nucleotide sequence did not result in any differences in the predicted am ino acid translation among specimens of H. mistroides (Figure 3-4). Specimens of H. postorchis included three localities: Hawa ii, Florida, and the Caribbean (Costa Rica). Four base pair positions of 270 were different (98.5% homology) between the Hawaiian specimens and H. postorchis from Florida and the Caribbean (Figure 3-2). No mitochondrial sequence could be amplified from th e Costa Rican samples, but the partial MCOI was sequenced for most Hawaiian and Floridian sa mples. Two slightly different variants in nucleotide sequence were identified among the Florida H. postorchis (Figure 3-3). One variant was represented by a single parasite and was di fferent at one base pos ition (>99% homology) that did not alter the predicted translation. The MCOI nucleic acid sequences for all Hawaiian H. postorchis were 100% homologous. Fl orida and Hawaiian MCOI nuc leotide sequences were different at 26/367 nucleotide po sitions (92.9% homology), which resulted in three amino acid differences (97.5% homology) (Figure 3-4). The pairwise distances between the MCOI nucleic acid sequences of Florida and Hawaii parasites were significantly greater than between individuals of the sa me locality (p<0.0001). All specimens identified as H. pambanensis had identical ITS2 se quences (Figure 3-2). Four variant partial MCOI sequences were amp lified and were different at one or two of 367 base positions (>99% homology) (Figure 3-3). Multiple (3) variants were identified in one of

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143 two turtles from which multiple parasites were examined. The predicted amino acid translation was 100% homologous for all specimens (Figure 3-4). The ITS2 was 100% homologous for four specimens of the novel Hapalotrema species (Figure 3-2). Identical ITS2 sequence was obtained from embolized eggs from the infected animal. The eggs exhibited the ty pical bipolar processes observed in Hapalotrema species. One specimen had a single nucleotide difference in the MCOI sequence (Figure 3-3) that did not alter the predicted amino acid sequence (Figures 3-4). Learedius learedi The ITS2 was sequenced from a total of 45 individual L. learedi from four localities, including Hawaii, Florida, and the two Caribbean localities (Table 3-1). The ITS2 sequences were greater than 99% homologous and consisted of two variants, designated A and B, that had two nucleotide differences over the 299 positions examined (Figure 3-5). Variant A included some Florida L. learedi all CTFL parasites, and the one TNP sample. The B variant was found in all of the Hawaiian L. learedi as well as parasites from two Florida C. mydas Both Florida turtles with the variant B also we re infected by parasite s with the variant A. Greater diversity was detected in the MCOI nucleic acid sequenc es, which included thirte en individual variants with as many as thirty-five nucleic acid differences over the 418 positions examined (91.6% homology) (Figure 3-6). In cont rast to the ITS2 sequence, repr esentatives of each MCOI variant were limited to one locality. Predicted translat ion of the MCOI resulte d in only two variants, designated A and B, that differed by two amino acids (98.6% homology) (F igure 3-7). Variant B was represented by three Hawaiian L. learedi and all remaining specim ens were variant A. Pairwise distances in MCOI nucleotide se quence were compared between Hawaiian and Floridian L. learedi and between ITS2 variants (Table 33, Figure 3-8). The median distance between individuals from different localities was significantly greater th an between individuals

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144 of the same locality (p<0.0001). Also, there was significantly more variation between individuals with different ITS2 sequences as compared to those with homologous sequence (p<0.0001). There was, however, significant ove rlap in the ranges of pairwise distances observed between and within local ities and ITS2 genotypes. When both ITS2 variant, locality, and MCOI variant were considered, the Florid a ITS variant B and Hawaiian MCOI variant A were most similar (Figure 3-8). Carettacola bipora The ITS2 was sequenced from five C. bipora from three different C. caretta (Table 3-1) and was found to be identical (Figure 3-9). A dditionally, the partial MC OI was sequenced from three parasites and all were 100% homologous (Figure 3-10). Neospirorchis species Adults or eggs from 234 examples of Neospirorchis species were collected from a total of 91 stranded tu rtles, including 60 C. caretta 28 C. mydas and three L. kempi (Table 3-4). Confident morphological identification was only po ssible in two circumstances. The first was original voucher specimens of N. schistosomatoides from the U.S. National Parasite Collection (Rand and Wiles, 1985). DNA was successfully extracted and amplified from two fragmented specimens. Second, several examples of N. pricei were observed in Florida C. caretta and were collected intact in condition suitable for morphological identification. The complete ITS2 and partial MCOI gene were sequenced for most specimens. The lengths of ITS2 sequences ranged from 271 to 298 base pairs, which did not include fifteen base pairs from the 5end that were part of the forward primer (Figure 3-11). An overlapping 429 partial nucleotide sequence of th e MCOI was obtained for most specimens (Figure 3-12) with a predicted translation of 142 amino acids (Figur e 3-13). Nineteen di fferent genotypes were identified in which there was minimal or no va riation between examples in either the ITS2

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145 and/or MCOI. As in the other examined spiror chiid genera, the nucleic acid sequence of the MCOI was much more variable than the ITS2. Much of this variation did not alter the amino acid sequence of the MCOI, which yielded dist ances similar to the ITS2 in pairwise comparisons. Parasites in which only partial sequence data were collected included Neogen-15 (incomplete ITS2, complete MCOI), Neogen-16 (ITS2 data only), and Neogen-19 (MCOI data only). In addition, shorter MCOI sequences were obtained for Neogen-17, 18, and 19. The ITS1 also was completely or partially se quenced for some examples (data not shown); however, many parasites had eviden ce of intragenomic variation due to variable numbers of internal repeats. Amplification of the ITS1 often yielded consiste nt patterns of products within genotypes when bands were visualized by electro phoresis (Figure 3-14). Some ITS1 versions were greater than 1,500 base pairs in length. This variation confounded sequencing efforts, alignments, and comparisons in many instances because selection of the appropriate variant could not be confidently determined. Thus, cl assification by genotype a nd phylogenetic analyses were based on the ITS2 and MCOI data. Phylogenetic Analysis For the ITS2, Bayesian phyl ogenetic analysis using the TCoffee alignment resulted in a greater harmonic mean of estimated marginal likelihoods as compared to ClustalW and MUSCLE alignments. The branching patterns were not significantly different. The Bayesian tree based on ITS2 sequence using the T-Coff ee alignment is shown in Figure 3-15. The Mtmam model of amino acid substitution for th e MCOI sequence was found to be the most probable with a posterior probability of 1.00 (C ao et al., 1998; Yang, Nielsen, and Hasegawa, 1998). The tree resulting from Bayesian analysis using the ClustalW alignments of the MCOI amino acid sequence is given in Figure 3-16.

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146 The most likely trees were determined from the ITS2 alignment (T-Coffee) and MCOI alignment (ClustalW) by ML analysis and thes e parameters were appl ied to the bootstrap analyses. The JTT model of amino acid substi tution was found to be the most likely for the MCOI sequence. As expected, the ML analyses produced a less resolved tree as compared the Bayesian analyses. The bootstrap values from ML analyses are shown on the ITS2 (Figure 3-15) and MCOI Bayesian trees (Figure 3-16). The ITS2 and MCOI produced similar trees with good support for a clade that included all Neospirorchis collected from the gast rointestinal tract (Neoge n-9 through 14). Support for this clade was weakest in the ML analysis of th e MCOI sequences. Both trees also demonstrated good support for grouping to two similar Neospirochis types (based on limited morphology and egg size) represented by Neogen-9 and 11. Anal ysis of the ITS2 also support grouping of Neogen-13 and 14, which were both amplified from distinctive eggs with surface projections, as will be discussed subsequently, which were not observed in other specimens. This grouping was not indicated in the MCOI tree. The distances estimated between the gastrointestinal Neospirorchis and other forms were comparable to that observed between the genera Hapalotrema and Learedius There was poor resolution of th e phylogeny of non-gastrointestinal Neospirorchis, with very weak support of an inclusiv e clade in the MCOI analysis. Further consideration of phylogenetic re sults is given under discussi on of individual genotypes. Genotypes by Microhabitat and Host Species The anatomic locations or microhabitats fr om which spirorchiids were collected and the results of genetic analyses revealed many consis tent relationships. Specific host microhabitats often were associated with genotypes represented by adult spirorchiids and/or localized egg deposition (Table 3-5). Furthermore, most genot ypes were only found in one host species. The general categories of microhabi tats observed were the subm ucosal vasculature of the

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147 gastrointestinal tract (GI) and ve ssels of non-gastrointestinal organs (NGI). Sites included under NGI included the central nervous system, endocrine organs, gonads, and heart. The degree of similarity among genotypes recovered from like microhabitats observed in the phylogenetic analyses were reflected in lower p-distances in both the ITS2 and MCOI (Table 3-6). In the following discussion, groups of genotypes recove red from similar microhabitats will be presented and compared by a pparent organ distribution. Neogen-1, 2, and 3, were found exclusively in th e leptomeninges of the CNS. These three genotypes were found in the 40 of 43 instances in which adult spirorchiids or locally deposited egg masses were removed from the CNS, and were not identified in parasites from other sites. These spirorchiids were observed as elongate, filiform adults and/ or discrete egg masses within leptomeningeal vessels, as is typical of ne urospirorchiidiasis in sea turtles. Neogen-1 corresponded to N. schistosomatoides, as supported by genetic charac terization of two individual voucher specimens, and was observed in 20 C. caretta and one L. kempi Anterior and posterior fragments from Florida parasites that were identified with the Neogen-1 genotype were compatible with N. schistosomatoides based on limited examination. Neogen-2 was restricted to C. caretta (n=14 turtles) and Ne ogen-3 was only found in C. mydas (n=4 turtles). The three examples of other genotypes iden tified in the central nervous syst em included two cases in which adult Neospirorchis of other NGI genotypes (Neogen-5 a nd 6) were recovered and one case where a single egg mass was examined (Neogen-18) but no adults were observed. There was no variation in either ITS2 or MCOI within th e Neogen-1 and Neogen-2 genotypes. Neogen-3 included one ITS2 variant (B) that had two s ubstitutions across 302 posi tions (>99% homology) as compared to the other three representatives (v ariant A) of this genotype. This difference corresponded to some variability in the MCOI nucleic acid sequence. Of the 429 nucleotide

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148 sequence of the MCOI examined, the parasite with variant B ITS2 had 14 nucleic acid differences (96.7% homology) as compared to va riant A examples, which were identical. The predicted MCOI amino acid sequence for Neogen-3, how ever, was identical for all examples. In pairwise comparisons between the three genotypes (Table 3-6), th ere were between fourteen and sixteen base pair differences (94.7 to 95.4% homology) in th e ITS2 and between 32 and 38 differences in the MCOI (90.9 to 92.3% homology). Four genotypes, Neogen-4, 5, 6, and 7, primarily were found in parasites collected from the vessels of endocrine organs, especially th e thyroid gland and adre nal glands, and thymus (Table 3-5). A few examples also were recovere d from the liver and testis. These spirorchiids were most often observed either as single or a fe w adult spirorchiids, discrete egg masses, or as infections with many adults and/or eggs resul ting in distention and obstruction of vessels and effacement of the host tissue. No other genotyp es were detected among th e 40 adult spirorchiids collected from endocrine organs, thymus, or gonad. Furthermore, only one other genotype, Neogen-16, was observed in one of 38 genetic char acterizations of eggs recovered from these sites. In four cases in which parasitism of the pineal gland was observed, Neogen-4 or 7 were identified in adults or egg masses in all specime ns. Notably, no other geno types, including those frequently found in the leptomeninges, were recovered from the pineal gland. Neogen-6 was characterized in the least number of samples and included only three adult specimens, which were recovered from very different anatomic locations. Two adult spirorchiids were flushed from hepatic vessels of one turtle and in anot her case, adults were found migrating within the optic nerves, suggesting that adults with the Ne ogen-6 may be relatively widespread. However, egg masses within thyroid and thymic vessels, as was frequently observe d in parasites with genotypes 4 and 5, also were identified with the Neogen-6 genotype in one case. The only other

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149 example of characterization of one of these genotypes outside the sites previously listed (other than diffusely embolized eggs) was one case in which Neogen-5 was amplified from a single adult collected from th e leptomeninges of a C. caretta Neogen-4 and 6 were recovered exclusively from C. caretta and Neogen-7 was only found in C. mydas Neogen-5 was found in both C. caretta and L. kempi The ITS2 and MCOI sequences were identical for all specimens of Neogen-4 (n=46), Neogen-5 (n=14) and Neogen-7 (n=36). Slight variation in the ITS2 was observed in two examples of Neogen-6 from the same individual host and consisted of a single substitution across the ITS2 (302 base pair sequence). Unfortunately, repeated attempts to sequence the MCOI from this material were unsuccessful; however, this variant was 100% homologous with another Neogen-6 specimen acro ss a 444 base pair portion of the ITS1, which included the 3 region. Pairwise distances between genotypes ar e given in Table 3-6. There were between 11 and 16 nucleotide differences between ITS2 sequences (94.9% to 96.4% homology) of the different genotyp es that corresponded to 24 to 36 nucleotide differences in the MCOI sequence (94.2% to 91.3% homology). Neogen-8 was consistently detected in nine specimens identified as N. pricei. Adult N. pricei primarily were recovered from the cardi ac chambers and major vessels, which is consistent with the original description and only report of this para site (Manter and Larson, 1950). All examples were found in C. caretta The ITS2 was identical in all specimens and only single nucleotide differences in th e MCOI were observed in the tw o adult specimens. There was no alteration of the predicted amino acid sequence. The next several genotypes, Neogen-9, 10, 11, 12, 13, and 14, were obtained from adults found within the gastrointestinal submucosa associated with local ized egg deposition. Examples with genotypes 9, 10, 11, and 12 were observed gro ssly as black, serpiginous egg masses in the

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150 alimentary submucosa often with an associated adult that tended to course extensively through submucosal vessels. Some distinctive features were present in parasite s of some genotypes; however, adequate specimens could not be obtained for detailed morphological examination. Neogen-10 was consistently amplified from adult parasites noted to have a distinct yellow color and relatively large egg size (average of 59 x 47 m). Neogen-9 and 11 were detected from adults that were very similar in appearance and were characterized by white, very delicate adults and smaller egg size (average of 43 x 36 m) as compared to Neogen-10. Only one adult parasite was identified with the Neogen-12 genot ype and the remaining examples were obtained from egg masses. In contrast to these four genotypes, Neogen-13 and 14 appeared to have a more restricted distribution and were found as small discrete egg masses in the stomach (both) or colon (Neogen-14 only). The eggs of both genot ypes had distinctive surface processes visible by light and electron microscopy. Neoge n-10, 11, and 12 were recovered from C. caretta and Neogen-14 was only identified in C. mydas Two alimentary forms were identified in multiple hosts including Neogen-9, which was recovered from both C. caretta and L. kempi and Neogen13, which was found in both C. caretta and C. mydas Neogen-13 was the only genotype found in both C. caretta and C. mydas in this study. All individual s within each GI genotype had identical ITS2 sequences. Individuals of th ree genotypes, Neogen-10 (n=13), Neogen-11 (n=11), and Neogen-12 (n=4), also had identical MCOI sequences. One or two nucleotide differences were observed in the MCOI sequences of Ne ogen-9 (n=15), Neogen-13 (n=4), and Neogen-14 (n=4). The only resulting va riation in the predicted ami no acid sequence was for Neogen-14, which was represented by two variants with tw o amino acid differences. Pairwise distances between genotypes are shown in Table 3-6. Neogen -9 and 11 were the most similar with only 4 nucleotide differences (98.7% homology) in the IT S2 and 16 to 17 nucleotide differences in the

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151 MCOI (95.9% to 96.2% homology) th at translated into one predicted amino acid difference. Neogen-13 and 14 were the next most similar, with 14 differences (95.3% homology) in the ITS2 sequence, 42 to 46 differences (87.2% to 88.4% homology) in MCOI nucleotide sequence and ten and eleven differences in predicte d amino acid sequence ( 92.3% to 93.0% homology). The remaining GI genotypes had between 31 a nd 51 nucleotide differences (83.1% to 89.6% homology) in ITS2 sequences a nd relatively similar differences in the MCOI sequences that ranged from 44 to 69 nucleotide differences (83. 4% to 96.1% homology) and seven to 19 amino acid differences (86.6% to 95.1% homology). The remaining genotypes were amplified from e ggs and no examples were associated with adult parasites. Also, most of these genotypes were found in only a few cases. The most distinctive distribution of eggs was observed in specimens identified with Neogen-15 (n=5), which was amplified from small discrete egg masses in the cloaca and urinary bladder of C. caretta This genotype was most similar to the GI forms based on available ITS2 sequence, which included a short sequence of 173 nucleotid es. Attempts to sequence the remaining 3 fraction of the ITS2 were unsuccessful, thus this genotype was not included in the ITS2 phylogenetic analysis. The MCOI, however, was successfully sequenced and had equivalent distances to both the GI and NGI forms, as reflected in its exclusion from the GI clade in the MCOI phylogenetic analysis. With the exception of Neogen-17 (n=1), which was found in a small egg mass in the leptomeninges of a single C. mydas the remaining genotypes were detected in diffusely embolized eggs. Neogen16 (n=1), 18 (n=4), and 19 (n=2) were only found in C. mydas Both Neogen-18 and 19 were found in eggs from multiple sites in infected turtles. The p-distance data and phyloge netic analysis support that the genotypes Neogen16 through 19 are most similar to NGI forms (Table 3-6).

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152 Mixed Infections and Embolized Egg Data Parasites from multiple sites were collected and sequenced from 62 turtles. Of these, eighteen had only one genotype, twenty-three had two genotypes, eleven had three genotypes, and ten had four or five genotypes. Multiple para site samples, either multiple adults, adults and eggs, or multiple egg samples, from the same specific site (not including different areas within the intestine) were examined in six instances, an d concordant results were obtained in five. The discordant result was sequence obtained from egg masses from the leptomeninges of an infected loggerhead that yielded mixed results and evid ence of three different genotypes. Mixed sequence data were observed in on ly two other examples, and in both cases the source materials also were egg masses, which are easily comprised of eggs from multiple sources. Despite this risk of mixed samples in DNA extractions from egg masses, unambiguous sequence data were collected from 117 of 120 examples of source materials containing eggs. Mixed infections by genotypes with similar apparent tropisms were documented in some cases. Most commonly observed was recovery of multiple different genotypes associated with adult Neospirorchis and or egg masses in the gastrointestinal tract. Of the eleven turtles from which multiple samples were collected from the submucosa, multiple genotypes were detected in seven. In most instances, it was elected to se quence multiple parasites because the adults had a dissimilar sub-gross appearance, e.g. yellow or wh ite coloration, and/or different egg sizes were observed. Infection by multiple genotypes with a tropism for the endocrine organs and nongastrointestinal organs, including Neogen-4 thro ugh 7, was less frequently observed. Individual genotypes were detected from multiple sites, typi cally either the thyroid and thymus or multiple other endocrine organs, in twenty -eight cases. Mixed infections were dete cted in only four turtles. One turtle had mixed sequence includi ng both Neogen-4 and 5 in the thyroid gland. Another turtle had embolized eggs in the lung from which Neogen 6 was amplified and Neogen-

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153 4 was detected from three other sites. A third example had Neogen-4 amplified from adult Neospirorchis from the thyroid gland, thymus, tes tis and adrenal gla nd, whereas two adult Neospirorchis flushed from the liver had the Neogen-6 genotype. In the fourth example, Neogen-5 was detected in adults from the thyr oid gland and testis and Neogen-4 was amplified from a single adult in the leptomeninges. Eggs that were diffusely embolized to organs were examined in 26 turtles. Twenty two were characterized as spirorchiids with endocri ne organ microhabitats (N eogen-4, 5, 6 or 7), two were Neogen-8 ( N. pricei), two were Neogen-18, and one was Ne ogen-19. In half of these cases, all of which infected with spirorchiids of the genotypes Neogen-4, 5, 6, or 7, adult Neospirorchis sp. or localized egg deposition with the same genotype was identified in endocrine organs. Consistency of genotype between the spirorchiid a dults recovered and eggs found in distant sites was most readily observed in a group of C. mydas that died during a hypot hermic stunning event. Seven turtles had adult Neospirorchis with the genotype Neogen-7 in the thyroid gland, and all had eggs of the same size and with same genotype detected in a diffusely embolized pattern in one or more organs, including the lungs, gastric mucosa, and/or ente ric mucosa. Only four cases had discordant results in which a genotype with a similar orga n tropism was detected, but the embolized eggs yielded a different genotype. Congruence of result s could not be determined in nine additional cases. In these cases, either a single specimen was examined or embolized eggs represented the genotype, i.e. the location of adult parasites is unknown (Neogen-16 through 19). Discussion Application of Genetic Fi ndings and Implications In a review of the use of ribosomal DNA in studies of Digenea, Nolan and Cribb (2005) recommended several guidelines for effective molecular approaches to trematode studies. Efforts were made to incorporate these guidelines to whatever extent was possible. Specifically,

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154 relatively large numbers of individual parasites were examined and a broad range of specimens was sought to include three host species and any parasites that appeared to exhibit distinct morphological features or use of different microhabitats. Also, specimens of some species were kindly provided from other regions. Replic ates were found for many of the discovered genotypes and all genetic sequences were carefully scrutinized and differen ces were investigated by repeated PCR and sequencing. In addition, two different genetic markers were used to investigate results. Furthermore, as discussed be low, the genetic results were evaluated in the context of available biological and morphological data. The lim itations of this study were largely due to the relative restrictions of wo rking with protected, ocean-going species and the problematic morphological features of the genus Neospirorchis. Limitations that must be considered included the limited unde rstanding of spirorchiids in terms of morphology (especially Neospirorchis), host range, life cycle, and geographic di stribution, as well as the inability to collect adequate voucher specimens for Neospirorchis. With exception of one Hapalotrema species, the data collected duri ng the study do not meet the cla ssical requirement for species designation, which is morphological examination of sexually mature adult trematodes. Thus, new species were not proposed nor were there attemp ts to delimit individual species within the genetic data. Rather, these findings are pres ented as evidence of previously unrecognized diversity in marine spirorchiids with the fo llowing relevant applic ations: 1) a basis for hypotheses regarding regional differences and biological species diversity; 2) a comparative genetic reference for other spirorchiid species an d parasites from other geographic regions; 3) an alternative means of speci es identification for partial specimens eggs, or immature forms; and 4) an important consideration in di sease studies, parasito logical surveys, and clinical diagnosis.

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155 Comparison of Genetic Diversity with Other Studies of Digenea Although it is not proposed to part ition the genetic data from th is study into species or to divide recognized species, ther e is ample support for the presence of clearly distinct forms, as well as more subtle differences that require further examination. Comparison of genetic variation across taxa, referred to as the genetic yardstick approach, is not appropriate for delimiting species, but is useful for formulati ng hypotheses and identifying parasites that may possess unrecognized or cryptic diffe rences (Vilas et al., 2005). In the following discussion, results of genetic characterizati on of the various marine spirorch iids will be compared with observations in other digenean trematodes with an emphasis on significance of genetic diversity. The ITS is one of the most commonly used ge netic markers in trematode studies and is useful because limited or no variation has been obse rved within species, with few exceptions. In a review of 63 studies that inco rporated the use of the ITS to study digenean trematodes, almost all examples of intraspecific variation were susp ected to be due to error, either in sample identification or labeling, or failure to recogni ze multiple species, as was clearly evidenced in multiple examples (Nolan and Cribb, 2005). Anot her potential source of erroneous interpretation is variable numbers of tandem repeats in the ITS1, as was observed in multiple Neospirorchis genotypes in the current study. These data were largely omitted from analysis due to the uncertainly that appropriate vari ants were being compar ed. Some actual intr aspecific differences may occur due to geographical variation, which has been investigated in Schistosomatidae, Fasciolidae, and Paragonimidae (reviewed by No lan and Cribb, 2005). Blair et al. (2005) recommended subspecific status under a Paragonimus skrjabini species complex for several recognized species based on geogr aphic origin, morphological, a nd molecular findings. Most studies, however, have found no variation from dist ant localities and fewer have found variation

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156 that was interpreted as evidence of multiple species based on host usage and/or egg morphology (Nolan and Cribb, 2005). Intraspecific variation is gr eater within the mitochondrial genes nicotinamide adenine dinucleotide dehydrogenase subunit I (NDI) and MCOI (Vilas et al., 2005). In a study of four different genera and seven species of digenea, maximum intraspecific differences observed were less than or equal to 2.3% in the NDI, which is reported to exhibit more divergence than the MCOI. It is suggested that di stances of greater than 5% w ithin mitochondrial sequences of specimens from the same population should be further investigated (Vilas et al., 2005). Diversity of Spirorchiids of Sea turtles in Florida Hapalotrema Learedius and Carettacola No variation in the ITS2 and minimal or no variation MCOI was found in four of the spirorchiid species examined, including H. mistroides, H. pambanensis, a novel Hapalotrema species, and C. bipora, in which identification was supported by confident morphological study. Hapalotrema mistroides, H. pambanensis and C. bipora were represented by parasites from multiple turtle hosts that stranded in different area s of the Atlantic and Gulf Coasts of Florida. The apparent variation in organization of the testes of H. pambanensis was not reflected in any observed genetic difference, thus this observation is regarded as either artifact or phenotypic variation. Genetic differences were observed, ho wever, in the two species in which specimens from distant localities were examined. Multiple morphological voucher specimens from Hawaii, Florida, and Costa Rica were identified as H. postorchis The ITS2 was identical within localities of H. postorchis but minor differences of four nucleotides (98.5% hom ology) were observed between Hawaiian and Atlantic-Caribbean samples. Greater variation (93.0% homol ogy) was observed in the MCOI nucleotide sequence between regions. Two possi ble explanations for these findings are that

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157 differences reflect the presence of two different species or geographical va riation. The pairwise distances of both the ITS2 and MCOI were co mparable to that observed between different species of Schistosoma and Paragonimus (Vilas et al., 2005). Either explanation would not be surprising given that Atlantic-M editerranean and Indian-Pacific C. mydas are estimated to have been isolated for as long as 1.5 to 3.0 million years (Bowen et al., 1992). Detailed correlative morphological studies of H. postorchis from both regions are nece ssary to investigate these different possibilities. However, assessment of othe r important biological ch aracteristics, such as morphology of immature stages and intermediate host usage, will be limited until the life cycle is discovered. Similar results were obtained from Atlantic and Pacific L. learedi although the emergent picture was somewhat more complicated. Two IT S2 variants that differed by two nucleic acid positions were found in parasites identified as L. learedi but did not correspond to the region of origin for the turtle host. Both variants were found in Florida C. mydas and in both examples these turtles were co-infected by Learedius with the two different vari ants. The ITS2 of variant B was 100% homologous to that obtained from all Hawaiian L. learedi which were identical. The MCIO data were informative in segregating specimens identified as L. learedi in Florida turtles. Individuals with different ITS2 varian ts had much greater pair wise distances (6.9% to 7.4%) as compared to individuals with like ITS2 sequences (less than 1%). This degree of difference in the MCOI is greater than the intr aspecific variation reported in other digeneans from the same population (Vilas et al., 2005). In the case of L. learedi in the present study, the different genotypes actually were collected from the same individual host in two instances. Thus, there is evidence that L. learedi in Florida turtles may actua lly be two closely related species. Interestingly, one of the turtles from which both variants were identified was noted to

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158 have very two different sizes of eggs on fecal examination that were both consistent in morphology with Learedius or Hapalotrema species. No Hapalotrema were recovered from this animal. No obvious differences were seen in the voucher specimens; however, some vouchers had been previously frozen and were not id eal for examination. More detailed study is necessary. Variability also was observed MCOI sequences obtained from Hawaiian L. learedi Pairwise distances as high as 5.7% were observed between samples and resulted in two amino acid differences in three individua l parasites. Hawaiian sequences could be segregated into two groups based on the predicted amino acid sequence of the MCOI with some overlap in pairwise distances. This degree of variability within Hawaiian samples was comparable to that observed between H. postorchis from the Atlantic and Pacific and is higher than observed within other digenean species from the same populations (Vilas et al., 2005). Although the ITS2 was identical for Hawaiian L. learedi the ITS2 may not be the most sensitive indicator of species differences, especially cryptic species (Nol an and Cribb, 2005). Examination of more L. learedi from additional C. mydas from Hawaii and other Pacific lo calities clearly is needed. When Florida and Hawaiian genotypes of L.learedi were compared, Florida parasites with the variant B were more similar to the Hawaiian variant A than either genotype was to the other variant from the same region. The Florida vari ant A and Hawaii variant B had identical ITS2 sequences and very similar MCOI sequences (2.4% to 4.5% homology) This degree of variation in the MCOI was comparable to that observed with in species of other trem atode genera (Villas et al., 2005, Blair et al., 2005). We hypothesize that the Learedius represented by the Florida A variant and Hawaiian B variant is the same species, although there is evidence of a much more complicated population structur e in both regions that requi res further investigation.

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159 Neospirorchis species The genetic data obtained from Neospirorchis sp. support that the diversity of this genus is far greater than previously recognized and memb ers fall into two general groups, those with an apparent tropism for the vasculature of the gastrointestinal tract and those found in vessels of non-gastrointestinal sites, incl uding the nervous system and e ndocrine organs. Phylogenetic analyses support that the gastrointestinal group comprise a well-supported monophyletic clade that is different from other Neospirorchis, as indicated by the observe d branch lengths. These differences are greater than that observed between the genera Hapalotrema and Learedius, suggesting that gastrointestinal forms may actually belong to a different genus. The remaining non-gastrointestinal forms, i.e. those in whic h adults and/or locali zed egg deposition was observed outside of the gastrointe stinal tract or eggs were diffu sely embolized, are more closely related and there was little stat istical support for additional phyl ogenetic structure based on ITS2 and MCOI sequences. The observations in Nolan and Cribbs extens ive review of studies involving the ITS in digenean trematodes support that an y differences in the ITS should be taken as an indicator of potential species diversity because current eviden ce of intraspecific variation was either suspect or, in the case of geographical variation, rarely observed. Consid ering the ITS data alone in the present study, fourteen distinct homol ogous genotypes were identified (Neogen1,2,4,5,7,8,9,10,11,12,13,14,15,18) that were represented by multip le parasite specimens. Of those parasites that belonged within these fourte en distinct ITS2 genotypes that were represented by multiple examples, eight (Neogen-1,2,4,7,10,11,12,18) of the fourteen had identical MCOI sequences. Thus, eight of the Neospirorchis genotypes represented by multiple specimens had 100% homologous ITS2 and MCOI sequences. Less th an 2% variation in th e MCOI nucleic acid was observed in the remaining six ITS2 ge notypes (Neogen-5,8,9,13,14,15), four of which only

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160 had one or two nucleotide differences. This leve l of variation is less than the intraspecific differences documented in other digeneans (Vilas et al., 2005). Furthermore, resulting variation in the predicted amino acid sequence was only obs erved in Neogen-14. Therefore, fourteen of the ninteen Neospirorchis genotypes identified in this st udy had identical ITS2 sequences and exhibited either no variation or minimal variation in the mitochondrial sequence. Additional studies are needed of the remaining five observed genotypes (Neogen3,6,16,17,19). Slight variation in the ITS2 was obser ved in one of four examples of Neogen-3 and one of ten examples of Neogen-6. The va riant observed within the Neogen-3, which was different at 2/302 positions in the ITS2, also was different at 14/429 nucleic acid positions (3.3%) in the MCOI sequence that did not result in any amino acid differences. Our interpretation is that it is premature to incl ude the single Neogen-3 vari ant with the other three examples given the many unknown characteristics of these parasites and limited examination of only a small number of infected C. mydas Careful examination of more meningeal Neospirorchis from C. mydas is needed. All four examples of Neogen-3 were collected from adult C. mydas a nesting female in the case of the observed variant, which migrate long distances from foraging grounds to nesting beaches It is possible that the observed variation reflects some degree of geographi cal variation. With regard to the variant in the Neogen-6, which had a single nucleic acid difference over 302 positions in the ITS2, this specimen was 100% homologous across 445 base pairs of the IT S1 as compared with other examples. Unfortunately, the MCOI sequence could not be am plified from the example with the variant, thus corroborative data from a second genetic ma rker were not available. This genotype was represented by very few adult parasites, whic h were found in diverse locations, including the liver and the perineurium of the optic nerve. Thus, Neogen-6 requires furt her characterization in

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161 multiple respects, as do those genotypes that were identified from embolized eggs only or in which adults have not been collected (Neogen-15,16,17,18,19). Two distinct ITS2 genotypes (Neogen-16,17) were observed in sing le examples, both of which were from C. mydas, thus it cannot be stated whether or not the ITS2 is trul y conserved; furthermore, the MCOI could not be amplified from Neogen-16. The MCOI sequence of Neogen-17; however, had distances ranging from 6.2% to 18.1% from other Neospirorchis, supporting that it is diffe rent. Lastly, the ITS2 could not be amplified from Neogen-19, which is represented by identical MCOI sequences from embolized eggs (2 different sites) from a C. mydas As with Neogen-17, the distances observed, 7.2% to 19.6%, also sugges t that this spirorchii d is a distinct form. The next point of discussion is comparison of distances observed between genotypes and the evidence for biological or morphological di fferences between similar genotypes. It was previously mentioned that any differences in the ITS sequences between individuals warrant further investigation. Regardi ng the MCOI, one study that examin ed variation in mitochondrial sequences within and between species of multiple digenean genera suggested the rough guideline of distances greater than 5% as an indicator that multiple species may be present. In the present study, pairwise distances in MCOI sequence were below this mark in two examples, Neogen-3 and 5, and Neogen-9 and 11. In the case of Neoge n-3 and 5, representatives with this genotype displayed differences in both the microhabitat and host species from which they were collected. Neogen-3 was only found in the leptomeninges of C. mydas (n=4), whereas Neogen-5 was found primarily in endocrine organs (n=13) and, in one instance, the leptomeninges and only in C. caretta Furthermore, Neogen-3 was never detected in the 41 instances in which parasites were sequenced from the leptomeninges of C. caretta and Neogen-5 was not identified from any of the seventeen specimens obtained fr om the endocrine organs of C. mydas These findings support

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162 biological differences between para sites with relatively little a ssociated genetic distance in MCOI sequences (4.5%). Nota bly, although pairwise distances between MCOI sequences were relatively low, there was no support for phylogenetic grouping of these genotypes in either the ITS2 or MCOI analyses. No such detectable biological differences were observed between Neogen-9 and 11, which formed a well-supported cluster in phylogenetic analyses of both genetic targets. There were four nucleotide differences in the ITS2 and 16 to 17 nucleotide differences in the MCOI between these genotypes. Spirorchiids with both genotypes were found in the enteric submucosa, the adults exhibited the same sle nder, white morphology, eggs were indistinguishable, and both were found in C. caretta in comparable numbers. Stranded turtles from which parasites were collected were from multiple zones on the Atlantic and Gulf coasts and in one instance both genotypes were found in th e same turtle. Further examination of these apparently highly similar forms will require additional morphological and biological data. Genotypes and Microhabitat The association between spirorchiid genotype a nd select distribution in host organs, i.e. microhabitat use, was a remarkable finding in the pr esent study. A lack of site fidelity has been noted in Spirorchis species and has been re ported most often in the spirorchiid literature (Holliman, 1971; Platt, 1993; Goodchild and Kirk, 1960), although tropism for gastroenteric arterioles was described for Spirorchis elephantis and the heart and major vessels for Vasotrema robustum in early studies (Wall, 1941; Wall, 1951). Selective microhabitat use, however, is well-recognized for many blood flukes. For example, adults of both Schistosoma mansoni and S. japonicum migrate to mesenteric veins to mate and S. haematobium traffics to the vessels of the urinary bladder (Marquardt et al., 2000). Also, the bird parasite Trichobilharzia regenti migrates within nervous tissue to reach the nasal cavity (Blazov and Hork, 2005). Similarly, there was a strong association between most of the genot ypes observed in this study and intrahost

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163 distribution. One notable exception that conflictes with prev ious reports was N. schistosomatoides. The publication correspond ing to the voucher specimen (Rand and Wiles, 1985) describes adults within the heart and th e original description (Price, 1934) was from visceral blood vessels, whereas the associated genotype, Neogen-1, was consistently found in the CNS in Florida C. caretta It is possible that the infectio ns encountered by others were early and the parasites ultimately would have migrated to meningeal vessels. Furthermore, these specimens were collected from a C. mydas whereas all Neogen-1 examples in the present study were found in C. caretta thus species differences in host-parasite interaction are another consideration. These apparent tropisms are relevant to the biology of the parasite, as well as the implications on the health of the host. Propaga tion strategy is not read ily apparent in some examples where sites of intense egg deposition ar e seemingly remote from access to the external environment, such as in spirorchiids found with in endocrine organs and the central nervous system. One group of turtles included in this study provided some insight. Several immature C. mydas that died from hypothermic stunning were found to be infected by spirorchiids of the Neogen-7 genotype that has only been observed this far in the thyroid gland and pineal gland. Adults and eggs were observed in the thyroid gl and in these turtles; however, the most notable finding was large numbers of embolized eggs within the gastroenteric submucosa and pulmonary vessels. The eggs were all of comparable size an d genetic analysis demonstrated that they were the same genotype as the adults found in the thyroid gland. The resu lts provided a clearer indication of embolization patterns than was observed in C. caretta or older C. mydas which were often found to be infected by multiple spirorchiid types. It appears that Neogen-7 very effectively disseminates eggs to sites where they may be expectorated and/or passed out in the

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164 feces. The thyroscapular vein drains thyroid gland and surrounding region, flows into the precava, and empties into the sinus venosus (Wyneken, 2001). By this route, widespread egg embolization may occur. A similar strategy may be used by the CNS forms, although the vascular pathway is not as direct or in as close proximity to central venous flow. It also was considered that adults may migrate and oviposit within the nasal submucosa, as observed in T. regenti in waterfowl and S. nasalis in cattle; however, neither a dults nor abundant eggs have been recovered from the nasal mucosa in turtle s with neurospirorchiid iasis. The propogation strategy for the GI forms is more obvious and seemingly the most direct Based on observations in necropsied turtles and genetic analysis of embolized eggs, mo st of the eggs produced by the GI forms appear to be locally deposited. One form represented by Neogen-15 has a similar egg distribution to S. haematobium The adults correlating to thes e eggs have not been identified, although based on distribution, the vessels of the urinary bladder and co lon should be closely examined. The discovery of association be tween microhabitat and genotype is relevant to associated pathological lesions, effects on th e host, and detection. Neurospi rorchiidiasis is a concern in Florida turtles based on anecdotal evidence from clinical cases en countered in rehabilitation (C. Manire personal communication) and the poten tial role of these parasites as a confounding health problem during a mass mortality event (Jacobson et al., 2006). Two genotypes were associated with most neurological infections in Florida C. caretta one of which was found in voucher specimens of N. schistosomatoides. The other genotype was genetically similar but was characterized by 16 nucleotide differences in the ITS (94.7% homo logy) and 30 position differences (92.0% homology) in the MCOI. Based on our findings, these forms can be genetically identified when specimens for morphology are unavailable, as is most often the case,

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165 and can be targeted by specific detection methods The same applications are true for other forms, such as those genotypes associated with endocrine organ tropisms. Severe injury to the thyroid gland and thymus was obs erved in 12.0% (9/75) stranded C. caretta and has been observed in some C. mydas The findings of the present stud y support that genetically distinct spirorchiids have an association with these site s and will have to be specifically targeted for detection and further disease stud ies. Currently available antemo rtem diagnostic tests, which are limited to fecal examination and crude antigen ELISA will not detect or account for the diversity observed in this study. Adaptati on of genetic data to molecular diagnostics is the next most logical step in disease studies. Genotypes and Host Species The other notable finding in this st udy was apparent restriction of some Neospirorchis genotypes to either C. caretta or C. mydas which mirrored the apparent host restriction in some Hapalotrema species and L. learedi The strongest evidence in the present study was observed in the genotypes recovered from the CNS, endocri ne tissues, and gastrointe stinal tract. In 37 examples in which adult spirorchiids or e gg masses were sequenced from the brains of C. caretta none were identified as Neogen-3. Likewise none of the four examples of neurological parasitism in C. mydas were associated with genotypes recovered from C. mydas In conflict with these findings were the voucher specimens of N. schistosomatoides (Neogen-1), which were recovered from a C. mydas Neogen-1 was the most common (21/37) genotype associated with neurospirorchiidiasis in Florida C. caretta in the present study. Examination of more spirorchiids from the CNS of C. mydas is necessary. With regard to the genotypes recovered from the endocrine organs and thymus, three genotypes, Neogen-4, 5, a nd 6, were consistently recovered from these sites in C. caretta in 62 examples, whereas Neogen-7 was not found in C. caretta Neogen-7, and only this genotype, was recovered from endocrine organs of C. mydas in

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166 17 examples. Many of the GI forms, with the exception of Neogen-13, were limited to one or the other host species. All of the genotypes a ssociated with larger submucosal egg masses, Neogen-9-12, were only observed in C. caretta The Neogen-15 also was only found in C. caretta In summary, of the nineteen obser ved genotypes, eleven were found in C. caretta and seven were found in C. mydas and only one example, Neogen 13, was observed in both species. Minimally, there is a significant difference in prevalence of th ese parasites among different host species, at least in Florida waters. The other observations regarding host sp ecies are that three genotypes observed in C. caretta also were found in single examples in L. kempi which are the firs t observations of spirorchiidiasis in this species. It was intere sting that all three examples were shared with C. caretta which has a similar diet and habitat. Coevolution and Colonization: Potentia l Influences on Parasite Diversity The diversity of genetically distinct Neospirorchis observed within individual host species in this study was surprising in hosts or iginating from a single geographical region. The number of similar studies of marine parasites, however, are relatively limited, thus comparable examples are likely to emerge as molecular tools ar e applied in the marine environment. At this time, there are critical gaps in the data needed to test specific hypotheses regarding mechanisms of diversification of spirorchii d trematodes. Foremost is the limited number of spirorchiid taxa available for co-phylogeny studies, which make it impossible to determine whether these parasites truly co-evolved with sea turtles or it parasites have diversified, at least in some instances, by host switching/capture (colonization) (Snyder, 2004). Associations that support a hypothesis of coevolution, such as congruent phy logenies and a high degree of cospeciation (Hoberg and Klassen, 2002) have yet to be demo nstrated in sea turtles and spirorchiids. Nonetheless, co-speciation and colonization likely have influenced marine spirorchiid diversity

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167 and processes, such as intrahost speciation and host-switching between rela ted or different hosts (including intermediate hosts), must be investigated. Intrahost speciation is a form of co-specia tion that occurs when speciation evolves in a parasite, but not the host. This process suspec ted when multiple congeneric species are found in the same host (Hoberg, 2005). The diversity of Neospirorchis genotyes observed in the present study is suggestive of such a pattern; however evidence for intrahost speciation requires phylogenetic analysis to identify parasites as si ster species and to asse ss for evidence of cophylogeny verses colonization (Hobe rg, 2005). A more complete re presentation of spirorchiid taxa from other regions and from freshwater and brackish chelonians is necessary for these analyses. There are two commonly proposed mech anisms of intrahost speciation that would produce essentially identical phylog enetic patterns, and thus woul d be impossible to distinguish without adequate supporting biogeographical hi story. The first mechanism is allopatric speciation resulting from cryptic isolation events. It is proposed that parasite fauna can serve as cryptic indicators of isol ation events for host species and may reflect unrecognized biogeographical events (Hoberg, 2005) Isolation events may be sufficient in duration to result in speciation in parasites, but too brief to produce speci ation in the host due to faster parasite generation times and higher mutation rates. The second mechanism by which intrahost speciation may occur is symp atric speciation through special ization to occupy different microhabitats within or on the host. To our knowledge, the only proposed examples of niche specialization or sympatric spec iation in a host are ectoparasi tes (Hoberg, 2005; Dabert and Mironov, 1999). Microhabitat sp ecialization may offer selec tive advantages of avoiding competition (nutrient procurement and disper sal) and promoting reproduction between like

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168 types. With regard to Neospirorchis the microhabitat fidelity observed in the various genotypes may reflect intrahost niche specialization. Colonization is a common process in the diversification of helminths of marine vertebrates (Hoberg, 2005). Morphological evidence of host switching between sea turtles and other marine taxa has been demonstrated for Pronocephalidae (Prez-Ponce de Leon and Brooks, 2005), which is a species-rich family of digenean trematodes of sea turtles. Thus, host-switching may have influenced spiror chiid evolution as well. Diversity of spirorchiids in sea turtle s also may reflect the colonization of new intermediate hosts or co-speciation with intermediate host species. Much of the literature on host switching and co-speciation of digenean tremat odes pertains to definitive hosts; however, the same evolutionary mechanisms of diversif ication apply to parasite-intermediate host relationships. The older paradigm that the great host specificity of digenean gastropod interaction would make hostswitching improbable has been challenged by several key exceptions. Selective pressures driving adaptation to novel intermediate hosts include avoidance of intrahost competition and access to new ecolo gical niches, which may in turn lead to speciation events. For example, Paragonimus species exhibit intermediate host specificity at the superfamily level (as opposed to species specificity) and it is proposed that ecological niche partitioning may be a key influence on evolution of this parasite genus (Wilke, 2000). With regard to spirorchiids, the marine environm ent is inhabited by an estimated 43,000 living gastropod species as compared to around 12,000 fre shwater species (Nicol 1969). The diversity of gastropod fauna within sea turtle habitats, such as seagrass beds and reefs, dwarfs that of freshwater systems. Furthermore, marine sangui nicolids (blood flukes of fish) are unique among digeneans in that they can util ize bivalves and annelids as prim ary intermediate hosts (reviewed

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169 by Smith 1997a), thus vastly increasing the numbe rs of potential intermediate hosts. Such adaptation also may have evol ved in marine spirorchiids. Conclusions The genetic diversity observed in mari ne spirorchiids has many implications on taxonomic and biogeographical relationships, parasite -host interacti on, and future parasitological studies, especially those that incorporate molecu lar approaches. First, the genetic divergence between Atlantic and Pacific H. postorchis and L. learedi minimally may be regarded as geographical variation, but also may reflect divi sion of these taxa into separate species. Furthermore, the two genetically dis tinct genotypes observed in Florida L. learedi suggest the presence of a second closely-rela ted species, possibly a cryptic sp ecies. Both findings would greatly benefit from careful mo rphological studies, which, as of yet, have been limited to relatively small numbers of voucher specimens. The next important finding was evidence for great diversity in the genus Neospirorchis, as supported by the iden tification of many genotypes distinct from one another and from th e two currently recognized species, N. schistosomatoides and N. pricei The selective use of host organs and tissue by many forms, differences in host species, and limited morphological data support the ex istence of many distinct forms that likely will be eventually translated into novel species. In addition, the gastrointestinal forms were demonstrated to be a monophyletic group and genetically different from the other Neospirorchis to the extent that these spirorchiids ultimately may be regarded as a separate genus. In seems likely that application of this information will progress in advance of any formal taxonomic revision at the species level. The full compli ment of biological and morphological data needed for ideal assessment of biological species diversit y and required for formal recognition of species is unlikely to become available in the near future for many of the parasite s characterized in this study.

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170 Although we were not able to propose cha nges in currently assigned species or new species, with the exception of one Hapalotrema many of our findings must be considered in future studies, especially investigations involving health and disease in sea turtles. The selective organ tropisms exhibited by distinct genotypes corre lates with pathological lesions, thus specific methods must be used to detect and identify spirorchiids and to associate specific parasites with any effect on the host. The library of genetic data from spirorchiids of Florida sea turtles obtained during this study may be used as a comparative reference for identification of parasites and for the correlative morphological studies needed to further define these many forms described herein. Furthermore, genetic characterization is an a lternative if morphologica l identification is not possible and hopefully can be used to increase the number of publications especially disease studies, in which spirorchiids are specifically id entified. Also, it hoped that further efforts will be made to better define spirorchiid fauna and di versity in other geographi cal regions to elucidate some of the questions regarding host restriction, geneti c diversity, and host-parasite interaction.

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171 Figure 3-1. Primer sequences and binding locations for amplification of the internal transcribed spacer regions of the ribosomal gene.

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172 H.postorchis .Atlantic.Caribb. CGATGCACATTTAGTCGTGGATTGGATGAGTGCCTGCCGGCGTTGTTATC 50 H.postorchis .Hawaii CGACGCACATTTAGTCGTGGATTGGATGAGTGCCTGCCGGCGTTGTTATC 50 H.mistroides CGACGCACATTTAGTCGTGGATTGGATGAGTGCCTGCCGGCGTTGTTATC 50 Hapalotrema .novel.sp. CGACGCACATTTAGTCGTGGATTGGATGTGTGCCTGCCGGCGTTGTTACC 50 H.pambanensis CGGCGCACATTTAGTCGTGGATTGGATGAGTGCCTGCCGGCGTTGTTGCC 50 ** ************************ ****************** H.postorchis .Atlantic.Caribb. CGTATACCTAA-TCGGATTGCTGGTCATAGGCTCCTTCCTAATTTGTCCG 99 H.postorchis .Hawaii CGTATACCTAA-TCGGATTGCTGGTCAAAGGCTCCTTCCTAATTTGTCCG 99 H.mistroides CGTATACCTAA-TCGGATTGCTGGTCAAAGGCTCCTTCCTAATTTTTCCG 99 Hapalotrema .novel.sp. CGCATATCAAA-TCGGGTTGCTGGTCCAAGGCTCCTTCCTAATTTGTCCG 99 H.pambanensis CGTATAACAAAATCGGGTTGCTGGTCAAAGGCTCCTTCCTAATTTGTCCG 100 ** *** ** **** ********* ***************** **** H.postorchis .Atlantic.Caribb. GTGCAGCCTAATCCGGT--------TTACCAGGTTGAGTTGCTGCAATGG 141 H.postorchis .Hawaii GTGCAGCCTAATTCGGT--------TTACCAGGTTGAGTTGCTGCAATGG 141 H.mistroides GTGCAGCCAAGTCCGGT--------TTACCAGGTTGAGTTGCTGCAATGG 141 Hapalotrema .novel.sp. GCGCAGCCTAGTCCGGTGTTATTGTTTACCAGATTGAGTTGCTGCGGTGG 149 H.pambanensis GTGCAGCCTAGTCCAGT--------TTACCAGGTTGAGTTGCTGCG-TGG 141 ****** ** ******* ************ *** H.postorchis .Atlantic.Caribb. GTATTGCTCGAGTCGTGGCTTAATGCTTTGTTTCATGCTCGAGGC----186 H.postorchis .Hawaii GTAGTGCTCGAGTCGTGGCTTAATGCTTTGTTTCATGCTCGAGGC----186 H.mistroides GTAATGCTCGAGTCGTGGCTTAATGCTTTGGTTCATGCTCGAGGC----186 Hapalotrema .novel.sp. GTTATGCTCGGGTCGTGGCTTAATGTATTATTTCATGCTCGAGGCAGTTG 199 H.pambanensis GTTGTGCTCGAGTCATGGCTTAATACTTTGTTGCATGCTCGAGAC----186 ** ****** *** ********* ** ********** H.postorchis .Atlantic.Caribb. ----CTATTGTGCGGCA-TATTTACACTTGATCTTGGTTTAACTGCTGTG 231 H.postorchis .Hawaii ----CTATTGTGCGGCA-TATTTACACTTGATCTTGGTTTAACTGCTGTG 231 H.mistroides ----CTATTGTGCGGCA-TATTTACACTTGATCTTGGTTTTACTGGTGCG 231 Hapalotrema .novel.sp. AAACCTATCGTATGCTAATGTTTACACTTGATCTTGGTTCTACTGGCTAG 249 H.pambanensis ----CTATCGTG-GGCATAACTTACACCTTGTCTTGGTTTTGCTGACAGG 231 **** ** ****** ******** *** H.postorchis .Atlantic.Caribb. CATGTACTGTAGGTGTGTATCACACA--ATTTATTTGACCC 270 H.postorchis .Hawaii CATGTACTGTAGGTGTGTAGCACACA--GTCTATTTGACCC 270 H.mistroides CAGGTACTGTGGGTGTGTGTCACACA--ATCTATTTGACCC 270 Hapalotrema .novel.sp. TATGTGCTGTAGGTGTGTATCGCACATGATTCTATTGACCC 290 H.pambanensis CGTGTGCTGCAGATGTGCACTGCACATAATTTATTAGACCC 272 ** *** **** **** ***** Figure 3-2. Alignment of internal transcribed spacer 2 sequences of Hapalotrema species. Base pair differences between Hawaiian an d Atlantic-Caribbean specimens of H. postorchis are shaded.

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173 H.pambanensis .var2(1) TCTGTATGACGTTGAGTAATAAAGATTCCCCGTTTGGTTATTATGGGCTT 50 H.pambanensis .var3(1) TCTGTATGACGTTGAGTAATAAAGATTCCCCGTTTGGTTATTATGGGCTT 50 H.pambanensis .var1(2) TTTGTATGACGTTGAGTAATAAAGATTCCCCGTTTGGTTATTATGGGCTT 50 H.pambanensis .var4(1) TTTGTATGACGTTGAGTAATAAAGATTCCCCGTTTGGTTATTATGGGCTT 50 H.mistroides .var3(2) TTTGTATGACTTTAAGAAAAAATGATTCTTCTTTTGGTTATTATGGTTTA 50 H.mistroides .var4(1) TTTGTATGACTTTAAGAAAAAATGATTCTTCTTTTGGTTATTATGGTTTA 50 H.mistroides .var1(8) TTTGTATGACTTTAAGAAAAAATGATTCTTCTTTTGGTTATTATGGTTTA 50 H.mistroides .var5(1) TTTGTATGACTTTAAGAAAAAATGATTCTTCTTTTGGTTATTATGGTTTA 50 H.mistroides .var2(1) TTTGTATGACTTTAAGAAAAAATGATTCTTCTTTTGGTTATTATGGTTTA 50 H.postorchis .FL.var1(6) TTTGTATGACATTGAGAAAAAATGATTCTTCCTTTGGTTATTATGGATTA 50 H.postorchis .FL.var2(1) TTTGTATGACATTGAGAAAAAATGATTCTTCCTTTGGTTATTATGGATTA 50 H.postorchis .HW.(10) TTTGTATGACGTTAAGGAATAATGATTCTTCATTTGGTTATTATGGGTTG 50 Hapalotrema .nov.sp.var1(3) TTTGCATGACGTTAAGAAAAAAAGATTCATCGTTTGGTTACTATGGTCTT 50 Hapalotrema .nov.sp.var2(1) TTTGCATGACGTTAAGAAAAAAAGATTCATCGTTTGGTTATTATGGTCTT 50 ** ***** ** ** ** ** ***** ******** ***** H.pambanensis .var2(1) GTATGTGCTATGGGTTCAATTGTTTGTTTAGGGAGAGTTGTATGGGCACA 100 H.pambanensis .var3(1) GTATGTGCTATGGGTTCAATTGTTTGTTTAGGGAGAGTTGTATGGGCACA 100 H.pambanensis .var1(2) GTATGTGCTATGGGTTCAATTGTTTGTTTAGGGAGAGTTGTATGGGCACA 100 H.pambanensis .var4(1) GTATGTGCTATGGGTTCAATTGTTTGTTTAGGGAGAGTTGTATGGGCACA 100 H.mistroides .var3(2) GTTTGTGCTATGGGTTCAATTGTCTGTTTAGGAAGTGTGGTTTGAGCTCA 100 H.mistroides .var4(1) GTTTGTGCTATGGGTTCAATTGTCTGTTTAGGAAGTGTGGTTTGAGCTCA 100 H.mistroides .var1(8) GTTTGTGCTATGGGTTCAATTGTCTGTTTAGGAAGTGTGGTTTGAGCTCA 100 H.mistroides .var5(1) GTTTGTGCTATGGGTTCAATTGTCTGTTTAGGAAGTGTGGTTTGAGCTCA 100 H.mistroides .var2(1) GTTTGTGCTATGGGTTCAATTGTCTGTTTAGGAAGTGTGGTTTGAGCTCA 100 H.postorchis .FL.var1(6) GTGTGTGCAATGGGTTCTATAGTGTGTTTAGGGAGTGTAGTTTGAGCTCA 100 H.postorchis .FL.var2(1)_ GTGTGTGCAATGGGTTCTATAGTGTGTTTAGGGAGTGTAGTTTGAGCTCA 100 H.postorchis .HW.(10) GTGTGTGCAATGGGTTCTATTGTGTGTTTAGGGAGTGTAGTTTGGGCTCA 100 Hapalotrema .nov.sp.var1(3) GTTTGTGCGATGGGATCTATAGTGTGTTTAGGTAGAGTTGTTTGGGCCCA 100 Hapalotrema .nov.sp.var2(1) GTTTGTGCGATGGGGTCTATAGTGTGTTTAGGTAGAGTTGTTTGGGCCCA 100 ** ***** ***** ** ** ** ******** ** ** ** ** ** ** H.pambanensis .var2(1) TCACATGTTTATGGTTGGGTTAGACGTAAAGACAGCAGTATTTTTTAGGT 150 H.pambanensis .var3(1) TCACATGTTTATGGTTGGGTTAGACGTAAAGACAGCAGTATTTTTTAGGT 150 H.pambanensis .var1(2) TCACATGTTTATGGTTGGGTTAGACGTAAAGACAGCAGTATTTTTTAGGT 150 H.pambanensis .var4(1) TCACATGTTTATGGTTGGGTTAGACGTAAAGACAGCAGTATTTTTTAGGT 150 H.mistroides .var3(2) TCATATGTTCATGGTTGGATTAGATGTGAAGACAGCTGTTTTTTTTAGAT 150 H.mistroides .var4(1) TCATATGTTCATGGTTGGATTAGATGTGAAGACAGCTGTTTTTTTTAGAT 150 H.mistroides .var1(8) TCATATGTTCATGGTTGGATTAGATGTGAAGACAGCTGTTTTTTTTAGAT 150 H.mistroides .var5(1) TCATATGTTCATGGTTGGATTAGATGTGAAGACAGCTGTTTTTTTTAGAT 150 H.mistroides .var2(1) TCATATGTTCATGGTTGGATTAGATGTGAAGACAGCTGTTTTTTTTAGAT 150 H.postorchis .FL.var1(6) TCATATGTTTATGGTGGGGTTAGATGTGAAGACAGCTGTTTTTTTTAGAT 150 H.postorchis .FL.var2(1) TCATATGTTTATGGTGGGGTTAGATGTGAAGACAGCTGTTTTTTTTAGAT 150 H.postorchis .HW.(10) TCACATGTTTATGGTAGGGTTAGATGTGAAGACAGCTGTTTTTTTTAGAT 150 Hapalotrema .nov.sp.var1(3) TCATATGTTTATGGTTGGTTTAGATGTGAAGACTGCAGTGTTTTTTAGTT 150 Hapalotrema .nov.sp.var2(1) TCATATGTTTATGGTTGGTTTAGATGTGAAGACTGCAGTGTTTTTTAGTT 150 *** ***** ***** ** ***** ** ***** ** ** ******** H.pambanensis .var2(1) CTGTCACGATGGTTATTGGTATTCCGACAGGGATCAAGGTTTTTTCGTGA 200 H.pambanensis .var3(1) CTGTCACGATGGTTATTGGTATTCCGACAGGGATCAAGGTTTTTTCATGA 200 H.pambanensis .var1(2) CTGTCACGATGGTTATTGGTATTCCGACAGGGATCAAGGTTTTTTCGTGA 200 H.pambanensis .var4(1) CTGTCACGATGGTTATTGGTATTCCGACAGGGATCAAGGTTTTTTCGTGA 200 H.mistroides .var3(2) CTGTGACTATGGTTATAGGTATTCCTACAGGGATAAAGGTGTTTTCTTGG 200 H.mistroides .var4(1) CTGTGACTATGGTTATAGGTATTCCTACAGGGATAAAGGTGTTTTCTTGG 200 H.mistroides .var1(8) CTGTGACTATGGTTATAGGTATTCCTACAGGGATAAAGGTGTTTTCTTGG 200 H.mistroides .var5(1) CTGTGACTATGGTTATAGGTATTCCTACAGGGATAAAGGTGTTTTCTTGG 200 H.mistroides .var2(1) CTGTGACTATGGTTATAGGTATTCCTACAGGGATAAAGGTGTTTTCTTGG 200 H.postorchis .FL.var1(6) CTGTAACTATGGTTATTGGTATACCTACAGGGATAAAGGTTTTTTCTTGA 200 H.postorchis .FL.var2(1) CTGTAACTATGGTTATTGGTATACCTACAGGGATAAAGGTTTTTTCTTGA 200 H.postorchis .HW.(10) CTGTAACGATGGTTATTGGAATACCTACTGGGATAAAGGTTTTTTCTTGG 200 Hapalotrema .nov.sp.var1(3) CTGTCACAATGGTTATAGGGATACCAACTGGGATTAAGGTTTTTTCTTGA 200 Hapalotrema .nov.sp.var2(1) CTGTCACAATGGTTATAGGGATACCAACTGGGATTAAGGTTTTTTCTTGA 200 **** ** ******** ** ** ** ** ***** ***** ***** ** Figure 3-3. Alignment of partial nucleotide seque nces of the mitochondria l cytochrome oxidase I gene of Hapalotrema species. Shaded positions are differences between specimens identified as the same species by morphology.

PAGE 174

174 H.pambanensis .var2(1) GTGTATATGCTTGGTGTCAGACGAGTGCGAATGAGAGATCCTATAGTTTG 250 H.pambanensis .var3(1) GTGTATATGCTTGGTGTCAGACGAGTGCGAATGAGAGATCCTATAGTTTG 250 H.pambanensis .var1(2) GTGTATATGCTTGGTGTCAGACGAGTGCGAATGAGAGATCCTATAGTTTG 250 H.pambanensis .var4(1) GTGTATATGCTTGGTGTCAGACGAGTGCGAATGAGAGATCCTATAGTTTG 250 H.mistroides .var3(2) GTGTATATGTTAGGTGTTAGACGAGTTCGGGCTAGTGATCCTATAGTGTG 250 H.mistroides .var4(1) GTGTATATGTTAGGTGTTAGACGAGTTCGGGCTAGTGATCCTATAGTGTG 250 H.mistroides .var1(8) GTGTATATGTTAGGTGTTAGACGAGTTCGGGCTAGTGATCCTATAGTGTG 250 H.mistroides .var5(1) GTGTATATGTTAGGTGTTAGACGAGTTCGGGCTAGTGATCCTATAGTGTG 250 H.mistroides .var2(1) GTGTATATGTTAGGTGTTAGACGAGTTCGGGCTAGTGATCCTATAGTGTG 250 H.postorchis .FL.var1(6) GTGTATATGTTGGGTGTTAGACGAGTTCGGGCAAGTGATCCTATAGTATG 250 H.postorchis .FL.var2(1) GTGTATATGTTGGGTGTTAGACGAGTTCGGGCAAGTGATCCTATAGTATG 250 H.postorchis .HW.(10) GTGTATATGTTAGGTGTTAGACGAGTTCGGGTGAGTGATCCTATAGTGTG 250 Hapalotrema .nov.sp.var1(3) GTTTATATGTTAGGGGTTAGACGAGTACGGATGAGGGATCCAATTGTTTG 250 Hapalotrema .nov.sp.var2(1) GTTTATATGTTAGGGGTTAGACGAGTACGGATGAGGGATCCAATTGTTTG 250 ** ****** ** ** ******** ** ** ***** ** ** ** H.pambanensis .var2(1) ATGGGTAGTAGGGTTTATTTTTCTATTTACTGTGGGGGGGGTTACTGGTA 300 H.pambanensis .var3(1) ATGGGTAGTAGGGTTTATTTTTCTATTTACTGTGGGGGGGGTTACTGGTA 300 H.pambanensis .var1(2) ATGGGTAGTAGGGTTTATTTTTCTATTTACTGTGGGGGGGGTTACTGGTA 300 H.pambanensis .var4(1) ATGGGTGGTAGGGTTTATTTTTCTATTTACTGTGGGGGGGGTTACTGGTA 300 H.mistroides .var3(2) ATGGGTTGTTGGGTTTATTTTTTTGTTTACGGTGGGGGGGGTTACTGGGA 300 H.mistroides .var4(1) ATGGGTTGTTGGGTTTATTTTTTTGTTTACAGTGGGGGGGGTTACTGGGA 300 H.mistroides .var1(8) ATGGGTTGTTGGGTTTATTTTTTTGTTTACTGTGGGGGGGGTTACTGGGA 300 H.mistroides .var5(1) ATGGGTTGTTGGGTTTATTTTTTTATTTACTGTGGGGGGGGTTACTGGGA 300 H.mistroides .var2(1) ATGGGTTGTTGGGTTTATTTTTTTGTTTACTGTAGGGGGGGTTACTGGGA 300 H.postorchis .FL.var1(6) ATGAGTGATAGGTTTTATATTTTTGTTTACGGTAGGGGGGGTGACTGGAA 300 H.postorchis .FL.var2(1) ATGAGTGATAGGTTTTATATTTTTGTTTACAGTAGGGGGGGTGACTGGAA 300 H.postorchis .HW.(10) ATGAGTAGTTGGATTTATATTTTTGTTTACTGTGGGGGGGGTTACTGGAA 300 Hapalotrema .nov.sp.var1(3) ATGGGTAGTAGGGTTTATATTTTTGTTCACTGTGGGAGGTGTAACGGGGA 300 Hapalotrema .nov.sp.var2(1) ATGGGTAGTAGGGTTTATATTTTTGTTCACTGTGGGGGGTGTAACGGGGA 300 *** ** ** ***** *** ** ** ** ** ** ** ** ** H.pambanensis .var2(1) TAGTTTTATCAGCATCTCTCTTGGATATAGTTTTTCACGATACTTGGTTT 350 H.pambanensis .var3(1) TAGTTTTATCAGCATCTCTCTTGGATATAGTTTTTCACGATACTTGGTTT 350 H.pambanensis .var1(2) TAGTTTTATCAGCATCTCTCTTGGATATAGTTTTTCACGATACTTGGTTT 350 H.pambanensis .var4(1) TAGTTTTATCAGCATCTCTCTTGGATATAGTTTTTCACGATACTTGGTTT 350 H.mistroides .var3(2) TAGTTTTATCTGCGTCGTTGTTGGATATTTTATTTCATGATACTTGGTTT 350 H.mistroides .var4(1) TAGTTTTATCTGCGTCGTTGTTGGATATTTTATTTCATGATACTTGGTTT 350 H.mistroides .var1(8) TAGTTTTATCTGCGTCGTTGTTGGATATTTTATTTCATGATACTTGGTTT 350 H.mistroides .var5(1) TAGTTTTATCTGCGTCGTTGTTGGATATTTTATTTCATGATACTTGGTTT 350 H.mistroides .var2(1) TAGTTTTATCTGCGTCGTTGTTGGATATTTTATTTCATGATACTTGGTTT 350 H.postorchis .FL.var1(6) TAGTTTTATCTGCATCTTTATTGGATATAATTTTTCATGATACTTGATTT 350 H.postorchis .FL.var2(1) TAGTTTTATCTGCATCTTTATTGGATATAATTTTTCATGATACTTGATTT 350 H.postorchis .HW.(10) TAGTTTTATCTGCGTCTTTATTAGATATAATTTTTCATGATACTTGGTTT 350 Hapalotrema .nov.sp.var1(3) TTGTTTTATCAGCTTCTTTGTTGGATGTTATTTTTCATGATACTTGATTC 350 Hapalotrema .nov.sp.var2(1) TTGTTTTATCAGCTTCTTTGTTGGATGTTATTTTTCATGATACTTGATTC 350 ******** ** ** ** *** ***** ******** ** H.pambanensis .var2(1) GTGATAGCTCATTTTCA 367 H.pambanensis .var3(1) GTGATAGCTCATTTTCA 367 H.pambanensis .var1(2) GTGATAGCTCATTTTCA 367 H.pambanensis .var4(1) GTGATAGCTCATTTTCA 367 H.mistroides .var3(2) GTTATTGCTCATTTTCA 367 H.mistroides .var4(1) GTTATTGCTCATTTTCA 367 H.mistroides .var1(8) GTTATTGCTCATTTTCA 367 H.mistroides .var5(1) GTTATTGCTCATTTTCA 367 H.mistroides .var2(1) GTTATTGCTCATTTTCA 367 H.postorchis .FL.var1(6) GTTATTGCTCATTTTCA 367 H.postorchis .FL.var2(1) GTTATTGCTCATTTTCA 367 H.postorchis .HW.(10) GTTATAGCTCATTTTCA 367 Hapalotrema .nov.sp.var1(3) GTAATAGCTCATTTTCA 367 Hapalotrema .nov.sp.var2(1) GTAATAGCTCATTTTCA 367 ** ** *********** Figure 3-3. Continued.

PAGE 175

175 H.pambanensis .MCOI CMTLSNNDSPFGYYGLVCAMGSIVCLGSVVWAHHMFMVGLDVKTAVFFSS 50 H.mistroides .MCOI CMTLSNNDSSFGYYGLVCAMGSIVCLGSVVWAHHMFMVGLDVKTAVFFSS 50 H.postorchis .FL.MCOI CMTLSNNDSSFGYYGLVCAMGSMVCLGSVVWAHHMFMVGLDVKTAVFFSS 50 H.postorchis .HW.MCOI CMTLSNNDSSFGYYGLVCAMGSIVCLGSVVWAHHMFMVGLDVKTAVFFSS 50 Hapalotrema .nov.sp.MCOI CMTLSNNDSSFGYYGLVCAMGSMVCLGSVVWAHHMFMVGLDVKTAVFFSS 50 *********.************:*************************** H.pambanensis .MCOI VTMVIGIPTGIKVFSWVYMLGVSRVRMSDPMVWWVVGFIFLFTVGGVTGM 100 H.mistroides .MCOI VTMVMGIPTGMKVFSWVYMLGVSRVRASDPMVWWVVGFIFLFTVGGVTGM 100 H.postorchis .FL.MCOI VTMVIGMPTGMKVFSWVYMLGVSRVRASDPMVWWVMGFMFLFTVGGVTGM 100 H.postorchis .HW.MCOI VTMVIGMPTGMKVFSWVYMLGVSRVRVSDPMVWWVVGFMFLFTVGGVTGM 100 Hapalotrema .nov.sp.MCOI VTMVMGMPTGIKVFSWVYMLGVSRVRMSDPIVWWVVGFMFLFTVGGVTGI 100 ****:*:***:*************** ***:****:**:**********: H.pambanensis .MCOI VLSASLLDMVFHDTWFVMAHF 121 H.mistroides .MCOI VLSASLLDILFHDTWFVIAHF 121 H.postorchis .FL.MCOI VLSASLLDMIFHDTWFVIAHF 121 H.postorchis .HW.MCOI VLSASLLDMIFHDTWFVMAHF 121 Hapalotrema .nov.sp.MCOI VLSASLLDVIFHDTWFVMAHF 121 ********::*******:*** Figure 3-4. Alignment of partial predicted am ino acid sequence of m itochondrial cytochrome oxidase I gene of Hapalotrema species. Shaded positions are differences between specimens identified as the same species by morphology.

PAGE 176

176 L.learedi .variant A GTCGGCTTATTATCTATCACGGCGCACATTTAGTCGTGGATTGGATGAGT 50 L.learedi .variant B GTCGGCTTATTATCTATCACGGCGCACATTTAGTCGTGGATTGGATGAGT 50 ************************************************** L.learedi .variant A GCCTGCCGGCGTTGTTACCCGTATAACAAAATCGGGTTGCTGGTCAAAGG 100 L.learedi .variant B GCCTGCCGGCGTTGTTACCCGTATAACAAAATCGGGTTGCTGGTCAAAGG 100 ************************************************** L.learedi .variant A CTCCTTCCTAATTTGTCCGGCGCAGCCTAGTCCGGTTTATCAGGTTGAGT 150 L.learedi .variant B CTCCTTCCTAATTTGTCCGGCGCAGCCTTGTCCGGTTTATCAGGTTGAGT 150 **************************** ********************* L.learedi .variant A TGCTGCGGTGGGTTGTGCTCGAGTCGTGGCTTAATGCTTTGTTTCATGCT 200 L.learedi .variant B TGCTGCGGTGGGTTGTGCTCGAGTCGTGGCTTAATGCTTTGTTTCATGCT 200 ************************************************** L.learedi .variant A CGGGACCTATCGTGTGCATCATTTATGCCTCAGCTTGGTTTCACTGGCAG 250 L.learedi .variant B CGGGACCTATCGTGTGCATCATTTATGCCTCAGCTTGGTTTTACTGGCAG 250 ***************************************** ******** L.learedi .variant A GTATGTGCTGCAGGTGTGCATCACACATTTCCCAATTTGACCCTGACCT 299 L.learedi .variant B GTATGTGCTGCAGGTGTGCATCACACATTTCCCAATTTGACCCTGACCT 299 ************************************************* Figure 3-5. Alignment of internal tran scribed spacer 2 (ITS2) sequences of Learedius learedi

PAGE 177

177 L.learedi.MCOI.var1 ATCCTGAGGTTTATGTTTTAATTCTACCTGGATTTGGTGTGGTAAGACAC 50 L.learedi.MCOI.var2 ATCCTGAGGTTTATGTTTTAATTCTACCTGGATTTGGTGTGGTAAGACAC 50 L.learedi.MCOI.var3 ATCCTGAGGTTTATGTTTTAATTCTACCTGGATTTGGTGTGGTTAGACAC 50 L.learedi.MCOI.var4 ATCCTGAGGTTTATGTTTTAATTCTACCTGGATTTGGTGTGGTAAGACAC 50 L.learedi.MCOI.var5 ATCCTGAGGTTTATGTTTTAATTCTACCTGGATTTGGTGTGGTAAGGCAC 50 L.learedi.MCOI.var6 ATCCTGAGGTTTATGTTTTAATTCTACCTGGATTTGGTGTGGTAAGACAC 50 L.learedi.MCOI.var7 ATCCTGAGGTTTATGTTTTAATTCTACCTGGGTTTGGTGTGGTAAGACAT 50 L.learedi.MCOI.var8 ATCCTGAGGTTTATGTTTTAATTCTACCTGGGTTTGGTGTGGTAAGACAT 50 L.learedi.MCOI.var9 ATCCTGAGGTTTATGTTTTAATTCTACCTGGGTTTGGTGTGGTAAGACAT 50 L.learedi.MCOI.var10 ATCCTGAGGTTTATGTTTTAATTCTACCTGGATTTGGTGTGGTGAGACAT 50 L.learedi.MCOI.var11 ATCCTGAGGTTTATGTTTTAATTCTACCTGGATTTGGTGTGGTAAGACAT 50 L.learedi.MCOI.var12 ATCCTGAGGTTTATGTTTTAATTCTACCAGGGTTTGGAATAGTAAGTCAT 50 L.learedi.MCOI.var13 ATCCTGAGGTTTATGTTTTAATTCTGCCTGGGTTTGGGGTAGTAAGTCAT 50 ************************* ** ** ***** ** ** ** L.learedi.MCOI.var1 ATTTGTATGACTTTGAGAAATAATGATTCTACGTTTGGTTATTATGGTCT 100 L.learedi.MCOI.var2 ATTTGTATGACTTTGAGAAATAATGATTCTACGTTTGGTTATTATGGTCT 100 L.learedi.MCOI.var3 ATTTGTATGACTTTGAGAAATAATGATTCTACGTTTGGTTATTATGGTCT 100 L.learedi.MCOI.var4 ATTTGTATGACTTTGAGAAATAATGATTCTACGTTTGGTTATTATGGTCT 100 L.learedi.MCOI.var5 ATTTGTATGACTTTGAGAAATAATGATTCTACGTTTGGTTATTATGGTCT 100 L.learedi.MCOI.var6 ATTTGTATGACTTTGAGAAATAATGATTCTACGTTTGGTTATTATGGTCT 100 L.learedi.MCOI.var7 ATTTGTATGACTTTAAGGAATAATGACTCTACGTTTGGTTATTATGGGCT 100 L.learedi.MCOI.var8 ATTTGTATGACTTTAAGGAAAAATGACTCTACGTTTGGTTATTATGGGCT 100 L.learedi.MCOI.var9 ATTTGTATGACTTTAAGGAATAATGATTCTACGTTTGGTTATTATGGGCT 100 L.learedi.MCOI.var10 ATTTGTATGACTTTAAGTAATAATGATTCTACGTTTGGTTATTATGGACT 100 L.learedi.MCOI.var11 ATTTGTATGACTTTAAGTAATAATGATTCTACGTTTGGTTATTATGGACT 100 L.learedi.MCOI.var12 ATTTGTATGACTTTAAGTAATAATGATTCTACGTTTGGTTATTATGGACT 100 L.learedi.MCOI.var13 ATTTGTATGACCTTAAGTAATAATGATTCTACGTTTGGTTATTATGGACT 100 *********** ** ** ** ***** ******************** ** L.learedi.MCOI.var1 TGTTTGTGCTATGGGTTCAATAGTGTGTTTGGGTAGTGTGGTATGAGCTC 150 L.learedi.MCOI.var2 TGTTTGTGCTATGGGTTCAATAGTGTGTTTGGGTAGTGTGGTATGAGCTC 150 L.learedi.MCOI.var3 TGTTTGTGCTATGGGTTCAATAGTGTGTTTGGGTAGTGTGGTATGAGCTC 150 L.learedi.MCOI.var4 TGTTTGTGCTATGGGTTCAATAGTGTGTTTGGGTAGTGTGGTATGAGCTC 150 L.learedi.MCOI.var5 TGTTTGTGCTATGGGTTCAATAGTGTGTTTGGGTAGTGTGGTATGAGCTC 150 L.learedi.MCOI.var6 TGTTTGTGCTATGGGTTCAATAGTGTGTTTGGGTAGTGTGGTATGAGCTC 150 L.learedi.MCOI.var7 TGTTTGTGCTATGGGTTCAATAGTGTGCTTAGGAAGTGTAGTGTGGGCTC 150 L.learedi.MCOI.var8 TGTTTGTGCTATGGGTTCAATAGTGTGCTTAGGAAGTGTAGTGTGGGCTC 150 L.learedi.MCOI.var9 TGTTTGTGCTATGGGTTCAATAGTGTGCTTAGGAAGTGTAGTGTGGGCTC 150 L.learedi.MCOI.var10 TGTTTGTGCTATGGGTTCAATAGTCTGTTTAGGAAGTGTAGTGTGGGCTC 150 L.learedi.MCOI.var11 TGTTTGTGCTATGGGTTCAATAGTCTGTCTAGGAAGTGTAGTGTGGGCTC 150 L.learedi.MCOI.var12 TGTTTGTGCTATGGGTTCAATAGTGTGTTTAGGAAGCGTAGTATGGGCTC 150 L.learedi.MCOI.var13 TGTTTGTGCTATGGGTTCAATAGTGTGTTTAGGGAGTGTAGTGTGGGCTC 150 ************************ ** ** ** ** ** ** **** L.learedi.MCOI.var1 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCTGTTTTTTTTAGT 200 L.learedi.MCOI.var2 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCTGTTTTTTTTAGT 200 L.learedi.MCOI.var3 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCTGTTTTTTTTAGT 200 L.learedi.MCOI.var4 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCTGTTTTTTTTAGT 200 L.learedi.MCOI.var5 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCTGTTTTTTTTAGT 200 L.learedi.MCOI.var6 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCTGTTTTTTTTAGT 200 L.learedi.MCOI.var7 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCAGTTTTTTTTAGT 200 L.learedi.MCOI.var8 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCAGTTTTTTTTAGT 200 L.learedi.MCOI.var9 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCAGTTTTTTTTAGT 200 L.learedi.MCOI.var10 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCAGTTTTTTTTAGT 200 L.learedi.MCOI.var11 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCAGTTTTTTTTAGT 200 L.learedi.MCOI.var12 ATCATATGTTTATGGTGGGTTTGGATGTTAAGACTGCAATATTTTTTAGT 200 L.learedi.MCOI.var13 ATCATATGTTTATGGTTGGTTTAGATGTTAAGACTGCAGTTTTTTTTAGT 200 **************** ***** ************** ********* L.learedi.MCOI.var1 TCTGTAACTATGGTTATTGGGATCCCCACAGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var2 TCTGTAACTATGGTTATTGGGATCCCCACGGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var3 TCTGTAACTATGGTTATTGGGATCCCCACGGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var4 TCTGTAACTATGGTTATTGGGATCCCCACAGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var5 TCTGTAACTATGGTTATTGGGATCCCCACAGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var6 TCTGTAACTATGGTTATTGGGATTCCCACAGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var7 TCTGTAACTATGGTTATTGGAATTCCTACTGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var8 TCCGTAACTATGGTTATTGGAATTCCTACTGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var9 TCTGTCACTATGGTTATTGGAATTCCTACTGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var10 TCTGTAACTATGGTTATTGGAATTCCTACTGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var11 TCTGTAACTATGGTTATTGGAATTCCTACTGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var12 TCTGTAACTATGGTTATTGGAATTCCTACAGGAATAAAGGTTTTTTCTTG 250 L.learedi.MCOI.var13 TCTGTAACTATGGTTATTGGAATTCCTACGGGAATAAAGGTTTTTTCTTG 250 ** ** ************** ** ** ** ******************** Figure 3-6. Mitochondrial cyto chrome oxidase I gene partial nucleotide sequence for Learedius learedi

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178 L.learedi.MCOI.var1 AATATATATGCTTGGAGTTAGTCGAGTGCGGGCTAAAGATCCCATAGTTT 300 L.learedi.MCOI.var2 AATATATATGCTTGGAGTTAGTCGAGTGCGGGCTAAAGATCCCATAGTTT 300 L.learedi.MCOI.var3 AATATATATGCTTGGAGTTAGTCGAGTGCGGGCTAAAGATCCCATAGTTT 300 L.learedi.MCOI.var4 AATATATATGCTTGGAGTTAGTCGAGTGCGGGCTAAAGATCCCATAGTTT 300 L.learedi.MCOI.var5 AATATATATGCTTGGAGTTAGTCGAGTGCGGGCTAAAGATCCCATAGTTT 300 L.learedi.MCOI.var6 AATATATATGCTTGGAGTTAGTCGAGTGCGGGCTAAAGATCCCATAGTTT 300 L.learedi.MCOI.var7 AATATATATGCTTGGTGTTAGTCGAGTACGGGCTAATGATCCTATAGTTT 300 L.learedi.MCOI.var8 AATATATATGCTTGGTGTTAGTCGAGTACGGGCTAATGATCCTATAGTTT 300 L.learedi.MCOI.var9 AATATATATGCTTGGTGTTAGTCGAGTACGGGCTAATGATCCTATAGTTT 300 L.learedi.MCOI.var10 AATATACATGCTTGGTGTTAGTCGAGTGCGAGCTAATGATCCTATAGTTT 300 L.learedi.MCOI.var11 AATATACATGCTTGGTGTTAGTCGAGTGCGAGCTAATGATCCTATAGTTT 300 L.learedi.MCOI.var12 AATATATATGCTTGGAGTTAGTCGAGTACGGGCTAATGATCCTATAGTTT 300 L.learedi.MCOI.var13 GATATATATGCTTGGAGTTAGTCGAGTACGGGCTAATGATCCTATAGTTT 300 ***** ******** *********** ** ***** ***** ******* L.learedi.MCOI.var1 GATGAATAGTTGGTTTTATTTTTTTGTTTACGGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var2 GATGAATAGTTGGTTTTATTTTTTTGTTTACGGTGGGGGGGGTGACTGGT 350 L.learedi.MCOI.var3 GATGAATAGTTGGTTTTATTTTTTTGTTTACGGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var4 GATGAATAGTTGGTTTTATTTTTTTGTTTACGGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var5 GATGAATAGTTGGTTTTATTTTTTTGTTTACGGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var6 GATGAATAGTTGGTTTTATTTTTTTGTTTACGGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var7 GATGAATAGTTGGATTTATTTTTTTATTTACTGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var8 GATGAATAGTTGGATTTATTTTTTTATTTACTGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var9 GATGAATAGTTGGATTTATTTTTTTATTTACCGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var10 GATGAATAGTTGGATTTATTTTTTTATTTACTGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var11 GATGAATAGTTGGATTTATTTTTTTATTTACTGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var12 GATGAATAGTTGGGTTTATTTTTTTATTTACGGTGGGGGGGGTAACTGGT 350 L.learedi.MCOI.var13 GATGAATAGTTGGGTTTATTTTTTTATTTACTGTGGGGGGGGTAACAGGT 350 ************* *********** ***** *********** ** *** L.learedi.MCOI.var1 ATTGTTTTATCTGCATCTTTGTTGGATATATTATTCCATGATACTTGGTT 400 L.learedi.MCOI.var2 ATTGTTTTATCTGCATCTTTGTTGGATATATTATTCCATGATACTTGGTT 400 L.learedi.MCOI.var3 ATTGTTTTATCTGCATCTTTGTTGGATATATTATTCCATGATACTTGGTT 400 L.learedi.MCOI.var4 ATTGTTCTATCTGCATCTTTGTTGGATATATTATTCCATGATACTTGGTT 400 L.learedi.MCOI.var5 ATTGTTCTATCTGCATCTTTGTTGGATATATTATTCCATGATACTTGGTT 400 L.learedi.MCOI.var6 ATTGTTCTATCTGCATCTTTGTTGGATATATTATTCCATGATACTTGGTT 400 L.learedi.MCOI.var7 ATTGTTTTATCTGCATCTCTTTTGGATATATTATTTCATGATACTTGATT 400 L.learedi.MCOI.var8 ATTGTTCTATCTGCATCTCTTTTGGATATATTATTTCATGATACTTGATT 400 L.learedi.MCOI.var9 ATTGTTCTATCTGCATCTCTTTTGGATATATTATTTCATGATACTTGATT 400 L.learedi.MCOI.var10 ATTGTTTTATCTGCATCTCTTTTGGATATATTATTTCATGATACTTGATT 400 L.learedi.MCOI.var11 ATTGTTTTATCTGCATCTCTTTTGGATATATTATTTCATGATACTTGATT 400 L.learedi.MCOI.var12 ATTGTTTTATCTGCGTCTCTTTTGGATATATTATTTCATGATACTTGATT 400 L.learedi.MCOI.var13 ATTGTTTTATCTGCATCTCTTTTAGATATATTATTTCATGATACTTGATT 400 ****** ******* *** ** *********** *********** ** L.learedi.MCOI.var1 TGTGATAGCTCATTTTCA 418 L.learedi.MCOI.var2 TGTGATAGCTCATTTTCA 418 L.learedi.MCOI.var3 TGTGATAGCTCATTTTCA 418 L.learedi.MCOI.var4 TGTGATAGCTCATTTTCA 418 L.learedi.MCOI.var5 TGTGATAGCTCATTTTCA 418 L.learedi.MCOI.var6 TGTGATAGCTCATTTTCA 418 L.learedi.MCOI.var7 TGTAATAGCTCATTTTCA 418 L.learedi.MCOI.var8 TGTAATAGCTCATTTTCA 418 L.learedi.MCOI.var9 TGTAATAGCTCATTTTCA 418 L.learedi.MCOI.var10 TGTAATAGCTCATTTTCA 418 L.learedi.MCOI.var11 TGTAATAGCTCATTTTCA 418 L.learedi.MCOI.var12 TGTGATAGCTCATTTTCA 418 L.learedi.MCOI.var13 CGTGATAGCTCATTTTCA 418 ** ************** Figure 3-6. Continued.

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179 -0.01 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 HWA-BHWA-AHWB-BFLA-AFLA-BFL-B-BHWA-FLAHWA-FLBHWB-FLAHWB-FLB P-distance L.learedi .MCOI.variant A PEVYVLILPGFGVVSHICMTLSNNDSTFGYYGLVCAMGSMVCLGSVVWAH 50 L.learedi .MCOI.variant B PEVYVLILPGFGMVSHICMTLSNNDSTFGYYGLVCAMGSMVCLGSVVWAH 50 ************:************************************* L.learedi .MCOI.variant A HMFMVGLDVKTAVFFSSVTMVIGIPTGMKVFSWMYMLGVSRVRANDPMVW 100 L.learedi .MCOI.variant B HMFMVGLDVKTAMFFSSVTMVIGIPTGMKVFSWMYMLGVSRVRANDPMVW 100 ************:************************************* L.learedi .MCOI.variant A WMVGFIFLFTVGGVTGIVLSASLLDMLFHDTWFVMAHF 138 L.learedi .MCOI.variant B WMVGFIFLFTVGGVTGIVLSASLLDMLFHDTWFVMAHF 138 ************************************** Figure 3-7. Alignment of partial amino acid sequ ences of the mitochondria l cytochrome oxidase I gene of Learedius learedi. Figure 3-8. Box and whiskers pl ot of pairwise distances in the mitochondrial cytochrome oxidase I gene within and be tween genotype variants of L.learedi from Florida and Hawaii. Outliers are denoted by a plus sign. Note the significantly lower pairswise distances between Hawaii vari ant A and Florida variant B as compared to genotypes from the same locality. Region and Genotype

PAGE 180

180 05-51.C.bipora.ITS2 CGATGTACATTAAGTCGTGGATTGGATGTGTGCCTGCCGGCAGTTATACT 50 05-58.C.bipora.ITS2 CGATGTACATTAAGTCGTGGATTGGATGTGTGCCTGCCGGCAGTTATACT 50 06-12.C.bipora.1.ITS2 CGATGTACATTAAGTCGTGGATTGGATGTGTGCCTGCCGGCAGTTATACT 50 06-12.C.bipora.3.ITS2 CGATGTACATTAAGTCGTGGATTGGATGTGTGCCTGCCGGCAGTTATACT 50 06-12.C.bipora.2.ITS2 CGATGTACATTAAGTCGTGGATTGGATGTGTGCCTGCCGGCAGTTATACT 50 ************************************************** 05-51.C.bipora.ITS2 CGTATATCAACGCGAGTTGTCGGTCTAAGGCTCCTTCCTAATTTGTCCGG 100 05-58.C.bipora.ITS2 CGTATATCAACGCGAGTTGTCGGTCTAAGGCTCCTTCCTAATTTGTCCGG 100 06-12.C.bipora.1.ITS2 CGTATATCAACGCGAGTTGTCGGTCTAAGGCTCCTTCCTAATTTGTCCGG 100 06-12.C.bipora.3.ITS2 CGTATATCAACGCGAGTTGTCGGTCTAAGGCTCCTTCCTAATTTGTCCGG 100 06-12.C.bipora.2.ITS2 CGTATATCAACGCGAGTTGTCGGTCTAAGGCTCCTTCCTAATTTGTCCGG 100 ************************************************** 05-51.C.bipora.ITS2 CTCAGCCTAGTCCGCAATGAAGACCAGACTGAATTGTTACAGTGGGTTGT 150 05-58.C.bipora.ITS2 CTCAGCCTAGTCCGCAATGAAGACCAGACTGAATTGTTACAGTGGGTTGT 150 06-12.C.bipora.1.ITS2 CTCAGCCTAGTCCGCAATGAAGACCAGACTGAATTGTTACAGTGGGTTGT 150 06-12.C.bipora.3.ITS2 CTCAGCCTAGTCCGCAATGAAGACCAGACTGAATTGTTACAGTGGGTTGT 150 06-12.C.bipora.2.ITS2 CTCAGCCTAGTCCGCAATGAAGACCAGACTGAATTGTTACAGTGGGTTGT 150 ************************************************** 05-51.C.bipora.ITS2 GCTTGAGTCATGGGTTAATGTTGATATACATGCTCGCACAGTAAGCCCCT 200 05-58.C.bipora.ITS2 GCTTGAGTCATGGGTTAATGTTGATATACATGCTCGCACAGTAAGCCCCT 200 06-12.C.bipora.1.ITS2 GCTTGAGTCATGGGTTAATGTTGATATACATGCTCGCACAGTAAGCCCCT 200 06-12.C.bipora.3.ITS2 GCTTGAGTCATGGGTTAATGTTGATATACATGCTCGCACAGTAAGCCCCT 200 06-12.C.bipora.2.ITS2 GCTTGAGTCATGGGTTAATGTTGATATACATGCTCGCACAGTAAGCCCCT 200 ************************************************** 05-51.C.bipora.ITS2 ACTGTTTTCATCTTGGTTCTGAAACGGTCTATGGCTTGTACCGAGAGTGC 250 05-58.C.bipora.ITS2 ACTGTTTTCATCTTGGTTCTGAAACGGTCTATGGCTTGTACCGAGAGTGC 250 06-12.C.bipora.1.ITS2 ACTGTTTTCATCTTGGTTCTGAAACGGTCTATGGCTTGTACCGAGAGTGC 250 06-12.C.bipora.3.ITS2 ACTGTTTTCATCTTGGTTCTGAAACGGTCTATGGCTTGTACCGAGAGTGC 250 06-12.C.bipora.2.ITS2 ACTGTTTTCATCTTGGTTCTGAAACGGTCTATGGCTTGTACCGAGAGTGC 250 ************************************************** 05-51.C.bipora.ITS2 ATAAGGCACAGTGTCTATTTTATCC 275 05-58.C.bipora.ITS2 ATAAGGCACAGTGTCTATTTTATCC 275 06-12.C.bipora.1.ITS2 ATAAGGCACAGTGTCTATTTTATCC 275 06-12.C.bipora.3.ITS2 ATAAGGCACAGTGTCTATTTTATCC 275 06-12.C.bipora.2.ITS2 ATAAGGCACAGTGTCTATTTTATCC 275 ************************* Figure 3-9. Alignment of internal tran scribed spacer 2 (ITS2) sequences of Carettacola bipora.

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181 06-12.C.bipora.1.MCOI TTTGGGCATCCTGAGGTTTATGTTTTAATTTTACCTGGGTTTGGTATGGT 50 06-12.C.bipora.3.MCOI TTTGGGCATCCTGAGGTTTATGTTTTAATTTTACCTGGGTTTGGTATGGT 50 06-12.C.bipora.2.MCOI TTTGGGCATCCTGAGGTTTATGTTTTAATTTTACCTGGGTTTGGTATGGT 50 ************************************************** 06-12.C.bipora.1.MCOI TAGACATATTTGTATGAGGTTGAGTAAAAATGATTCTTCTTTTGGGTATT 100 06-12.C.bipora.3.MCOI TAGACATATTTGTATGAGGTTGAGTAAAAATGATTCTTCTTTTGGGTATT 100 06-12.C.bipora.2.MCOI TAGACATATTTGTATGAGGTTGAGTAAAAATGATTCTTCTTTTGGGTATT 100 ************************************************** 06-12.C.bipora.1.MCOI ATGGGTTGGTGTGTGCTATGGGGGCTATAGTATGTTTGGGGAGTGTGGTT 150 06-12.C.bipora.3.MCOI ATGGGTTGGTGTGTGCTATGGGGGCTATAGTATGTTTGGGGAGTGTGGTT 150 06-12.C.bipora.2.MCOI ATGGGTTGGTGTGTGCTATGGGGGCTATAGTATGTTTGGGGAGTGTGGTT 150 ************************************************** 06-12.C.bipora.1.MCOI TGAGCGCATCATATGTTTATGATTGGTTTAGATATTAAGACTGCTGTGTT 200 06-12.C.bipora.3.MCOI TGAGCGCATCATATGTTTATGATTGGTTTAGATATTAAGACTGCTGTGTT 200 06-12.C.bipora.2.MCOI TGAGCGCATCATATGTTTATGATTGGTTTAGATATTAAGACTGCTGTGTT 200 ************************************************** 06-12.C.bipora.1.MCOI TTTTAGTTCAGTTACTATGGTAATAGGGATTCCTACTGGGATAAAGATAT 250 06-12.C.bipora.3.MCOI TTTTAGTTCAGTTACTATGGTAATAGGGATTCCTACTGGGATAAAGATAT 250 06-12.C.bipora.2.MCOI TTTTAGTTCAGTTACTATGGTAATAGGGATTCCTACTGGGATAAAGATAT 250 ************************************************** 06-12.C.bipora.1.MCOI TTTCTTGATTGTATATGCTTGGTGTTAGTAATATTCGTGTTAATGATCCA 300 06-12.C.bipora.3.MCOI TTTCTTGATTGTATATGCTTGGTGTTAGTAATATTCGTGTTAATGATCCA 300 06-12.C.bipora.2.MCOI TTTCTTGATTGTATATGCTTGGTGTTAGTAATATTCGTGTTAATGATCCA 300 ************************************************** 06-12.C.bipora.1.MCOI ATTGTTTGGTGGATTTTAGGGTTTATTTTTTTATTTACTATTGGTGGGGT 350 06-12.C.bipora.3.MCOI ATTGTTTGGTGGATTTTAGGGTTTATTTTTTTATTTACTATTGGTGGGGT 350 06-12.C.bipora.2.MCOI ATTGTTTGGTGGATTTTAGGGTTTATTTTTTTATTTACTATTGGTGGGGT 350 ************************************************** 06-12.C.bipora.1.MCOI TACTGGGATTGTTTTATCTGCTTCAGTTTTGGATAGTTTGTTTCATGATA 400 06-12.C.bipora.3.MCOI TACTGGGATTGTTTTATCTGCTTCAGTTTTGGATAGTTTGTTTCATGATA 400 06-12.C.bipora.2.MCOI TACTGGGATTGTTTTATCTGCTTCAGTTTTGGATAGTTTGTTTCATGATA 400 ************************************************** 06-12.C.bipora.1.MCOI CTTGGTTTATAATTGCTCATTTTCATT 427 06-12.C.bipora.3.MCOI CTTGGTTTATAATTGCTCATTTTCATT 427 06-12.C.bipora.2.MCOI CTTGGTTTATAATTGCTCATTTTCATT 427 *************************** HPEVYVLILPGFGMVSHICMSLSNNDSSFGYYGLVCAMGAMVCLGSVVWAHHMFMIGLDIKTAVFFSSVTMVMGIPTGMKMFSWLYMLGVSNIRVND PIVWWILGFIFLFTIGGVTGIVLSASVLDSLFHDTWFMIAHFH Figure 3-10. Mitochondrial cytoch rome oxidase I gene partial nucleotide sequence and predicted amino acid sequence for Carettacola bipora

PAGE 182

182Neogen1 CGACGCACAATTA-GTCGTGGCTTGGATGCTGTGCCAACCGGCGTGTTACTCTCACATAC 59 Neogen2 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCGGCATGTTGCTCTCACATAC 59 Neogen3 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCTGCATGTTACTCTCACATAC 59 Neogen4 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCGGCGTGTTACTCTCACATAC 59 Neogen5 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCGGCATGTTACTCTCACATAC 59 Neogen6 CGACGCACAACTA-GTCGTGGCTTGGATGTTGTGCCAACTGGCATGTTACTCTCACATAC 59 Neogen7 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCGGCATGTTACTCTCACATAC 59 Neogen8 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCGGCTTGTTGCTCTCACATAC 59 Neogen9 CGGCGCACAATTT-GTCGTGGCTTGGATGCTGTGCCAACCGGCCTGTTTATCTCACGT-57 Neogen10 CGACGCACAATTT-GTCGTGGCTTGGATGCTGTGCCAACCGGCCTGCTTTGCTCACTT-57 Neogen11 CGGCGCACAATTT-GTCGTGGCTTGGATGCTGTGCCAACCGGCCTGTTTATCTCACGT-57 Neogen12 CGACGCACAATTT-GTCGTGGCTTGGATGTTGTGCCAACCAGCCTGAT-TTCTCA----53 Neogen13 CGACGCACAATTT-GTCGTGGCTTGGATGCTGTGCCAACCGGCCTGTTTCCCCCATGTAT 59 Neogen14 CGACGCACAATTTTGTCGTGGCTTGGATGCTGTGCCAACCGGCCTGTTTCCCCCATGTAT 60 Neogen15 CGACGCACAAATT-GTCGTGGCTTGGATGCTGTGCCAACCGGCATGGTTAGCTCAATTGT 59 Neogen16 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCGGCATGTTACTCTCACATAC 59 Neogen17 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCGGCATGTTACTCTCACATAC 59 Neogen18 CGACGCACAATTA-GTCGTGGCTTGGATGTTGTGCCAACCGGCATGTTACTCTCACATAC 59 ** ******* *************** ********* ** ** ** Neogen1 TGT---------GGTTGAATGTATGAGTTTGAGAGTTGCTGGTTTAATGGCTCCATCCCA 110 Neogen2 TGT---------GGTTGAATGTATGAGTTTGAGAGTCGCTGGTTTAATGGCTCCATCCCA 110 Neogen3 TGT---------GGTTGAATGTATGAGTTTGAGAGTTGCTGGTTTAATGGCTCCATCCCA 110 Neogen4 TGTTGTTTTTGTTGTTGAATGTATGAGTTTGAGAGTTGCTGGTTTAATGGCTCCATCCCA 119 Neogen5 TGT---------GGTTGAATGTATGAGTTTGAGAGTTGCTGGTTTAATGGCTCCATCCCA 110 Neogen6 TGT---------GGTTGAATGTATGAGTTTGAGAGTTGCTGGTTTAATGGCTCCATCCCA 110 Neogen7 TGT------TGTTGTTGGATGTATGAGTTTGAGAGTTGCTGGTTTAATGGCTCCATCCCA 113 Neogen8 TGT---------GATTGGATGTGTGTAATTGAGAGTTGCTGGTTTAATGGCTCCATCCCA 110 Neogen9 -------------GTCCATGGTATACGGTTGTGGGATGCTGGTTTAATGGCTCCATCCTA 104 Neogen10 -------------GTCCGTCGTATACGGTTGTGAGATGCCGGTTTAATGGCTCCATCCTA 104 Neogen11 -------------ATCCATGGTATACGGTTGTGGGATGCTGGTTTAATGGCTCCATCCTA 104 Neogen12 -------------------TGTGTAAGGTTGTGGGAAGCTGGTTTAATGGCTCCATCCTA 94 Neogen13 CCG--------TAGTGCAATGTGCGGTTGTGGG---TGCTGGTTTAATGGCTCCATCCTA 108 Neogen14 CCG--------TGGTATAATGTGCGGTTGTGGG---TGCTGGTTTAATGGCTCCATCCCA 109 Neogen15 CCG--------TGGTATAATATACGG--TTGTGGGCTGCCGGTTTAATGGCTCCATCCTA 109 Neogen16 TGT---------GGTTGAATGTATGAGTTTGAGAGTTGCTGGTTTAATGGCTCCATCCCA 110 Neogen17 TGT---------GGTTGAATGTATGAGTTTGAGAGTTGCTGGTTTAATGGCTCAAGCCCA 110 Neogen18 TGT---------GATT-AATGTATGAGTTTGAGAGTTGCTGGTTTCATGGCTCCATCCCA 109 ** ** **** ******* ** Neogen1 ATGTGTCCGGTTGCAACGTTGTTCGGGTCGAGTGGCTCGTCTGGGTTGTGACGTTGGGTT 170 Neogen2 ATGTGTCCAGTTGCAACGTTGTTCGGGTCGGGTGGCTCGTCTGGGTTGTGGCGATGGGTT 170 Neogen3 ATGTGTCCAGTTGCAACGTTGTTCGGGTCGGGTGGCTCGTCTGGGTTGTGGCGATGGGTT 170 Neogen4 ATGTGTCCAGTTGCAACGTTGTTCGGGTCGGGTGGCTCGTCTGGGTTGTGACGGTAGGTT 179 Neogen5 ATGTGTCCAGTTGCAACATTGTTCGGGTCGGGCGGCTCGTCTGGGTTGTGACGATGGGTT 170 Neogen6 ATGTGTCCGGTTGCAACGTTGTTCGGGTCGGATGGCTCGTCTGGGTTGTGACGTTGGGTT 170 Neogen7 ATGTGCCCAGTTGCAACGTTGTTCGGGTCGGGTGGCTCGTCTGGGTTGTGACGGTGGGTT 173 Neogen8 ATGTGTCCAGTTGCAACGTTGTTCGGGTCGGATGGCTTGACTGGGTTGTGGCGATGGGTT 170 Neogen9 ATGTGTCCAGCTGCAACATTGTTCGGGTCTGATGGCTCGTTTGGGTTGTAGCGATGGGTT 164 Neogen10 ATGTGTCCAGCTGCAACGTTGCTTGGGTCTGATGACTTGTTTGGGTTGTGGCGTTGGGTT 164 Neogen11 ATGTGTCCAGCTGCAACATTGTTCGGGTCTGATGGCTCGTTTGGGTTGTAGCGATGGGTT 164 Neogen12 ATGTGTCCAGCTGCAACATTGCTTGGGTCTTGTGGCTCGTTTGGGTTGTGGCGGTGGGTT 154 Neogen13 ATGTGTCCAGCTGCAACATTACTCGGGTCTGATGGCCTGTTTGGGTTGTGGCGATGGGTT 168 Neogen14 ATGTGTCCAGCTGCAACATTGTTCGGGTCTGATGGCCTGTTGGGGTTGTGGCGATGGGTT 169 Neogen15 ATGTGTCCAGCTGCAAGATTGCCCAGATCTAATGGTTTGATTGAATTCTGGCGGCGGGCT 169 Neogen16 ATGTGTCCGGTTACAACGTTGTTCGGGTCGGGTGGCTCGTCTGGGTTGTGACGTTGGGTT 170 Neogen17 ATGTGTCCGGTTGCAACGTTGTTCGGGTCGGGTGGCTCGTCTGGGTTGTGACGTTGGGTT 170 Neogen18 ATGTGTCCAGTTGCAACGTTGTTCGGGTCGGGTGGCTCGTCTGGGTTGTGGCGGTGGGTT 169 ***** ** *** ** ** ** ** ** Figure 3-11. Alignment of internal transcribed spacer 2 sequences of Neospirorchis specimens.

PAGE 183

183 Neogen1 GTGCACCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTATAATCTGTCGTTTGC 230 Neogen2 GTGCCCCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTATAAGCTGTCGTTTGT 230 Neogen3 ATGCACCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTGTAGTCTGTCGTTTGC 230 Neogen4 TTACGCCTTGTCGTGGCTCAATGATTTGGATCACGTTTGGTGTTATAATCTGTCGTTTGC 239 Neogen5 GTGCACCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTATGATCTGTCGTTTGT 230 Neogen6 GTGCACCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTGCAATCTGTCGTTTGT 230 Neogen7 GTGCACCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTATAATCTGTCGTTTGT 233 Neogen8 GTGCGCCTTGTCGTGGCTCAATGTTTTGGATCACGCTTGGTGTTATGATCTGTTGTTCAT 230 Neogen9 TTGC-CCTTGTCGTGGCTCAATGTTGTTGATCACGCTTGGTGTCATGATCTGTCGTTCAT 223 Neogen10 GTGC-CCTTGTCGTGGCTCAATGTTTT-GATCACGCTTGGTGTCATGGTCCATCGTTCAT 222 Neogen11 TTGC-CCTTGTCGTGGCTCAATGTTGTTGATCACGCTTGGTGTCTTGATCTGTCGTTCAT 223 Neogen12 GTGC-CCTTGTCGTGGCTCAATGTTGTTGATCACGCTTGGTGTCATGGTCTGTCGTTCAT 213 Neogen13 ATGC-CCTTGTCGTGGCTCAATGTTTTTGATCACGCTTGGTGTCATGGTCTGTCGTCTCA 227 Neogen14 ATGC-CCTTGTCGTGGCTCAATGTTTTTGATCACGCTTGGTGTCATGATCTGTCGTCTCA 228 Neogen15 ATAC-------------------------------------------------------173 Neogen16 ATGCGCCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTTTAATCTGTCGTTTAT 230 Neogen17 GTGCGCCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTATAATCTGTCGTTTGT 230 Neogen18 ATGCTCCTTGTCGTGGCTCAATGATTTGGATCACGCTTGGTGTTGTAATTTGTCGTTTGT 229 ****************** ******* ******* ** Neogen1 GACCTTTTCTTGCTATTCTGATT-TGGCAATTGTTTGTGGCTGATGAGCTT-TAAAAGCT 288 Neogen2 GGCCCTTTCTTGCTATTCTGATT-TGGCAATTGTTTGTGGCTGATGAGCTT-TGAAAGCT 288 Neogen3 AACTCTTTCTTGCTATTCTGATT-TAGCAATTGTTTGTGGCTGATGAGCTT-TGAAAGCT 288 Neogen4 GCCCCTTTCTTGCTATTCGGACTGTGGCAATTGTTTGTGGCTGATGAGCTT-TGAAAGCT 298 Neogen5 GACTCTTTCTTGCTATTCTGATT-TGGCAATTGTTTGTGGCTGATGAGCTT-TGAAAGCT 288 Neogen6 GACCCTTTCTTGCTATTCTGATT-TGGCAATTGTTTGTGGCTGATGAGCTT-TGAAAGCT 288 Neogen7 GATCCTTTCTTGCTATTCGGACTGTGGCAATTGTTTGTGGCTGAAGAGCTT-TGAAAGCT 292 Neogen8 GGCTCTCTCTTGCTGTTTTGATT-TGGCAATTGTCTGTGGCTGATGAGCTT-TGAAAGCT 288 Neogen9 GGCTCTTTCTTGCTATTATGATC-TGAAAATCCCTTGCAGTTGATGAGCATTTAAAAGCT 282 Neogen10 AGCTCTTTCTTGTTATTTTGATC-CGGGAATCGCTTGTGGCTGATGAGCATTTGGAAGCT 281 Neogen11 GGCTCTTTCTTGCTATTATGATC-TGGGAATCCCTTGCAGTTGATGAGCATTTAAAAGCT 282 Neogen12 ATCTCTTTCTTGCTATTATGATC-TGGCAGTGGCTTGTGGCTGATGAGCTT-TGAAAAGC 271 Neogen13 TTCTCTTTCTTGCTATTATGATC-TGGGAATTGCTTGTGGCTGATGAGCTCCTGAGAGCT 286 Neogen14 TTCTCTTTCTTGCCATTATGATC-TGGGAATTGTTTGTGGTTGATGGGCTCCTGAGAGCT 287 Neogen15 -----------------------------------------------------------Neogen16 GGCCCTTTCTTGCTATTCTGCCT-TGGCAATTGTTTGTGGCTGATGAGCTT-TGAAAGCT 288 Neogen17 GACCTTTTCTTGCTATTCTGATT-TGGCAACTGTTTGTAGCTGATGAGCTT-TAAAAGCT 288 Neogen18 GACCCTTTCTTGCTATTCTGATT-TGGCAATTGTTTGTGGCTGATGAGCTT-TTGAAGCT 287 ******* ** *** ** Neogen1 GTT--TCTGTTGACCC 302 Neogen2 AGT--TCTGTTGACCC 302 Neogen3 ATT--TCTGTTGACCC 302 Neogen4 ATT--TCTGTTGACCC 312 Neogen5 ATT--TCTGTTGACCC 302 Neogen6 ATT--TCTGTTGACCC 302 Neogen7 ATT--TCTGTTGACCC 306 Neogen8 AAT--ATTGTTGACCC 302 Neogen9 TTA-ACCTGTTGACCC 297 Neogen10 TTTATCTTGTTGACCC 297 Neogen11 TTA-ACCTGTTGACCC 297 Neogen12 TTTCATTTGTTGACCC 287 Neogen13 TTA--TCTGTTTATCC 300 Neogen14 TTA--TCTGTTGACCC 301 Neogen15 ---------------Neogen16 ATT--TCTGTTGACCC 302 Neogen17 ATT--TCTGTTGACCC 302 Neogen18 ATT--TATGTTGACCC 301 **** ** Figure 3-11. Continued.

PAGE 184

184 NEOGEN8.I.MCOI.NTD(6) TGAGTCATATATGTA 15 NEOGEN8.II.MCOI.NTD(1) TGAGTCATATATGTA 15 NEOGEN8.III.MCOI.NTD(1) TGAGTCATATATGTA 15 NEOGEN1.MCOI.NTD(18) GGTTTATGTTTTAATTCTTCCAGGGTTTGGAATGGTTAGTCATATTTGTA 50 NEOGEN17.MCOI.NTD(1) ATATTTGTA 9 NEOGEN7.MCOI.NTD(36) GGTTTATGTGTTAATTCTTCCTGGGTTTGGGATGGTTAGTCATATTTGTA 50 NEOGEN4.MCOI.NTD(42) GGTTTATGTATTAATTCTTCCAGGGTTTGGAATGGTTAGTCATATTTGTA 50 NEOGEN2.MCOI.NTD(13) GGTTTATGTTTTAATTCTTCCAGGTTTTGGAATGGTTAGTCATATTTGTA 50 NEOGEN3.I.MCOI.NTD(3) GGTTTATGTTTTAATTCTTCCCGGGTTTGGTATGGTTAGTCATATTTGTA 50 NEOGEN3.II.MCOI.NTD(1) GGTTTATGTTTTAATTCTTCCTGGGTTTGGGATGGTTAGTCATATTTGTA 50 NEOGEN6.MCOI.NTD(1) GGTTTATGTTTTGATTCTTCCAGGGTTTGGTATGGTTAGTCATATTTGTA 50 NEOGEN19.MCOI.NTD(2) GGTTTATGTTTTGATTCTTCCTGGATTTGGTATGGTTAGTTACATTTGTA 50 NEOGEN18.MCOI.NTD(4) GGTTTATGTTTTAATTCTTCCAGGGTTTGGGATGGTTAGTCATATTTGTA 50 NEOGEN5.I.MCOI.NTD(3) GGTTTATGTTTTGATTCTTCCAGGTTTTGGAATGGTTAGTCATATTTGTA 50 NEOGEN5.II.MCOI.NTD(8) GGTTTATGTTTTGATTCTTCCGGGTTTTGGGATGGTTAGTCATATTTGTA 50 NEOGEN9.I.MCOI.NTD(9) GGTTTATGTTTTGATAATTCCTGGTTTTGGTATGGTTAGACATATATGTA 50 NEOGEN9.II.MCOI.NTD(2) GGTTTATGTTTTGATAATTCCTGGTTTTGGTATGGTTAGACATATATGTA 50 NEOGEN11.MCOI.NTD(8) GGTTTATGTTTTGATAATTCCTGGTTTTGGTATGGTTAGACATATATGTA 50 NEOGEN13.I.MCOI.NTD(2) GGTTTATGTGTTGATTCTACCTGGTTTTGGTATGGTTAGTCATATATGTA 50 NEOGEN13.II.MCOI.NTD(1) GGTTTATGTGTTGATTTTACCTGGTTTTGGTATGGTTAGTCATATATGTA 50 NEOGEN14.I.MCOI.NTD(2) GGTTTATGTTTTAATTCTTCCTGGTTTTGGTATGGTTAGTCATATTTGTA 50 NEOGEN14.II.MCOI.NTD(3) GGTTTATGTTTTAATTCTTCCTGGTTTTGGTATGATTAGTCATATTTGTA 50 NEOGEN10.MCOI.NTD(11) GGTTTATGTTTTAATTCTTCCAGGGTTTGGTATGGTTAGACATATATGTA 50 NEOGEN15.I.MCOI.NTD(1) GGTTTATGTTTTAATTCTTCCTGGGTTTGGGATGGTTAGACATATATGTA 50 NEOGEN15.II.MCOI.NTD(3) GGTTTATGTTTTAATTCTTCCTGGGTTTGGGATGGTTAGACATATATGTA 50 NEOGEN12.MCOI.NTD(4) GGTTTATGTTTTGATTCTTCCTGGTTTTGGTATGGTTAGTCACATATGTA 50 ********* ** ** ** ** ***** ***** ** ** **** NEOGEN8.I.MCOI.NTD(6) TGACTTTGAGTAATAAAGAGTCTATGTTTGGTTATTTTGGTTTAGTGTGT 65 NEOGEN8.II.MCOI.NTD(1) TGACTTTGAGTAACAAAGAGTCTATGTTTGGTTATTTTGGTTTAGTGTGT 65 NEOGEN8.III.MCOI.NTD(1) TGACTTTGAGTAATAAAGAGTCTATGTTTGGTTATTTTGGTTTAGTGTGT 65 NEOGEN1.MCOI.NTD(18) TGACTTTAAGAAAAAGAGAGTCTTTGTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN17.MCOI.NTD(1) TGACTTTGAGAAAAAAAGAGTCTTTGTTTGGTTATTTTGGTTTGGTTTGT 59 NEOGEN7.MCOI.NTD(36) TGACTTTAAGGAATAATGAGTCATTGTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN4.MCOI.NTD(42) TGACTTTGAGGAATAATGAATCGTTGTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN2.MCOI.NTD(13) TGACTTTGAGGAATAATGAGTCGTTGTTTGGTTACTTTGGTTTGGTTTGT 100 NEOGEN3.I.MCOI.NTD(3) TGACTCTAAGAAATAATGAGTCATTGTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN3.II.MCOI.NTD(1) TGACTTTAAGAAATAATGAGTCGTTGTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN6.MCOI.NTD(1) TGACTTTAAGAAAAAAAGAGTCTTTGTTTGGTTATTTTGGTTTGGTTTGT 100 NEOGEN19.MCOI.NTD(2) TGACTTTAAGGAATAGTGAGTCGTTATTTGGTTATTTTGGTTTGGTTTGT 100 NEOGEN18.MCOI.NTD(4) TGACTTTAAGGAATAATGAGTCGTTGTTTGGTTATTTTGGTTTGGTTTGT 100 NEOGEN5.I.MCOI.NTD(3) TGACTTTAAGAAATAATGAGTCGTTGTTTGGTTATTTTGGTTTGGTTTGT 100 NEOGEN5.II.MCOI.NTD(8) TGACTTTAAGAAATAATGAGTCGTTGTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN9.I.MCOI.NTD(9) TGGTTTTAAGGAAAAGTGAGTCTGTATTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN9.II.MCOI.NTD(2) TGGTTTTAAGGAAAAGTGAGTCTGTATTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN11.MCOI.NTD(8) TGGTTTTAAGGAAAAGTGAGTCTGTGTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN13.I.MCOI.NTD(2) TGGTTTTAAGGAAAAATGAGTCGGTGTTTGGTTATTTTGGTTTAGTGTGT 100 NEOGEN13.II.MCOI.NTD(1) TGGTTTTAAGGAAAAATGAGTCGGTGTTTGGTTATTTTGGTTTAGTGTGT 100 NEOGEN14.I.MCOI.NTD(2) TGGTTTTAAGAAAAAATGAGTCGGTGTTTGGTTATTTTGGTTTGGTTTGT 100 NEOGEN14.II.MCOI.NTD(3) TGGTTTTAAGAAAAAATGAGTCGGTGTTTGGTTATTTTGGTTTGGTTTGT 100 NEOGEN10.MCOI.NTD(11) TGGTTTTAAGTAATAATGAGTCTGTGTTTGGTTATTTTGGTTTGGTTTGT 100 NEOGEN15.I.MCOI.NTD(1) TGACTTTAAGAAAAAGTGAGTCTGTTTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN15.II.MCOI.NTD(3) TGACTTTAAGAAAAAGTGAGTCTGTTTTTGGTTATTTTGGTTTAGTTTGT 100 NEOGEN12.MCOI.NTD(4) TGGTTTTGAGAAAAAGGGAGTGTGTGTTTGGTTATTTTGGATTGGTTTGT 100 ** ** ** ** ******** ***** ** ** *** Figure 3-12. Alignment of pa rtial nucleotide sequences of the mitochondria l cytochrome oxidase I gene for Neospirorchis specimens. Shaded positions are differences between like genotypes based on predicte d amino acid (mitoch ondrial cytochrome oxidase I) sequence and ribosomal ITS2 sequence.

PAGE 185

185 NEOGEN8.I.MCOI.NTD(6) GCTATGGGTGCTATTGTTTGTTTAGGTAGTATAGTTTGGGCTCATCATAT 115 NEOGEN8.II.MCOI.NTD(1) GCTATGGGTGCTATTGTTTGTTTAGGTAGTATAGTTTGGGCTCATCATAT 115 NEOGEN8.III.MCOI.NTD(1) GCTATGGGTGCTATTGTTTGTTTAGGTAGTATAGTTTGGGCTCATCATAT 115 NEOGEN1.MCOI.NTD(18) GCTATGGGTGCTATAGTTTGTCTAGGTAGAATAGTTTGGGCTCATCATAT 150 NEOGEN17.MCOI.NTD(1) GCTATGGGTGCTATTGTATGTTTAGGTAGGATAGTTTGAGCTCATCATAT 109 NEOGEN7.MCOI.NTD(36) GCTATGGGTGCTATTGTTTGTTTGGGTAGTATAGTTTGGGCTCATCATAT 150 NEOGEN4.MCOI.NTD(42) GCTATGGGTGCTATTGTTTGTTTGGGTAGTATAGTTTGGGCTCATCATAT 150 NEOGEN2.MCOI.NTD(13) GCTATGGGTGCTATTGTTTGTTTAGGTAGTATAGTTTGGGCTCATCACAT 150 NEOGEN3.I.MCOI.NTD(3) GCTATGGGTGCTATAGTTTGTTTAGGTAGAATAGTTTGAGCTCATCATAT 150 NEOGEN3.II.MCOI.NTD(1) GCTATGGGTGCTATAGTCTGTTTGGGTAGAATAGTTTGGGCTCATCATAT 150 NEOGEN6.MCOI.NTD(1) GCTATGGGTGCTATAGTTTGTTTGGGTAGAATAGTCTGGGCTCATCATAT 150 NEOGEN19.MCOI.NTD(2) GCTATGGGTGCTATTGTTTGTTTGGGTAGAATAGTTTGAGCTCATCATAT 150 NEOGEN18.MCOI.NTD(4) GCTATGGGTGCTATTGTTTGTTTGGGTAGGATAGTTTGAGCTCATCATAT 150 NEOGEN5.I.MCOI.NTD(3) GCTATGGGTGCTATAGTTTGTTTGGGTAGGATAGTTTGAGCTCATCATAT 150 NEOGEN5.II.MCOI.NTD(8) GCTATGGGTGCTATAGTTTGTTTGGGTAGAATAGTTTGAGCTCATCATAT 150 NEOGEN9.I.MCOI.NTD(9) GCTATGGGAGCAATAGTATGTTTAGGAAGGATAGTTTGAGCTCATCATAT 150 NEOGEN9.II.MCOI.NTD(2) GCTATGGGAGCAATAGTATGTTTAGGAAGGATAGTTTGAGCTCATCATAT 150 NEOGEN11.MCOI.NTD(8) GCTATGGGAGCGATAGTGTGTTTAGGTAGGATAGTTTGAGCTCATCATAT 150 NEOGEN13.I.MCOI.NTD(2) GCTATGGGTGCTATTGTGTGTTTGGGAAGTATAGTTTGAGCTCATCATAT 150 NEOGEN13.II.MCOI.NTD(1) GCTATGGGTGCTATTGTGTGTTTGGGAAGTATAGTTTGAGCTCATCATAT 150 NEOGEN14.I.MCOI.NTD(2) GCTATGGGGGCTATAGTATGTTTGGGTAGAATTGTTTGAGCCCATCACAT 150 NEOGEN14.II.MCOI.NTD(3) GCTATGGGGGCTATAGTATGTTTGGGTAGGATTGTTTGAGCTCATCACAT 150 NEOGEN10.MCOI.NTD(11) GCAATGGGAGCTATAGTGTGTTTAGGTAGAATAGTGTGGGCTCATCATAT 150 NEOGEN15.I.MCOI.NTD(1) GCTATGGGGGCTATAGTTTGTTTAGGTAGAATAGTTTGGGCGCATCATAT 150 NEOGEN15.II.MCOI.NTD(3) GCTATGGGGGCTATAGTTTGTTTAGGTAGAATAGTTTGGGCGCATCATAT 150 NEOGEN12.MCOI.NTD(4) GCTATGGGTTCAATTGTATGTTTGGGTAGTGTGGTTTGGGCGCATCATAT 150 ** ***** ** ** *** ** ** ** ** ** ***** ** NEOGEN8.I.MCOI.NTD(6) GTTTGTTGTTGGTATGGATATAAAGACTGCCGTTTTTTTTAGGTCTGTTA 165 NEOGEN8.II.MCOI.NTD(1) GTTTGTTGTTGGTATGGATATAAAGACTGCCGTTTTTTTTAGGTCTGTTA 165 NEOGEN8.III.MCOI.NTD(1) GTTTGTTGTTGGTATGGATATAAAGACTGCCGTTTTTTTTAGGTCTGTTA 165 NEOGEN1.MCOI.NTD(18) GTTTGTTGTTGGTATGGATATAAAGACTGCTGTGTTTTTTAGATCTGTTA 200 NEOGEN17.MCOI.NTD(1) GTTTGTTGTTGGTATGGATGTTAAGACTGCTGTGTTTTTTAGGTCAGTTA 159 NEOGEN7.MCOI.NTD(36) GTTTGTTGTGGGTATGGATATTAAGACTGCTGTGTTTTTTAGATCTGTTA 200 NEOGEN4.MCOI.NTD(42) GTTTGTTGTTGGTATGGATATTAAGACTGCTGTGTTTTTTAGATCTGTTA 200 NEOGEN2.MCOI.NTD(13) GTTTGTTGTGGGTATGGATATAAAGACTGCTGTGTTTTTTAGGTCTGTTA 200 NEOGEN3.I.MCOI.NTD(3) GTTTGTTGTGGGTATGGATATTAAGACTGCTGTGTTTTTTAGGTCTGTTA 200 NEOGEN3.II.MCOI.NTD(1) GTTTGTTGTGGGTATGGATATTAAGACTGCTGTATTTTTTAGATCTGTTA 200 NEOGEN6.MCOI.NTD(1) GTTTGTTGTTGGTATGGATATTAAGACTGCTGTGTTTTTTAGATCAGTTA 200 NEOGEN19.MCOI.NTD(2) GTTTGTTGTGGGTATGGATATTAAGACTGCTGTTTTTTTTAGGTCGGTTA 200 NEOGEN18.MCOI.NTD(4) GTTTGTTGTGGGTATGGATATTAAGACTGCTGTGTTTTTTAGGTCTGTTA 200 NEOGEN5.I.MCOI.NTD(3) GTTTGTTGTGGGTATGGATATAAAGACTGCTGTGTTTTTTAGGTCAGTTA 200 NEOGEN5.II.MCOI.NTD(8) GTTTGTTGTGGGTATGGATATAAAGACTGCTGTGTTTTTTAGGTCAGTTA 200 NEOGEN9.I.MCOI.NTD(9) GTTTGTGATTGGTATGGATATAAAGACTGCTGTTTTTTTTAGGTCTGTTA 200 NEOGEN9.II.MCOI.NTD(2) GTTTGTGATTGGTATGGATATAAAGACTGCTGTTTTTTTTAGGTCTGTTA 200 NEOGEN11.MCOI.NTD(8) GTTTGTGGTTGGGATGGATATAAAGACTGCTGTTTTTTTTAGATCTGTTA 200 NEOGEN13.I.MCOI.NTD(2) GTTTGTTGTTGGTATGGATATAAAGACTGCTGTGTTTTTTAGATCTGTTA 200 NEOGEN13.II.MCOI.NTD(1) GTTTGTTGTTGGTATGGATATAAAGACTGCTGTGTTTTTTAGATCTGTTA 200 NEOGEN14.I.MCOI.NTD(2) GTTTGTTGTTGGAATGGATATAAAGACTGCTGTGTTTTTTAGGTCTGTTA 200 NEOGEN14.II.MCOI.NTD(3) GTTTGTTGTTGGAATGGATATAAAGACTGCTGTGTTTTTTAGATCTGTTA 200 NEOGEN10.MCOI.NTD(11) GTTTGTGATTGGTATGGATATAAAGACTGCTGTGTTTTTTAGCTCTGTTA 200 NEOGEN15.I.MCOI.NTD(1) GTTTGTGGTTGGGATGGATGTTAAGACTGCTGTTTTTTTTAGTTCCGTTA 200 NEOGEN15.II.MCOI.NTD(3) GTTTGTGGTTGGGATGGATGTTAAGACTGCTGTTTTTTTTAGTTCCGTTA 200 NEOGEN12.MCOI.NTD(4) GTTTGTAATTGGGATGGATTTGAAGACTGCTGTATTTTTTAGTTCGGTTA 200 ****** ** ****** ******** ** ******** ** **** Figure 3-12. Continued.

PAGE 186

186 NEOGEN8.I.MCOI.NTD(6) CTATGATTATTGGTATTCCTACAGGTATTAAGGTATTCTCTTGATTATAT 215 NEOGEN8.II.MCOI.NTD(1) CTATGATTATTGGTATTCCTACAGGTATTAAGGTATTCTCTTGATTATAT 215 NEOGEN8.III.MCOI.NTD(1) CTATGATTATTGGTATTCCTACAGGTATTAAGGTATTCTCTTGATTATAT 215 NEOGEN1.MCOI.NTD(18) CTATGATAATTGGTATTCCTACAGGTATAAAGGTGTTTTCTTGGTTATAT 250 NEOGEN17.MCOI.NTD(1) CTATGATAATTGGTATTCCTACTGGTATAAAGGTGTTTTCTTGGTTGTAT 209 NEOGEN7.MCOI.NTD(36) CGATGATAATTGGAATTCCAACTGGTATAAAGGTGTTTTCCTGATTGTAT 250 NEOGEN4.MCOI.NTD(42) CAATGATAATTGGGATTCCGACTGGTATAAAGGTGTTTTCATGGTTATAT 250 NEOGEN2.MCOI.NTD(13) CTATGATTATTGGTATTCCGACTGGTATAAAGGTGTTTTCTTGATTGTAT 250 NEOGEN3.I.MCOI.NTD(3) CTATGATAATTGGTATTCCAACAGGAATAAAGGTGTTTTCTTGGTTATAT 250 NEOGEN3.II.MCOI.NTD(1) CTATGATAATTGGTATTCCAACAGGTATAAAGGTGTTTTCTTGGTTATAT 250 NEOGEN6.MCOI.NTD(1) CTATGATAATTGGTATTCCTACAGGTATAAAGGTTTTTTCTTGGTTGTAT 250 NEOGEN19.MCOI.NTD(2) CTATGATAATTGGTATTCCTACGGGGATAAAGGTGTTTTCTTGGTTGTAT 250 NEOGEN18.MCOI.NTD(4) CTATGATAATTGGTATTCCGACTGGTATAAAGGTGTTTTCTTGATTATAT 250 NEOGEN5.I.MCOI.NTD(3) CTATGATTATTGGTATTCCGACAGGTATAAAGGTATTTTCTTGGTTATAT 250 NEOGEN5.II.MCOI.NTD(8) CTATGATTATTGGTATTCCGACAGGTATAAAGGTGTTTTCTTGGTTATAT 250 NEOGEN9.I.MCOI.NTD(9) CTATGATTATTGGTGTTCCTACAGGTATAAAGGTTTTTTCTTGATTGTAT 250 NEOGEN9.II.MCOI.NTD(2) CTATGATTATTGGTGTTCCTACAGGTATAAAGGTTTTTTCTTGATTGTAT 250 NEOGEN11.MCOI.NTD(8) CGATGATTATTGGTGTTCCTACAGGTATAAAGGTTTTTTCTTGATTGTAT 250 NEOGEN13.I.MCOI.NTD(2) CGATGATTATTGGTATTCCTACAGGTATAAAGGTTTTTTCTTGGTTGTAT 250 NEOGEN13.II.MCOI.NTD(1) CGATGATTATTGGTATTCCTACAGGTATAAAGGTTTTTTCTTGGTTGTAT 250 NEOGEN14.I.MCOI.NTD(2) CTATGATTATAGGTGTTCCTACAGGTATAAAGGTTTTTTCTTGACTATAT 250 NEOGEN14.II.MCOI.NTD(3) CTATGATTATAGGCGTTCCTACAGGTATAAAGGTTTTTTCTTGATTATAT 250 NEOGEN10.MCOI.NTD(11) CTATGATTATTGGTATACCGACCGGGATTAAGGTTTTTTCTTGATTATAT 250 NEOGEN15.I.MCOI.NTD(1) CTATGATAATTGGTATACCTACGGGGATAAAGGTTTTTTCTTGATTGTTT 250 NEOGEN15.II.MCOI.NTD(3) CTATGATAATTGGTATACCTACGGGGATAAAGGTTTTTTCTTGATTGTTT 250 NEOGEN12.MCOI.NTD(4) CTATGATTATAGGTGTTCCGACCGGAATTAAGGTTTTTTCGTGGTTGTAT 250 ***** ** ** ** ** ** ** ***** ** ** ** NEOGEN8.I.MCOI.NTD(6) ATGCTTGGTAGAAGATATGTTCGGTTGGTTGATCCTGTGGTATGATGAAT 265 NEOGEN8.II.MCOI.NTD(1) ATGCTTGGTAGAAGATATGTTCGGTTGGTTGATCCTGTGGTATGATGAAT 265 NEOGEN8.III.MCOI.NTD(1) ATGCTTGGTAGAAGATATGTTCGGTTGGTTGATCCTGTGGTATGATGAAT 265 NEOGEN1.MCOI.NTD(18) ATGCTTGGTAGGAGATATGTTCGGTTGGTTGATCCTGTAGTTTGGTGGAT 300 NEOGEN17.MCOI.NTD(1) ATGCTTGGTAGTAGATATGTTCGGTTGGTTGATCCTGTAGTTTGGTGAAT 259 NEOGEN7.MCOI.NTD(36) ATGCTTGGTAGCAGATACGTTCGGTTAGTTGATCCGGTGGTTTGGTGAAT 300 NEOGEN4.MCOI.NTD(42) ATGCTTGGTAGGAGGTATGTTCGATTGGTTGATCCTGTGGTTTGATGAAT 300 NEOGEN2.MCOI.NTD(13) ATGCTTGGTAGAAGATATGTTCGGTTGGTTGATCCAGTGGTTTGGTGAAT 300 NEOGEN3.I.MCOI.NTD(3) ATGTTAGGTAGGAGGTATGTTCGATTAGTTGATCCAGTGGTTTGGTGAAT 300 NEOGEN3.II.MCOI.NTD(1) ATGTTAGGTAGTAGGTATGTTCGATTAGTTGATCCAGTGGTTTGGTGAAT 300 NEOGEN6.MCOI.NTD(1) ATGCTTGGTAGGAGGTATGTTCGTTTGGTTGATCCTGTGGTTTGGTGGGT 300 NEOGEN19.MCOI.NTD(2) ATGTTGGGTAGTAGGTATGTTCGACTGGTTGATCCGGTGGTTTGGTGGAT 300 NEOGEN18.MCOI.NTD(4) ATGCTTGGTAGGAGTTATGTTCGGTTGATTGATCCTGTGGTTTGATGAAT 300 NEOGEN5.I.MCOI.NTD(3) ATGCTTGGTAGGAGGTATGTTCGATTAGTTGATCCAGTGGTTTGGTGAAT 300 NEOGEN5.II.MCOI.NTD(8) ATGCTTGGTAGGAGGTATGTTCGATTAGTTGATCCAGTGGTTTGGTGAAT 300 NEOGEN9.I.MCOI.NTD(9) ATGCTTGGTGGGAGTAATGTTCGTTTATTTGATCCTGTGGTTTGATGAAT 300 NEOGEN9.II.MCOI.NTD(2) ATGCTTGGTGGGAGTAATGTTCGTTTATTTGATCCTGTGGTTTGATGAAT 300 NEOGEN11.MCOI.NTD(8) ATGCTTGGGGGAAGAAATGTTCGTTTATTTGATCCTGTGGTTTGATGAAT 300 NEOGEN13.I.MCOI.NTD(2) ATGCTTGGCGGTAGTGGTGTTCGATTATATGATCCTGTTGTATGGTGGGT 300 NEOGEN13.II.MCOI.NTD(1) ATGCTTGGCGGTAGTGGTGTTCGATTATATGATCCTGTTGTATGGTGGGT 300 NEOGEN14.I.MCOI.NTD(2) ATGCTTGGTGGGAGTGGTGTTCGTTTGTTTGATCCTGTGGTGTGGTGGGT 300 NEOGEN14.II.MCOI.NTD(3) ATGCTTGGTGGGAGTGGTGTTCGTTTGTTTGATCCTGTGGTGTGGTGGGT 300 NEOGEN10.MCOI.NTD(11) ATGCTTGGGAATAGTGGTGTTCGTTTATTAGATCCGGTGGTTTGGTGGGT 300 NEOGEN15.I.MCOI.NTD(1) ATGCTTGGTAGGAGGAATATTCGTGTGTATGATCCGATAGTTTGGTGAAT 300 NEOGEN15.II.MCOI.NTD(3) ATGCTTGGTAGGAGAAATATTCGTGTGTATGATCCGATAGTTTGGTGAAT 300 NEOGEN12.MCOI.NTD(4) ATGTTGGGTGGAAGAGGTGTTCGTGTTTATGATCCAGTTGTTTGGTGAAT 300 *** ** ** **** ***** ** ** ** Figure 3-12. Continued.

PAGE 187

187 NEOGEN8.I.MCOI.NTD(6) AATAGGTTTTATTTTTTTATTTACAGTGGGTGGGGTAACTGGCATAGTTT 315 NEOGEN8.II.MCOI.NTD(1) AATAGGTTTTATTTTTTTATTTACAGTGGGTGGGGTAACTGGCATAGTTT 315 NEOGEN8.III.MCOI.NTD(1) AATAGGTTTTATTTTTTTATTTACAGTGGGTGGGGTAACTGGGATAGTTT 315 NEOGEN1.MCOI.NTD(18) TGTGGGTTTTATTTTTTTGTTTACTATAGGTGGTGTGACTGGGATTGTTT 350 NEOGEN17.MCOI.NTD(1) TGTGGGTTTTATTTTTTTATTTACTATAGGCGGTGTAACTGGGATTGTTT 309 NEOGEN7.MCOI.NTD(36) TATAGGTTTTATTTTTTTATTTACGGTTGGTGGTGTTACTGGAATTGTTT 350 NEOGEN16.MCOI.NTD(3) TATAGGTTTTATTTTTTTATTTACGGTTGGTGGTGTTACTGGAATTGTTT 302 NEOGEN4.MCOI.NTD(42) TATAGGTTTTATTTTTTTATTTACAGTTGGTGGTGTTACTGGGATTGTTT 350 NEOGEN2.MCOI.NTD(13) TATAGGTTTTATTTTTTTGTTTACAATAGGTGGTGTTACTGGGATTGTTT 350 NEOGEN3.I.MCOI.NTD(3) CATAGGTTTTATTTTTTTGTTTACTGTTGGTGGGGTTACTGGAATTGTTT 350 NEOGEN3.II.MCOI.NTD(1) TATAGGTTTTATTTTTTTGTTTACTGTTGGTGGGGTTACCGGGATTGTTT 350 NEOGEN6.MCOI.NTD(1) TATAGGTTTTATTTTTTTATTTACTATAGGTGGTGTTACTGGAATTGTTT 350 NEOGEN19.MCOI.NTD(2) TATAGGTTTTATTTTTTTGTTTAC 324 NEOGEN18.MCOI.NTD(4) TATAGGTTTTATTTTTTTGTTTAC 324 NEOGEN5.I.MCOI.NTD(3) TATAGGTTTTATTTTTTTGTTTACGATAGGTGGTGTTACTGGGATTGTTT 350 NEOGEN5.II.MCOI.NTD(8) TATAGGTTTTATTTTTTTGTTTACAATAGGTGGTGTTACTGGGATTGTTT 350 NEOGEN9.I.MCOI.NTD(9) TGTTGGGTTTATATTTTTGTTTACTGTAGGAGGGGTTACTGGAATAGTGT 350 NEOGEN9.II.MCOI.NTD(2) TGTTGGGTTTATATTTTTGTTTACTGTAGGAGGGGTTACTGGAATAGTGT 350 NEOGEN11.MCOI.NTD(8) TGTTGGGTTTATATTTTTGTTTACTGTTGGGGGAGTTACTGGGATAGTGT 350 NEOGEN13.I.MCOI.NTD(2) GATTGGGTTTATATTTTTGTTTACAATTGGGGGTGTTACTGGTATAGTTT 350 NEOGEN13.II.MCOI.NTD(1) GATTGGGTTTATATTTTTGTTTACAATTGGGGGTGTTACTGGTATAGTTT 350 NEOGEN14.I.MCOI.NTD(2) TATTGGTTTTATATTTTTATTTGTTGTTGGTGGGGTTACTGGAGTTGTTT 350 NEOGEN14.II.MCOI.NTD(3) TATTGGTTTTATATTTTTATTTGTTGTTGGTGGGGTTACTGGTATTGTTT 350 NEOGEN10.MCOI.NTD(11) TGTTGGGTTTATATTTTTGTTTACTATTGGTGGTGTTACTGGTATAGTTT 350 NEOGEN15.I.MCOI.NTD(1) AATTGGATTTGTGTTTTTGTTTACGATTGGTGGGATTACTGGGGTTGTAT 350 NEOGEN15.II.MCOI.NTD(3) AATTGGATTTGTGTTTTTGTTTACGATTGGTGGGATTACTGGGGTTGTAT 350 NEOGEN12.MCOI.NTD(4) TTTAGGCTTTGTGTTTTTATTTACAATAGGTGGGGTTACTGGAATAGTTT 350 ** *** ***** *** ** ** ** ** ** NEOGEN8.I.MCOI.NTD(6) TGTCTGCGTCGGTTGTAGACTCTTTGTTTCATGATACATGATTTGTTGTG 365 NEOGEN8.II.MCOI.NTD(1) TGTCTGCGTCGGTTGTAGACTCTTTGTTTCATGATACATGATTTGTTGTG 365 NEOGEN8.III.MCOI.NTD(1) TGTCTGCGTCGGTTGTAGACTCTTTGTTTCATGATACATGATTTGTTGTG 365 NEOGEN1.MCOI.NTD(18) TATCTGCTTCGGTTATTGATTCTTTGTTTCATGATACTTGGTTTGTTGTT 400 NEOGEN17.MCOI.NTD(1) TGTCTGCTTCGGTTATTGATTCTTTGTTTCATGATACTTGATTTGTTGTT 359 NEOGEN7.MCOI.NTD(36) TGTCTGCTTCGGTTATTGATTCATTGTTTCATGATACTTGATTTGTTGTT 400 NEOGEN16.MCOI.NTD(3) TGTCTGCTT 311 NEOGEN4.MCOI.NTD(42) TGTCTGCTTCGATTATTGATTCGTTGTTTCATGATACTTGATTTGTTGTT 400 NEOGEN2.MCOI.NTD(13) TATCTGCTTCAGTGATTGATTCTTTGTTTCATGATACTTGATTTGTTGTT 400 NEOGEN3.I.MCOI.NTD(3) TATCTGCTTCGGTAGTTGATTCGTTATTTCATGATACTTGGTTTGTTGTT 400 NEOGEN3.II.MCOI.NTD(1) TATCTGCTTCGGTGGTTGATTCTTTATTTCATGATACTTGGTTTGTTGTT 400 NEOGEN6.MCOI.NTD(1) TATCTGCTTCTATTATTGATTCTTTG 376 NEOGEN19.MCOI.NTD(2) 324 NEOGEN18.MCOI.NTD(4) 324 NEOGEN5.I.MCOI.NTD(3) TATCTGCTTCGGTGGTTGATTCATTGTTTCATGATACTTGGTTTGTTGTT 400 NEOGEN5.II.MCOI.NTD(8) TATCTGCTTCGGTGGTTGATTCATTGTTTCATGATACTTGATTTGTTGTT 400 NEOGEN9.I.MCOI.NTD(9) TGTCTTCGTCTGTTTTAGATTCGTTATTTCATGATACTTGATTTGTTGTT 400 NEOGEN9.II.MCOI.NTD(2) TGTCTTCGTCTGTTTTAGATTCATTATTTCATGATACTTGATTTGTTGTT 400 NEOGEN11.MCOI.NTD(8) TGTCTTCGTCTGTTTTAGATTCGTTGTTTCATGATACTTGATTTGTTGTT 400 NEOGEN13.I.MCOI.NTD(2) TGTCTGCTTCAGTACTAGATT 371 NEOGEN13.II.MCOI.NTD(1) TGTCTGCTTCAGTGCTAGATT 371 NEOGEN14.I.MCOI.NTD(2) TATCTTCTTCTG 362 NEOGEN14.II.MCOI.NTD(3) TATCTTCTTCTG 362 NEOGEN10.MCOI.NTD(11) TGTCTGCTTCTGTTTTGGATTCATTATTTCATGATACTTGGTTTGTTGTT 400 NEOGEN15.I.MCOI.NTD(1) TGTCTGCTTCTGTATTAGATTCATTGTTTCATGATACTTGGTTTGTTGTT 400 NEOGEN15.II.MCOI.NTD(3) TGTCTGCTTCTGTATTAGATTCATTGTTTCATGATACTTGGTTTGTTGTT 400 NEOGEN12.MCOI.NTD(4) TATCTGCTTCTGTATTAGATTCTTTATTTCATGATACATGGTTTGTTGTC 400 *** ** ** ** ** *********** ** ******** Figure 3-12. Continued.

PAGE 188

188 NEOGEN8.I.MCOI.NTD(6) GC 367 NEOGEN8.II.MCOI.NTD(1) GC 367 NEOGEN8.III.MCOI.NTD(1) GC 367 NEOGEN1.MCOI.NTD(18) GCTCATTTTCATTATG 416 NEOGEN17.MCOI.NTD(1) GCTCATTTTCATTATG 375 NEOGEN7.MCOI.NTD(36) GCTCATTTTCATTATG 416 NEOGEN16.MCOI.NTD(3) 311 NEOGEN4.MCOI.NTD(42) GCTCATTTTCATTATG 416 NEOGEN2.MCOI.NTD(13) GCTCATTTTCATTATG 416 NEOGEN3.I.MCOI.NTD(3) GCTCATTTTCATTATG 416 NEOGEN3.II.MCOI.NTD(1) GCTCATTTTCATTATG 416 NEOGEN6.MCOI.NTD(1) 376 NEOGEN19.MCOI.NTD(2) 324 NEOGEN18.MCOI.NTD(4) 324 NEOGEN5.I.MCOI.NTD(3) GCTCATTTTCATTATG 416 NEOGEN5.II.MCOI.NTD(8) GCTCATTTTCATTATG 416 NEOGEN9.I.MCOI.NTD(9) GCTCATTTTCATTATG 416 NEOGEN9.II.MCOI.NTD(2) GCTCATTTTCATTATG 416 NEOGEN11.MCOI.NTD(8) GCTCATTTTCATTATG 416 NEOGEN13.I.MCOI.NTD(2) 371 NEOGEN13.II.MCOI.NTD(1) 371 NEOGEN14.I.MCOI.NTD(2) 362 NEOGEN14.II.MCOI.NTD(3) 362 NEOGEN10.MCOI.NTD(11) GCTCATTTTCATTATG 416 NEOGEN15.I.MCOI.NTD(1) GCTCATTTTCATTATG 416 NEOGEN15.II.MCOI.NTD(3) GCTCATTTTCATTATG 416 NEOGEN12.MCOI.NTD(4) GCTCATTTTCATTATG 416 **************** Figure 3-12. Continued.

PAGE 189

189NEOGEN18.MCOI.AA VYVLILPGFGMVSHICMTLSNNESLFGYFGLVCAMGAIVCLGSMVWA 47 NEOGEN19.MCOI.AA VYVLILPGFGMVSYICMTLSNSESLFGYFGLVCAMGAIVCLGSMVWA 47 NEOGEN3.MCOI.AA HPEVYVLILPGFGMVSHICMTLSNNESLFGYFGLVCAMGAMVCLGSMVWA 50 NEOGEN17.MCOI.AA ICMTLSNNESLFGYFGLVCAMGAIVCLGSMVWA 33 NEOGEN7.MCOI.AA HPEVYVLILPGFGMVSHICMTLSNNESLFGYFGLVCAMGAIVCLGSMVWA 50 NEOGEN4.MCOI.AA HPEVYVLILPGFGMVSHICMTLSNNESLFGYFGLVCAMGAIVCLGSMVWA 50 NEOGEN6.MCOI.AA HPEVYVLILPGFGMVSHICMTLSNNESLFGYFGLVCAMGAMVCLGSMVWA 50 NEOGEN1.MCOI.AA HPEVYVLILPGFGMVSHICMTLSNSESLFGYFGLVCAMGAMVCLGSMVWA 50 NEOGEN5.MCOI.AA HPEVYVLILPGFGMVSHICMTLSNNESLFGYFGLVCAMGAMVCLGSMVWA 50 NEOGEN2.MCOI.AA HPEVYVLILPGFGMVSHICMTLSNNESLFGYFGLVCAMGAIVCLGSMVWA 50 NEOGEN8.MCOI.AA HPEVYVLILPGFGMVSHMCMTLSNNESMFGYFGLVCAMGAIVCLGSMVWA 50 NEOGEN9.MCOI.AA HPEVYVLMIPGFGMVSHMCMVLSNSESVFGYFGLVCAMGAMVCLGSMVWA 50 NEOGEN11.MCOI.AA HPEVYVLMIPGFGMVSHMCMVLSNSESVFGYFGLVCAMGAMVCLGSMVWA 50 NEOGEN14.I.MCOI.AA HPEVYVLILPGFGMVSHICMVLSNNESVFGYFGLVCAMGAMVCLGSIVWA 50 NEOGEN14.II.MCOI.AA HPEVYVLILPGFGMISHICMVLSNNESVFGYFGLVCAMGAMVCLGSIVWA 50 NEOGEN10.MCOI.AA HPEVYVLILPGFGMVSHMCMVLSNNESVFGYFGLVCAMGAMVCLGSMVWA 50 NEOGEN12.MCOI.AA HPEVYVLILPGFGMVSHMCMVLSNSECVFGYFGLVCAMGSIVCLGSVVWA 50 NEOGEN13.MCOI.AA HPEVYVLILPGFGMVSHMCMVLSNNESVFGYFGLVCAMGAIVCLGSMVWA 50 NEOGEN15.MCOI.AA HPEVYVLILPGFGMVSHMCMTLSNSESVFGYFGLVCAMGAMVCLGSMVWA 50 *.***.*.:***********::*****:*** NEOGEN18.MCOI.AA HHMFVVGMDIKTAVFFSSVTMMIGIPTGMKVFSWLYMLGSSYVRLIDPVV 97 NEOGEN19.MCOI.AA HHMFVVGMDIKTAVFFSSVTMMIGIPTGMKVFSWLYMLGSSYVRLVDPVV 97 NEOGEN3.MCOI.AA HHMFVVGMDIKTAVFFSSVTMMIGIPTGMKVFSWLYMLGSSYVRLVDPVV 100 NEOGEN17.MCOI.AA HHMFVVGMDVKTAVFFSSVTMMIGIPTGMKVFSWLYMLGSSYVRLVDPVV 83 NEOGEN7.MCOI.AA HHMFVVGMDIKTAVFFSSVTMMIGIPTGMKVFSWLYMLGSSYVRLVDPVV 100 NEOGEN4.MCOI.AA HHMFVVGMDIKTAVFFSSVTMMIGIPTGMKVFSWLYMLGSSYVRLVDPVV 100 NEOGEN6.MCOI.AA HHMFVVGMDIKTAVFFSSVTMMIGIPTGMKVFSWLYMLGSSYVRLVDPVV 100 NEOGEN1.MCOI.AA HHMFVVGMDMKTAVFFSSVTMMIGIPTGMKVFSWLYMLGSSYVRLVDPVV 100 NEOGEN5.MCOI.AA HHMFVVGMDMKTAVFFSSVTMIIGIPTGMKVFSWLYMLGSSYVRLVDPVV 100 NEOGEN2.MCOI.AA HHMFVVGMDMKTAVFFSSVTMIIGIPTGMKVFSWLYMLGSSYVRLVDPVV 100 NEOGEN8.MCOI.AA HHMFVVGMDMKTAVFFSSVTMIIGIPTGIKVFSWLYMLGSSYVRLVDPVV 100 NEOGEN9.MCOI.AA HHMFVIGMDMKTAVFFSSVTMIIGVPTGMKVFSWLYMLGGSNVRLFDPVV 100 NEOGEN11.MCOI.AA HHMFVVGMDMKTAVFFSSVTMIIGVPTGMKVFSWLYMLGGSNVRLFDPVV 100 NEOGEN14.I.MCOI.AA HHMFVVGMDMKTAVFFSSVTMIMGVPTGMKVFSWLYMLGGSGVRLFDPVV 100 NEOGEN14.II.MCOI.AA HHMFVVGMDMKTAVFFSSVTMIMGVPTGMKVFSWLYMLGGSGVRLFDPVV 100 NEOGEN10.MCOI.AA HHMFVIGMDMKTAVFFSSVTMIIGMPTGIKVFSWLYMLGNSGVRLLDPVV 100 NEOGEN12.MCOI.AA HHMFVIGMDLKTAVFFSSVTMIMGVPTGIKVFSWLYMLGGSGVRVYDPVV 100 NEOGEN13.MCOI.AA HHMFVVGMDMKTAVFFSSVTMIIGIPTGMKVFSWLYMLGGSGVRLYDPVV 100 NEOGEN15.MCOI.AA HHMFVVGMDVKTAVFFSSVTMMIGMPTGMKVFSWLFMLGSSNIRVYDPMV 100 *****:*** ***********::*:***:******:***.* :*: **:* NEOGEN18.MCOI.AA WWIMGFIFLF 107 NEOGEN19.MCOI.AA WWIMGFIFLF 107 NEOGEN3.MCOI.AA WWIMGFIFLFTVGGVTGIVLSASVVDSLFHDTWFVVAHFHYV 142 NEOGEN17.MCOI.AA WWIVGFIFLFTMGGVTGIVLSASVIDSLFHDTWFVVAHFHY 124 NEOGEN7.MCOI.AA WWIMGFIFLFTVGGVTGIVLSASVIDSLFHDTWFVVAHFHYV 142 NEOGEN4.MCOI.AA WWIMGFIFLFTVGGVTGIVLSASIIDSLFHDTWFVVAHFHYV 142 NEOGEN6.MCOI.AA WWVMGFIFLFTMGGVTGIVLSASIIDSLFHDTWFVVAHFHYV 142 NEOGEN1.MCOI.AA WWIVGFIFLFTMGGVTGIVLSASVIDSLFHDTWFVVAHFHYV 142 NEOGEN5.MCOI.AA WWIMGFIFLFTMGGVTGIVLSASVVDSLFHDTWFVVAHFHYV 142 NEOGEN2.MCOI.AA WWIMGFIFLFTMGGVTGIVLSASVIDSLFHDTWFVVAHFHYV 142 NEOGEN8.MCOI.AA WWMMGFIFLFTVGGVTGMVLSASVVDSLFHDTWFVVAHFHYV 142 NEOGEN9.MCOI.AA WWIVGFMFLFTVGGVTGMVLSSSVLDSLFHDTWFVVAHFHYV 142 NEOGEN11.MCOI.AA WWIVGFMFLFTVGGVTGMVLSSSVLDSLFHDTWFVVAHFHYV 142 NEOGEN14.I.MCOI.AA WWVIGFMFLFVVGGVTGVVLSSSVLDSLFHDTWFVVAHFHYV 142 NEOGEN14.II.MCOI.AA WWVIGFMFLFVVGGVTGIVLSSSVLDSLFHDTWFVVAHFHYV 142 NEOGEN10.MCOI.AA WWVVGFMFLFTIGGVTGMVLSASVLDSLFHDTWFVVAHFHYV 142 NEOGEN12.MCOI.AA WWILGFVFLFTMGGVTGMVLSASVLDSLFHDTWFVVAHFHYV 142 NEOGEN13.MCOI.AA WWVIGFMFLFTIGGVTGMVLSASVLDSLFHDTWFVVAHFHYV 142 NEOGEN15.MCOI.AA WWMIGFVFLFTIGGITGVVLSASVLDSLFHDTWFVVAHFHYV 142 ** ** *** ** ** ** ***************** Figure 3-13. Alignment of partial predicte d amino acid sequences of the mitochondrial cytochrom oxidase I gene for Neospirorchis specimens. Shaded positions are differences between like genotypes based on predicted amino acid (mitochondrial cytochrome oxidase I) sequence and ribosomal ITS2 sequence.

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190 Figure 3-14. Gel electrophoresis of products obtained by PCR am plification of the ITS1. A forward primer within the 3 end of the 18s and a reverse primer within the ITS2 produced amplicons of multiple sizes. Side-b y-side lanes represent replicates of the same sample. Differences in band sizes are due to variable numbers of tandem repeats. With the exception of some varia tion in intensity, band patterns are nearly identical between like genotypes. Samples 1 and 2 = Neogen-9, Sample3 = Neogen10, Sample 4, 5, and 6 = Neogen-11.

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191 Figure 3-15. Bayesian phylogene tic tree of 270 to 306 nucleotides of the internal transcribed spacer region 2 of the ribosomal gene of Neospirorchis based on T-Coffee alignment. Bayesian posterior probabilities (italics) as percentages are given below branch nodes. Only branches with a probability of greater than 60% are shown. The ML bootstrap values are based on 200 re-samplings and are given above each node. Other marine spirorchiids, H. mistroides and L. learedi were used as an outgroup. Brackets indicate the anatomic locations from which specimens within groups were collected.

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192 Figure 3-16. Bayesian phylogene tic tree of 142 partial amino aci d sequence of the mitochondrial cytochrome oxidase I gene of Neospirorchis based on ClustalW alignment. Bayesian posterior probabilities (italics) as percen tages are given below branch nodes. Only branches with a probability of greater than 60% are shown. The ML bootstrap values are based on 100 re-samplings and are given above each node. Other marine spirorchiids, H. mistroides and L. learedi were used as an outgroup. The anatomic locations from which specimens within groups were collected are indicated by brackets.

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193 Table 3-1. Hapalotrema Learedius learedi and Carettacola bipora specimens from by locality, anatomic location, host species, sample number, and gene sequence obtained. Host ID Genus & species Locality Location Host No. ITS2 MCOI Hawaii 1-10 H. postorchis Hawaii Unknown CM 10 X X H.post.CR1-4 H. postorchis Caribbean Unknown CM 4 X 05-64 H. postorchis Florida Left aorta CM 7 X X 05-26 H. pambanensis Florida Heart CC 1 X X 06-72 H. pambanensis Florida Heart CC 2 X X 07-51 H. pambanensis Florida Liver CC 2 X X 07-51 H. pambanensis Florida Left aorta CC 1 X X 06-51 H. pambanensis Florida Heart CC 1 X X 05-45 H. mistroides Florida Left aorta CC 2 X X 05-45 H. mistroides Florida Mes. artery CC 1 X X 05-51 H. mistroides Florida Left aorta CC 1 X X 05-57 H. mistroides Florida Left aorta CC 1 X X 05-83 H. mistroides Florida Left aorta CC 1 X X 06-07 H. mistroides Florida Left aorta CC 1 X X 06-18 H. mistroides Florida Mes. artery CC 1 X X 07-13 H. mistroides Florida Mes. artery CC 2 X X 07-51 H. mistroides Florida Left aorta CC 1 X X 06-32 Hap. novel sp. Florida Liver CC 4 X X 07-45 L. learedi Florida Heart CM 10 X X 05-64 L. learedi Florida Heart CM 4 X X 08-96 L. learedi Florida Heart CM 10 X X Hawaii1-10 L. learedi Hawaii Heart CM 10 X X CTF1-10 L. learedi Caribbean Heart CM 10 X X L. lear.CR1 L. learedi Caribbean Heart CM 1 X 05-51 C. bipora Florida Liver CC 1 X 05-58 C. bipora Florida Liver CC 1 X 06-12 C. bipora Florida Multiple CC 3 X X

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194 Table 3-2. Internal transcribed sp acer 2 pairwise distances between Hapalotrema Learedius and Carettacola specimens. 1 2 3 4 5 6 7 1 H. post -A/C 2 H. post -HW 0.026 3 H. mistroides 0.053 0.053 4 H. pambanensis 0.154 0.154 0.165 5 Hap. novel sp. 0.139 0.147 0.143 0.158 6 L. learedi (varA) 0.143 0.135 0.147 0.113 0.128 7 L. learedi (varB) 0.147 0.139 0.147 0.113 0.128 0.008 8 C. bipora 0.335 0.327 0.338 0.323 0.316 0.327 0.335 Shaded cells reflect pairwise comparisons between species of different locality ( H. postorchis ) or cases in which variation in the ITS2 was observed ( L.learedi ).

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195Table 3-3. Mitochondrial cytochro me oxidase I gene and pairwise distances (nucleotide) between Hapalotrema Learedius and Carettacola specimens. 1 2 3 4 5 6 7 8 9 1 H. post -A/Ca 2 H. post -HW 0.082 3 H. mistroidesa 0.106 0.106 4 H. pambanensisa 0.170 0.146 0.169 5 Hap. novel sp.a 0.169 0.158 0.168 0.168 6 L. learedi (FL.varA) a 0.142 0.157 0.136 0.162 0.171 7 L. learedi (FL.varB) a 0.149 0.141 0.140 0.149 0.174 0.077 8 L. learedi (HW.varA) a 0.149 0.134 0.142 0.156 0.167 0.076 0.029 9 L. learedi (HW.varB) 0.149 0.150 0.144 0.152 0.174 0.073 0.046 0.045 10 C. bipora 0.196 0.202 0.172 0.222 0.207 0.186 0.179 0.182 0.193 aPairwise distances reflect an average of p-distances observed within the each designated type due to sequence variation. Shaded cells reflect pairwise comparisons between species of different locality ( H. postorchis ) or cases in which variation in the ITS2 was observed ( L.learedi ).

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196 Table 3-4. Neospirorchis specimens by host, anatomic location, sample type, gene sequence obtained, and genotype. SampleID Host species Location Sample type ITS2 MCOI Genotype Nervous system N05-31 CC Meninges Eggs X X NEOGEN-1 N05-44 CC Meninges Adult X X NEOGEN-1 N05-58 CC Meninges Eggs X NEOGEN-1 N05-81 CC Meninges Eggs X X NEOGEN-1 N05-83 CC Meninges Eggs X X NEOGEN-1 N05-89 CC Meninges Eggs X X NEOGEN-1 N06-07 CC Meninges Adult X X NEOGEN-1 N06-12 CC Meninges Adult X X NEOGEN-1 N06-20 CC Meninges Adult X X NEOGEN-1 N06-51 CC Meninges Adult X NEOGEN-1 N06-56 CC Meninges Adult X X NEOGEN-1 N07-46 CC Meninges Adult X X NEOGEN-1 CXO2006112401 CC Meninges Adult X X NEOGEN-1 NME2006090301 CC Meninges Adult X X NEOGEN-1 NMY2006101801 CC Meninges Eggs X NEOGEN-1 T5;6/20/06 CC Meninges Adult X X NEOGEN-1 T6;6/20/06 CC Meninges Adult X NEOGEN-1 T7;6/20/06 CC Meninges Adult X X NEOGEN-1 T8;6/20/06 CC Meninges Adult X X NEOGEN-1 N05-07 CC Meninges Adult X X NEOGEN-2 N05-45 (A) CC Meninges Adults & Eggs X X NEOGEN-2 N05-45 (B) CC Meninges Adult X NEOGEN-2 N05-45 (C) CC Meninges Eggs X NEOGEN-2 N05-57 CC Meninges Adults & Eggs X X NEOGEN-2 N05-63 CC Meninges Eggs X X NEOGEN-2 N06-18 CC Meninges Adult X X NEOGEN-2 N06-37 CC Meninges Adult X X NEOGEN-2 N06-44 CC Meninges Adult X X NEOGEN-2 N06-50 CC Meninges Adult X X NEOGEN-2 N06-53 CC Meninges Adult X X NEOGEN-2 T1;11/15/05 CC Meninges Adult X X NEOGEN-2 T1;6/20/06 CC Meninges Adult X X NEOGEN-2 T1;7/19/05 CC Meninges Adult X X NEOGEN-2 T2;11/15/05 CC Meninges Adult X NEOGEN-2 T4;11/15/05 CC Meninges Adult X X NEOGEN-2 N05-59 CM Meninges Adult X X NEOGEN-3 N05-64 CM Meninges Adult s & Eggs X X NEOGEN-3 N07-45 CM Meninges Adult X X NEOGEN-3 T9;6/20/06 CM Meninges Adult X X NEOGEN-3 BAO2006092101 CM Meninges Eggs X X NEOGEN-17 N06-68 CC Meninges Adult X NEOGEN-5

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197 Table 3-4. Continued. SampleID Host species Location Sample type ITS2 MCOI Genotype N05-91 CC Meninges Eggs X NEOGEN-8 N05-10 CC Meningesa Eggs X X Mixed N05-26 CC Meningesa Eggs X NEOGEN-4 N05-06 Meningesa Eggs X NEOGEN-8 MCK2006100801 LK Olfact. N Adult X X NEOGEN-1 AMF2006091101 CC Optic n. Adult X X NEOGEN-6 Endocrine organs GST0803 CM Thyroid Adult X X NEOGEN-7 GST0814 CM Thyroid Adult X X NEOGEN-7 GST0820 CM Thyroid Adult X X NEOGEN-7 GST0821 CM Thyroid Adult X X NEOGEN-7 GST0824 CM Thyroid Adult X X NEOGEN-7 GST0825 CM Thyroid Adult X X NEOGEN-7 GST0825 CM Thyroid Eggs X X NEOGEN-7 GST0828 CM Thyroid Eggs X X NEOGEN-7 GST0831 CM Thyroid Adult X X NEOGEN-7 N05-06 CC Thyroid Eggs X X NEOGEN-4 N05-26 CC Thyroid Eggs X X NEOGEN-4 N05-45 CC Thyroid Adults & Eggs X NEOGEN-4 N05-46 CC Thyroid Adult X X NEOGEN-4 N05-51 CC Thyroid Adult X X NEOGEN-4 N05-57 CC Thyroid Eggs X X NEOGEN-4 N06-13 CC Thyroid Adult X X NEOGEN-4 N06-13 CC Thyroid Eggs X NEOGEN-4 N06-18 CC Thyroid Eggs X X NEOGEN-4 N06-32 CC Thyroid Adult X X NEOGEN-4 N06-33 CC Thyroid Adult X X NEOGEN-4 N06-37 CC Thyroid Adult X X NEOGEN-4 N06-44 CC Thyroid Adult X X NEOGEN-4 N06-68 CC Thyroid Adult X X NEOGEN-4 N06-69 CC Thyroid Eggs X X NEOGEN-4 N06-81 CC Thyroid Eggs X X Mixed N07-13 CC Thyroid Adult X X NEOGEN-4 T1;7/19/05 CC Thyroid Adult X X NEOGEN-4 T1;7/19/05 CC Thyroid Eggs X NEOGEN-4 T2;7/19/05 CC Thyroid Eggs X X NEOGEN-4 T3;6/20/06 CC Thyroid Eggs X X NEOGEN-4 T4;11/15/05(A) CC Thyroid Eggs X X NEOGEN-4 T4;11/15/05(B) CC Thyroid Eggs X X NEOGEN-4 T5;11/15/05 CC Thyroid Adult X X NEOGEN-4 T5;6/20/06 CC Thyroid Eggs X X NEOGEN-4 T6;6/20/06 CC Thyroid Adult X X NEOGEN-4 N05-10 CC Thyroid Adult X NEOGEN-5 N05-31 CC Thyroid Eggs X X NEOGEN-5 N05-58 CC Thyroid Adult X X NEOGEN-5 N05-88 CC Thyroid Adult X X NEOGEN-5 N05-91 CC Thyroid Eggs X X NEOGEN-5

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198 Table 3-4. Continued. SampleID Host species Location Sample type ITS2 MCOI Genotype N06-12 CC Thyroid Adults & Eggs X X NEOGEN-5 N06-63 LK Thyroid Adults & Eggs X X NEOGEN-5 T1;11/15/05 CC Thyroid Adult X X NEOGEN-5 T1;6/20/06 CC Thyroid Eggs X NEOGEN-5 T7;6/20/06 CC Thyroid Adult X X NEOGEN-5 N05-83 CC Thyroid Eggs X NEOGEN-6 N05-48 CC Thyroid Eggs X X NEOGEN-6(99) N05-59 CM Thyroid Eggs X X NEOGEN-7 N05-64 CM Thyroid Eggs X X NEOGEN-7 N06-46 CM Thyroid Adult X X NEOGEN-7 N06-89 CM Thyroid Eggs X X NEOGEN-7 N05-07 CC Adrenal Adult X X NEOGEN-4 N05-46 CC Adrenal Adult X X NEOGEN-4 N06-13 CC Adrenal Adult X X NEOGEN-4 N06-37 CC Adrenal Adult X X NEOGEN-4 N06-56 CC Adrenal Adult X X NEOGEN-4 T4;6/20/06 CC Adrenal Adult X X NEOGEN-4 T5;6/20/06 CC Adrenal Adult X X NEOGEN-4 N05-58 CC Adrenal Adult X X NEOGEN-5 N06-12 CC Adrenal Adult X X NEOGEN-5 T7;6/20/06 CC Adrenal Adult X X NEOGEN-5 N05-49 CM Adrenal Eggs X X NEOGEN-7 N06-53 CC Pineal gland Eggs X X NEOGEN-4 N06-46 CM Pineal gland Eggs X X NEOGEN-7 RAB2006070901 CM Pineal gland Adult X X NEOGEN-7 T5;7/19/05 CM Pineal gland Eggs X X NEOGEN-7 Thymus N05-07 CC Thymus Eggs X X NEOGEN-4 N05-26 CC Thymus Eggs X X NEOGEN-4 N05-46 CC Thymus Adult X NEOGEN-4 N05-51 CC Thymus Eggs X X NEOGEN-4 N05-57 CC Thymus Eggs X X NEOGEN-4 N06-13 CC Thymus Eggs X X NEOGEN-4 N06-18 CC Thymus Adult X X NEOGEN-4 N06-32 CC Thymus Adult X X NEOGEN-4 N06-37 CC Thymus Eggs X X NEOGEN-4 N06-53 CC Thymus Eggs X X NEOGEN-4 N06-69 CC Thymus Eggs X X NEOGEN-4 T3;6/20/06 CC Thymus Eggs X X NEOGEN-4 N05-83 CC Thymus Eggs X X NEOGEN-6 Heart & Major arteries N05-10 (A) CC Heart Adult X X NEOGEN-8 N05-10 (B) CC Heart Adult X X NEOGEN-8 N06-12 CC Heart Adult X NEOGEN-8 N06-20 (A) CC Heart Adult X X NEOGEN-8 N06-20 (B) CC Heart Adult X X NEOGEN-8

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199 Table 3-4. Continued. SampleID Host species Location Sample type ITS2 MCOI Genotype N06-20 (C) CC Heart Adult X X NEOGEN-8 N06-51 (A) CC Heart Adult X X NEOGEN-8 N06-51 (B) CC Heart Adult X X NEOGEN-8 N07-51 CC Heart Adult X NEOGEN-8 N05-44 CC Artery Adult X X NEOGEN-8 N06-12 CC Artery Adult X NEOGEN-8 Gastrointestinal tract GST0822 CM Stomach Eggs X X NEOGEN-14 N06-06 CM Stomach Eggs X X NEOGEN-13 N07-45 CM Stomach Eggs X X NEOGEN-13 N06-21 CM Stomach Eggs X X NEOGEN-14 N06-89 CM Stomach Eggs X X NEOGEN-14 N07-65 CM Stomach Eggs X X NEOGEN-14 GST0805 CM Stomacha Eggs X X NEOGEN-18 GST0813 CM Stomacha Eggs X X NEOGEN-7 GST0820 CM Stomacha Eggs X X NEOGEN-7 GST0824 CM Stomacha Eggs X X NEOGEN-7 GST0827 CM Stomacha Eggs X X NEOGEN-7 N05-10 CC Intestine Adult X X NEOGEN-9 N05-10 (A) CC Intestine Eggs X NEOGEN-9 N05-10 (B) CC Intestine Eggs X NEOGEN-9 N05-26 CC Intestine Adult X X NEOGEN-9 N05-44 CC Intestine Adult X X NEOGEN-9 N05-90 CC Intestine Adult X X NEOGEN-9 N06-18 CC Intestine Adult X X NEOGEN-9 N06-26 LK Intestine Eggs X X NEOGEN-9 N06-51 CC Intestine Adult X NEOGEN-9 N06-53 CC Intestine Adult X X NEOGEN-9 N06-67 CC Intestine Eggs X X NEOGEN-9 N06-69 CC Intestine Eggs X X NEOGEN-9 N06-72 CC Intestine Adult X X NEOGEN-9 N07-13 CC Intestine Eggs X NEOGEN-9 T8;6/20/06 CC Intestine Adult X X NEOGEN-9 N05-31 CC Intestine Adult X X NEOGEN-10 N05-51 CC Intestine Adult X X NEOGEN-10 N05-57 CC Intestine Adult X X NEOGEN-10 N05-58 CC Intestine Adult X X NEOGEN-10 N06-12 CC Intestine Eggs X NEOGEN-10 N06-13 CC Intestine Adult X X NEOGEN-10 N06-33 CC Intestine Adult X X NEOGEN-10 N06-51 CC Intestine Adult X NEOGEN-10 N06-53 CC Intestine Adult X X NEOGEN-10 N06-68 CC Intestine Adult X X NEOGEN-10 N06-81 CC Intestine Adult X X NEOGEN-10 T2;6/20/06 CC Intestine Adult X X NEOGEN-10 N05-26 CC Intestine Adult X X NEOGEN-11 N05-30 CC Intestine Adult X X NEOGEN-11

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200 Table 3-4. Continued. SampleID Host species Location Sample type ITS2 MCOI Genotype N06-12 CC Intestine Adult X NEOGEN-11 N06-20 CC Intestine Eggs X NEOGEN-11 N06-32 CC Intestine Adult X X NEOGEN-11 N06-32 CC Intestine Adult X X NEOGEN-11 N06-37 CC Intestine Adult X X NEOGEN-11 N06-44 CC Intestine Adult X X NEOGEN-11 N06-81 CC Intestine Adult X X NEOGEN-11 T4;11/15/05 CC Intestine Adult X X NEOGEN-11 N05-30 CC Intestine Adult X X NEOGEN-12 N05-51 CC Intestine Adult X X NEOGEN-12 GST0810 CM Intestinea Eggs X X NEOGEN-7 GST0813 CM Intestinea Eggs X X NEOGEN-7 GST0814 CM Intestinea Eggs X X NEOGEN-7 GST0816 CM Intestinea Eggs X X NEOGEN-18 GST0821 CM Intestinea Eggs X X NEOGEN-7 GST0823 CM Intestinea Eggs X X NEOGEN-19 GST0825 CM Intestinea Eggs X X NEOGEN-7 GST0828 CM Intestinea Eggs X X NEOGEN-7 GST0831 CM Intestinea Eggs X X NEOGEN-7 N05-63 CC Intestinea Eggs X NEOGEN-5 N05-84 CC Intestinea Eggs X NEOGEN-5 N05-83 CC Intestinea Eggs X NEOGEN-6 N05-88 CC Intestinea Eggs X NEOGEN-6 N06-12 CC Intestinea Eggs X NEOGEN-10 N06-81 CC Intestinea Eggs X NEOGEN-9 N06-13 CC Colon Adult X X NEOGEN-12 N06-81 CC Colon Adult X X NEOGEN-12 N05-30 CC Colon Eggs X X NEOGEN-13 N05-57 CC Colon Eggs X X NEOGEN-13 N05-48 CC Colon* Eggs X NEOGEN-6(99) N05-30 CC Cloaca Eggs X NEOGEN-11 N06-20 CC Cloaca Eggs X X NEOGEN-15 N06-51 CC Cloaca Eggs X X NEOGEN-15 N06-53 CC Cloaca Eggs X X NEOGEN-15 N06-56 CC Cloaca Eggs X X NEOGEN-15 Lungs N06-06 CM Lunga Eggs X NEOGEN-16 GST0802 CM Lunga Eggs X X NEOGEN-7 GST0803 CM Lunga Eggs X X NEOGEN-7 GST0805 CM Lunga Eggs X X NEOGEN-18 GST0810 CM Lunga Eggs X X NEOGEN-7 GST0814 CM Lunga Eggs X X NEOGEN-7 GST0816 CM Lunga Eggs X X NEOGEN-18 GST0820 CM Lunga Eggs X X NEOGEN-7 GST0821 CM Lunga Eggs X X NEOGEN-7 GST0823 CM Lunga Eggs X X NEOGEN-19

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201 Table 3-4. Continued. SampleID Host species Location Sample type ITS2 MCOI Genotype GST0823 CM Lunga Eggs X X NEOGEN-19 GST0824 CM Lunga Eggs X X NEOGEN-7 GST0831 CM Lunga Eggs X X NEOGEN-7 N06-37 CC Lunga Eggs X NEOGEN-6 T5;6/20/06 CC Nasal mucosaa Eggs X X Mixed Testes N06-13 CC Testis Adults & Eggs X X NEOGEN-4 N06-68 CC Testis Eggs X X NEOGEN-4 Liver N06-13 (A) CC Liver Adult X X NEOGEN-6 N06-13 (B) CC Liver Adult X X NEOGEN-6 Urinary bladder N06-56 CC U. bladder Eggs X X NEOGEN-15 N05-45 CC U. bladdera Eggs X X NEOGEN-4 Voucher specimens N. schisosomatoides (A) CC Voucher Adult X X NEOGEN-1 N. schisosomatoides (B) CC Voucher Adult X X NEOGEN-1 aDerived from eggs with a diffusely embolized pattern

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202 Table 3-5. Neospirorchis genotypes by organ system and host species. Genotype Anatomic location(s) Host species No. parasite specimens No. turtle hosts NEOGEN-1 Meninges (n=20) Olfactory nerve (n=1) CC (n=20) LK (n=1) 21 21 NEOGEN-2 Meninges (n=16) CC 16 14 NEOGEN-3 Meninges (n=4) CM 4 4 NEOGEN-4 Thyroid gland (n=25) Thymus (n=12) Adrenal gland (n=7) Testis (n=2) Pineal gland (n=1) Meninges (n=1)a Urinary bladder (n=1)a CC 49 26 NEOGEN-5 Thyroid gland (n=10) Adrenal gland (n=3) Meninges (n=1) Intestine (2)* CC (n=15) LK (n=1) 16 13 NEOGEN-6 Thyroid gland (n=2) Liver (n=2) Optic nerve (n=1) Thymus (n=1) Intestine (n=2)a Colon (n=1)a Lung (n=1)a CC 10 6 NEOGEN-7 Thyroid gland (n=13) Pineal gland (n=3) Adrenal gland (n=1) Stomach (n=4)a Intestine (n=7)a Lung (n=8) a CM 36 19 NEOGEN-8 Heart (n=9) Major artery (n=2) Meninges (n=3)a CC 14 9 NEOGEN-9 Intestine (n=15) CC (n=12) LK (n=1) 15 13 NEOGEN-10 Intestine (n=13) CC 13 12 NEOGEN-11 Intestine (n=10) Cloaca (n=1) CC 11 9 NEOGEN-12 Intestine (n=2) Colon (n=2) CC 4 4 NEOGEN-13 Stomach (n=2) Colon (n=2) CC CM 4 4 NEOGEN-14 Stomach (n=4) CM 4 4 NEOGEN-15 Cloaca (n=4) Urinary bladder (n=1) CC 5 4 NEOGEN-16 Lung (n=1)* CM 1 1 NEOGEN-17 Meninges (n=1) CM 1 1 NEOGEN-18 Lung (n=2)a Stomach (n=1)a Intestine (n=1)a CM 4 2 NEOGEN-19 Lung (n=1)a Intestine (n=1)a CM 2 1 CC = Caretta caretta ; CM = Chelonia mydas ; LK = Lepidochelys kempi a Derived from eggs with a diffusely embolized pattern

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203 ITS2 Table 3-6. Mitochondrial cytochro me oxidase I gene and internal transcribed spa cer 2 pairwise distance s (nucleotide) between Neospirorchis specimens. Neogen 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 0.039 0.052 0.045 0.045 0.039 0.039 0.071 0.136 0.143 0.136 0.143 0.156 0.143 0.240 0.032 0.032 0.052 2 0.091 0.013 0.039 0.019 0.039 0.019 0.039 0.117 0.136 0.117 0.123 0.136 0.123 0.234 0.032 0.032 0.019 3 0.082 0.085 0.045 0.032 0.052 0.032 0.052 0.123 0.149 0.123 0.130 0.136 0.123 0.234 0.032 0.045 0.019 4 0.091 0.072 0.080 0.045 0.058 0.032 0.071 0.136 0.162 0.136 0.143 0.162 0.149 0.240 0.045 0.052 0.039 5 0.078 0.062 0.045 0.072 0.045 0.026 0.058 0.123 0.156 0.123 0.130 0.143 0.130 0.240 0.039 0.039 0.039 6 0.058 0.098 0.082 0.080 0.069 0.039 0.065 0.143 0.149 0.143 0.156 0.162 0.149 0.247 0.032 0.032 0.052 7 0.098 0.065 0.078 0.062 0.083 0.091 0.058 0.136 0.149 0.136 0.130 0.156 0.143 0.240 0.032 0.032 0.026 8 0.094 0.081 0.117 0.095 0.103 0.105 0.106 0.130 0.136 0.130 0.143 0.123 0.110 0.227 0.071 0.071 0.058 9 0.123 0.141 0.147 0.156 0.127 0.138 0.156 0.141 0.091 0.013 0.091 0.097 0.097 0.169 0.143 0.149 0.130 10 0.145 0.149 0.156 0.174 0.141 0.141 0.167 0.164 0.116 0.091 0.091 0.117 0.130 0.162 0.156 0.156 0.149 11 0.120 0.138 0.149 0.152 0.134 0.138 0.141 0.138 0.040 0.116 0.091 0.097 0.097 0.169 0.143 0.149 0.130 12 0.203 0.188 0.203 0.203 0.197 0.188 0.207 0.203 0.178 0.196 0.178 0.117 0.130 0.188 0.149 0.149 0.130 13 0.134 0.141 0.141 0.138 0.125 0.127 0.138 0.145 0.109 0.127 0.101 0.188 0.026 0.188 0.156 0.169 0.143 14 0.136 0.145 0.154 0.154 0.127 0.114 0.165 0.159 0.114 0.134 0.121 0.185 0.109 0.201 0.143 0.156 0.130 15 0.129 0.161 0.152 0.168 0.154 0.132 0.161 0.165 0.138 0.163 0.136 0.183 0.178 0.176 0.247 0.260 0.227 16 0.026 0.032 17 0.062 0.080 0.101 0.091 0.089 0.069 0.094 0.098 0.134 0.167 0.138 0.181 0.145 0.147 0.143 0.045 18 0.087 0.051 0.072 0.054 0.053 0.083 0.065 0.095 0.123 0.149 0.134 0.210 0.134 0.129 0.156 0.076 19 0.101 0.087 0.072 0.094 0.074 0.087 0.094 0.132 0.149 0.167 0.159 0.196 0.145 0.165 0.139 0.098 0.072 Mitochondrial cytochrome oxidase I *Shaded areas reflect distances of less than 0.1

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204 CHAPTER 4 MOLECULAR DETECTION OF MARINE SPIRORCHIIDS IN INTERMEDIATE HOSTS AND APPLICATION IN LIFE CYCLE STUDIES Introduction Ten genera and more than twenty different sp ecies of spirorchiids have been described in sea turtles; however, all of the life cycles remain unknown. Knowledge of parasite life cycles is essential to the understanding of virtually ever y aspect of host-parasi te interaction, including epidemiology of infections and influences on pr evalence and disease in the host. Three major issues complicate discovery of sp irorchiid life cycles: the diversity of habitats utilized by sea turtles, the diversity of gastropods within thos e habitats, and the probability of low prevalence among intermediate hosts. In addition, most pa rasites have aggregated or overdispersed distribution patterns within host populations (Rhode, 1993). Thus, concentrations of infected intermediate hosts can be missed during field sampling. There have been only two reported attempts to discover the life cycles of marine spirorchiids, both of which were unsuccessful (Greiner et al., 1980; Dailey et al., 1992) Although the life cycles of ma rine spirorchiids are unknown, major characteristics can be reasonably inferred from the known life cycles of other blood flukes, including spirorchiids of freshwater turtles. Both schi stosomes and freshwater spiror chiids utilize a single gastropod intermediate host and infect the definitive host by penetrating the skin or mucous membranes. None of the blood flukes studied t hus far infect their vertebrate hosts by in gestion. Infectious cercariae are short-lived and ar e not powerful swimmers, thus th e definitive and intermediate hosts likely must be in relative ly close proximity for transmission to occur. In addition, there is data to support that infection by at least some marine spirorchiids occurs in near shore habitat. Infections have been documented in captive-reared turtles at a coastal rehabilitation facility in

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205 Florida (L. Herbst, personal communication) an d at captive-breeding facility on Grand Cayman Island (Greiner et al., 1980). Also, serological evidence of exposure has been observed in captive C. mydas in Hawaii that were transferred into co astal seawater ponds (Work et al., 2005). Lastly, spirorchiid infections have not b een observed in necropsied pelagic phase C. caretta recovered in the Canary Islands, which are immature animals of th e Atlantic population (Oros et al., 2005). Thus, it is hypothesized that infection occurs afte r recruitment into the neritic habitat based on these observations Classic parasitology methods rely on monitori ng individual snails for cercarial emergence and microdissection to detect trematode infections. These methods are time consuming, do not detect early infections, and requi re that investigators sort thro ugh the many digenean taxa that may infect gastropods. With no advance knowledge to focus surveys, the effort required to investigate life cycles in the marine system by classical methods is daunting in terms of the number of different marine gastropod species and the numbers of individuals that must be screened. A molecular approach to spir orchiid life cycle investigation offers many potential advantages, including high thr oughput screening of large num bers of gastropods, greater sensitivity, and specificity. Furthermore, deve loping digeneans can be specifically identified using the appropriate comparativ e genetic data. The life cycle investigation aspect of these studies included the following objectives: 1) develop a PCR method for sensitive and specific detection of spirorchiid trematodes known to infect Florida sea turtles; 2) adaptation this method into a technique for screening gastropods using a laboratory model; 3) screen gastropods for spirorchiids at two f acilities where captive C. mydas are known acquire infections; 4) examine

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206 gastropods found in seagrass beds for spirorchiid and non-spirorchiid trematode parasites of wild sea turtles. With regard to the fourth objective, our hypot hesis that sea turtle parasite transmission, including infection by some spirorchiids, occu rs in seagrass beds is based on documented infections at one coastal facili ty and ecological features of se agrass habitat. First, captivehatched C. mydas were observed to become infected by Neospirorchis sp. at a rehabilitation facility in which the predominant gastropod ha bitat in the adjoining waters is seagrass, suggesting that the intermediate host may be a sp ecies found in seagrass beds. Second, seagrass beds are important foraging grounds for both C. caretta and C. mydas are globally distributed, and are very common coastal habita t in tropical and subtropical z ones. In addition, seagrass beds are dominated by a relatively small number of gastropod species in terms of biomass, and many of these species are found in high density (Fra nkovich and Zieman, 2005). Sea turtles spend time in reasonable proximity to these organisms wh ile foraging, and thus could become infected. Furthermore, C. mydas maintain distinct grazing plots wher e they may repeatedly encounter the same gastropod populations. The density of many gastropod populations, the relatively shallow depths of most seagrass beds, a nd importance of these areas to s ea turtles make this habitat a seemingly ideal location for parasite transmission. The findings of this study incl ude the following: a method for molecular screening of large numbers of gastropods for trematode infections, with many potential appl ications in trematode life cycle studies; tentative iden tification of a limpet species, Fissurella nodosa, as an intermediate host of the marine spirorchiid Learedius learedi ; and evidence that Modulus modulus a gastropod species commonly found in sea grass, is the intermed iate host of one, possibly two, alimentary trematode parasites of C. mydas These findings are the first significant

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207 advance in the elucidation of the life cycles of trematode parasites of sea turtles and hopefully will provide the means for the necessary confirmatory studies and discovery efforts in other regions. Methods and Materials Gastropod DNA Extraction Multiple methods of tissue lysis and DNA extr action were tested to devise a method by which DNA could be extracted fr om relatively large quantities of gastropod tissue. Albino Pomacea bridgesii were used to test extraction methods so that pigment would not interfere with spectrophotometry. The protocol th at best fulfilled our requirements in terms of sample volume, available equipment, DNA yield, and purity began with removal of gastr opods from the shell and separation of the hepatopancreas and gonad. Only these organs we re included in the DNA extraction, with the exception very small gastropods (less than 3 mm in greatest dimension) in which all soft tissue was processe d. Gastropod tissues were combin ed to a total wet weight of 1.5 grams. Wet weight rather than individual numbers defined methods so that the technique would be applicable to gastropods of differe nt sizes. The complete tissue lysis and DNA extraction protocol is given in Table 4-1. The lysis buffer wa s modified from Winnepenninckx et al. (1993) (Table 4-2). Incl uded in this protocol is an ad ditional step to remove melanin, which is abundant in the hepatopancreas and co-purifies with DNA. Multiple methods, including serial dilutions of DNA, were tested and the best re sult, in terms of PCR amplification and signal strength, was obtained by using the QIAquik PCR purification kit (Qiagen) for removal of pigment prior to PCR. Polymerase Chain Reaction Protoc ol for Detection of Trematode DNA Detection of trematode DNA was performe d using polymerase chain reaction (PCR) targeting the internal transcribed spacer 2 (ITS2) of the ribosomal gene. This target was selected

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208 because the ribosomal gene is present in the ge nome as a series of numerous repeats and thus supports sensitive detection. In addition, there is sufficient variation in the ITS2 to specific amplification. Several primers were designed an d tested using different primer combinations, magnesium concentrations, and reacti on conditions. Serial dilutions of Neospirorchis pricei and Hapalotrema mistroides DNA were prepared for testing of sensitivity. The DNA extracted from a non-spirorchiid digenean, Calycodes anthos initially was used to asse ss specificity. Given that the intended use of this protocol was life cycle discovery, it was anticipated that all amplicons would be sequenced, thus further ad dressing any issues of specificity. Four primers ultimately were selected for use in detection studies. The forward primer SPIR1 (5-GAGGGTCGGCTTATTATCTATCA-3) and outer reverse primer SPIR2 (5TCACATCTGATCCGAGGTCA-3) were consensus prim ers complementary to the 3 end of the 18s gene and 5-end of the 28s gene, respect fully. These primers amplify the ITS2 of a diverse variety of digenean trematodes. Two sp irorchiid-specific reverse primers were designed for use with SPIR1 that were complimentary to ar eas within the ITS2. The first reverse primer was NCS2 (5-CATTGAGCCACGACAAGG-3), which is complementary to the ITS2 of all Neospirorchis species and genotypes identified from Flor ida turtles to date. The second reverse primer, HLC4 (5-GCAGCAACTCAACCTGRTAAACC-3 ), was designed to amplify the ITS2 of Hapalotrema species known to infect Florida turtles ( H. mistroides H. postorchis and H. pambanensis ) and Learedius learedi. Initially, the SPIR1 and NS C2, and SPIR1 and HLC4 were used in two reactions for the det ection of spirorchiids. In later field stud ies conducted on Grand Cayman Island, the Marquesas Keys, and Florida Bay, a hemi-nested application was adopted to detect other trematode infecti ons, in addition to spirorchiids. This hemi-nested technique included initial amplification of trematode IT S2 using the SPIR1 and SPIR2, followed by two

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209 reactions using SPIR1 and the two specific revers e primers, NSC2 and HLC4. The Taqman PCR kit (Qiagen) was used for all reac tions, which were performed in a 20 l reaction volume according to standard protocol. The mixtures we re amplified in a thermal cycler (PCR Sprint, Thermo Hybaid, Franklin, MA). Reaction conditions for the first reaction included initial denaturation at 95C for 5 min, then 45 cycles of denaturation at 95C fo r 60 s; annealing at 50C for 45 s, and DNA extension at 72C for 120 s, followed by a final extension step at 72C for 10 min (Table 4-3) Similar conditions were used for the second reactions, except a higher annealing temperature of 56 C was used (Table 4-3). The PCR products were resolved in 1% agaros e gels and direct seque ncing identified all bands of interest. The expected amplicon size for the spirorchiid-specific primers was 180 to 225 base pairs. Amplicons produced by the cons erved primers typically were between 300 and 500 base pairs. The bands were excised and purified using the QIAquick gel extraction kit (Qiagen). Direct sequencing was performed using the Big-Dye Terminator Kit (Perkin-Elmer, Branchburg, NJ) and analyzed on ABI 377 auto mated DNA sequencers at the University of Florida Center for Mammalian Gene tics DNA Sequencing Facilities. Detection of Spirorchiids in Gastropods The threshold of detection for the gastropod DNA extraction and PCR technique was assessed by spiking P. bridgesii total extracts with known quant ities of spirorchiid DNA. Detection was measured using embryonated eggs as biological units. Eggs were obtained from the tissues of dead stranded turtles and counted into a 1.5 ml tube using a dissecting microscope and microcapillary tubes. To insure rupture of eggs and complete DNA extraction, eggs were placed into 180 l of deionized water, frozen at -80 C, thawed, and then sonicated for 1 minute. These steps were repeated three times, which re sulted in obvious rupture of approximately 90% of the eggs. Therefore, a 10% error rate wa s considered in egg DNA extractions and 110 eggs

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210 were included to obtain DNA from a target of 100. After sonication, DNA was extracted using the DNeasy kit. To obtain the desired volume needed to spike P. bridgesii extracts, the total elution volume was then divided by the number of eggs extracted (100). Using this technique, gastropod DNA was spiked with one to ten egg equivalents of spirorchiid DNA. For positive controls, one complete adult spirorchiid ( L. learedi or Neospirorchis sp.) was combined with 1.5 gm of snail tissues and extrac ted using the gastropod protocol. Field Studies: Gastropod Collections and Synopsis of Study Sites Gastropods were collected from four study sites: 1) coastal habitat adjoining The Turtle Hospital (TH), Marathon Florida; 2) coastal habi tat adjoining the Cayman Turtle Farm, Limited (CTFL), Grand Cayman Island, British West Indies ; 3) the Marquesas Keys region (MK); and 4) Florida Bay (FB). The Marine Conservation Board (Grand Cayman), the Key West National Wildlife Refuge (Marquesas Keys), and the Ever glades National Park (Florida Bay) provided permits for collections. All co llections were performed by snor keling or under SCUBA. Most of the collected gastropods were stored fro zen at -80C until extrac tion of DNA and PCR were performed. A small subset of gastropods was examined for trematode infection by microdissection. Preparation of gastropods and DNA extraction was performed in a separate laboratory from PCR. Positiv e controls included DNA from H. mistroides and Neospirorchis species (30 fg of template DNA). Any suspected positive results were re-examined by repeating the pigment removal step (QIAquick spin column ) using an additional aliquot of the original DNA extract and PCR. As in the model system, all amplicons of interest were sequenced for confirmation. Sequences were then compar ed with spirorchiid ITS2 sequences of Neospirorchis, Hapalotrema Learedius and Carettacola ITS2 sequences obtained from Florida turtles and from a limited set of ITS2 sequences available from non-spirorchiid trematode parasites of sea turtles. In addition, sequences were compared to those in the database s of GenBank (National

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211 Center for Biotechnology Information, Bethesda MD), EMBL (Cambridge, UK), and the Data Bank of Japan (Mishima, Shiuoka, Japan) using BLASTN (Altschul et al., 1990). The Turtle Hospital, Marathon Key, Florida The Turtle Hospital is on the Florida Bay side (north shore) of Mara thon Key (Figure 4-1). Much of the adjoining marine habitat is seagrass be ds with a depth of 1 to 2 meters. Sea turtles are brought to the TH for rehabilitation and hospi tal tanks utilize a simple flow-through system whereby seawater is pumped from the adjacent shallow inshore area. As previously mentioned, infection of captive-hatched C. mydas by Neospirorchis sp. has been was documented at the TH (L. Herbst, personal communication), which supports that the intermediate host occurs in the local habitat. Cayman Turtle Farm, Limite d (CTFL), Grand Cayman Island, British West Indies As of 2006, The CTFL has a population of approximately 10,000 C. mydas The facility has a flow-through filtration syst em whereby large volumes of seawater are pumped into the tanks via an open intake channel and the unproces sed effluent is discha rged approximately 215 meters from the intake area (Figure 4-2). The coastal habitat consists of a limestone shore (ironshore) and hard bottom that descends relati vely rapidly to a mini-wall (15 to 18 meters deep) and then to a shear main wall. The irons tone shore includes abundant tide pools (Figure 43) and the subtidal zone is extensively pocketed by burrows of rock-boring urchins ( Echinometra sp.) (Figure 4-4). The seafloor is relatively barren from the shore to the coral reef associated with mini-wall and main wall. Marquesas Keys region The Marquesas study site consiste d of two genera l areas, a large C. mydas grazing area located 2-4 kilometers west of actual islands, and the shallower Mooney Harbor lagoon within the island group and near shore zone on the outer southwestern-most islands (Figure 4-5). The

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212 grazing plots were identified by observing for grazi ng turtles, investigating lighter areas within seagrass beds visible from the surface, or by snorkeling (Figure 4-6). The typical grazing area was 3 to 4 m deep and tidal current were variab ly intense. The lagoon and near shore collection sites most often were less 1.5 m deep. Both C. caretta and smaller immature C. mydas are frequently observed in the lagoon and near shore areas. Florida Bay, Everglades National Park The Florida Bay sites were within the area surveyed during annual C. caretta captures conducted by the Florida Fish and Wildlife Comm ission (FWC) in partnership with the National Marine Fisheries Service (NM FS). Based on these studies, C. caretta are believed to relatively abundant in this part of Florid a Bay. The collection area in cluded Arsniker Basin, Rabbit Key Basin, and Twin Key Basin (Fig 4-7). Field Studies: Ancillary Data Cayman Turtle Farm, Limited Prior to gastropod collections, an advance site visit was conducted in December 2005 to obtain current prevalence data from C. mydas harvested for human consumption. The heart and major vessels of 30 turtles were examined for the presence of L. learedi All chambers were opened and the blood was washed into a #45 seive. Adults were observed using a dissecting microscope and counted. Fecal examinations were performed on all 30 tu rtles using a standard detergent/sedimentation technique. In addition, the mucosa of re presentative sections of the intestine was examined for 22 of the 30 turtles. Lastly, fecal examinations were performed on the single resident C. caretta on multiple occasions in 12/2005, 7/2007, and 4/2008. During gastropod collections in October 2006 and July 2007, the seawater coming into the CTFL via the intake channel was sampled usi ng a vertical tow plankton net (Wildco) with a 154 m mesh catch bucket. The net was suspended in middle of the channel approximately 0.3

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213 m from the surface and initially was emptied every twelve hours. Filtrates were examined for cercariae using a dissecting microscope. To minimize the filtrate volume and improve the viability of captured microorganisms, filtrates also were examined at three or four hour intervals on multiple occasions. Following examination, the remaining filtrate was frozen and transported to the University of Florida where DNA was extract ed using the gastropod protocol and screened for the presence of L. learedi by PCR. Marquesas Keys Free-floating fecal samples were obtained from foraging C. mydas during all four gastropod collecting trips to the Marquesas grazing area. Fecal samples were consistent with that of C. mydas based on appearance and frequent a ssociation with feeding turtles. Parasitological examinations were performed using standard floata tion and sedimentation techniques. Results Method for Screening Gastropods for Spirorchiid Trematodes by PCR By testing serial dilutions of spirorchiid DNA, the PCR protocol using the SPIR1/NSC2 and SPIR1/HLC4 primer combinations in two re actions was found to detect spirorchiid DNA in template quantities as low as a few femtograms, which equates to the level of a single copy. A magnesium concentration of 2.5 mM and the previously descri bed reaction conditions produced the greatest sensitivit y and did not detect C. anthos Although there is in evitable error in this estimation of analytical sensitivity, this th reshold of detection, although approximated, was decided to be suitable for us e in the gastropod model. The gastropod DNA extracti on technique using albino P. bridgesii yielded between 1.85 to 1.87 mg of DNA from 1.5 grams of starting material. Spectrophotometric analyses of 100fold dilutions of DNA yielded 260/280 ratios of 1.73 to 1.76, indicating removal of most

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214 contaminants. Thus, the purity of the extract ed DNA using this method was appropriate for use in PCR. Detection of L. learedi ITS2 was attempted using the primers SPIR1/HLC4 and several different quantities of template DNA (10, 50, 100, 500, and 1000 ng) from snail tissues that were extracted with a single adult L. learedi Snail tissues spiked with the L. learedi yielded single bright bands using all template DNA quantities, whereas P. bridgesii tissues that were not spiked were PCR negative. Based on these results, 100 ng of was selecting as the amount of template DNA used in each PCR reacti on. Further testing of P. bridgesii spiked with various amounts of DNA from embryonated eggs found that as little as one egg equivale nt could be detected using the DNA extraction protocol and SPIR1/HLC4 a nd SPIR1/NCS2 primer pairs (Figure 4-8). Furthermore, a brighter amplicon was detected wi th the use of the Qiagen PCR clean-up kit to remove melanin from wild-type P. bridgesii DNA, as compared to simply diluting the DNA (Figure 4-8). This level of sensitivity is compar able the earliest possible stage of infection in gastropods. Given host tissues of infected gast ropods often are severely effaced by developing stages of trematodes, this level of detection was deemed suitable for application in field studies. In addition, sensitivity was further increased when the consen sus primers (SPIR1 and SPIR2) were used in an initial reaction, followed by he mi-nested application of SPIR1 and the specific reverse primers. Field Studies The Turtle Hospital, Marathon, Florida Gastropod collections were performed at Th e Turtle Hospital (TH) in June and August 2005 and 2006. All of the species collected were common inhabitants of seagrass beds (Table 44). No evidence spirorchiid infection was found in any of the 2,100 gastropods examined.

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215 Cayman Turtle Farm, Limi ted, Grand Cayman Island, British West Indies In advance of the life cycle studies, the prevalence of L. learedi in examined harvested C. mydas was found to be very high. All 30 turtles examined had either adult L. learedi in the cardiovascular system (29/30) or embolized eggs visible in the enteri c submucosa (20/22). Numbers of adult L. learedi ranged from one to 57, with an average of 12.3 per turtle; however, L.learedi eggs were observed in the feces of only 10% (3/30) of infected turtles. Similarly, egg numbers in examined submucosal samples also were very small. The single resident C. caretta at the facility had negative fecal results on all three instances (2005, 2006, and 2007) in which samples were collected. Gastropods were collected at the CTFL dur ing October 2006, July 2007, and April 2008. A list of collected species is gi ven in Table 4-5. A variety of tidal, intertidal, and subtidal species were examined. Almost all gastropods were collected within 5 mete rs of the shoreline; however, the surveyed habitat extended from the tidal pools within the iron shore to the nearshore mini-wall to the reef system of the upper main wall with a depth of approximately 15 to 18 meters. A single night collection was performed, but did yield si gnificantly different species from daytime collecti ons, with the exception of two Cowry species. A total of 3,883 gastropods from the CTFL were screened by PCR. A single positive PCR result was obtained from a limpet species, Fissurella nodosa, collected in July 2007 (Figure 4-9). Multiple amplicons from three separate PCR runs were sequenced and identified as L. learedi ITS2. The positive result was from a pooled sample of thirteen individual limpets. A total of 550 F. nodosa were examined, including 413 that were collected during April 2008 in efforts targeting this species after the original positive result was obtained (Figure 4-10). No additional positive results were detected. Cercarial development was observed in 2.3% (10/438) of F. nodosa examined by microdissection, which include d 25 individuals in July 2007 and 413 from

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216 April 2008. All of these cercari ae were cotylomicrocercous and sequence obtained using the consensus trematode primers indicated greate st homology with an opocoelid species (82% identify), which is consistent with the obs erved cercarial morphology. No cercariae were observed that had morphology consiste nt with developing blood flukes. Of the 528 gastropods of other species examined by microdissection, cercarial development was only observed in four (0.76%). Infected species included Fissurella barbadensis Diodora listeri Nodolittorina dilatata and Cenchritas muricatus. None of these cercariae exhibited morphology consistent with sp irorchiids. Additional trematode infections (unidentified taxa) were detected in Nodilittorina augustior, Nodilittorina mespillum Nerita peloronta and Cerithium littoratum by consensus PCR. All plankton net filtrates te sted by PCR were positive for L. learedi No free-swimming cercariae were observed in the plankton net filtra tes; however, the myriad of different organisms made examination for soft-bodied forms, such as cercariae, very difficult to detect. There is considerable mixing of facility e ffluent with seawater in the near shore zone. The effluent plume occasionally was observed to flow back directly into the intake channel. Although plankton net collections were not performed during these times the positive PCR result from intake filtrates may reflect a combination of parasite material in the effluent, as well as any cercariae being discharged from infected intermediate hosts. Marquesas Keys Four gastropod collecting tr ips were conducted in the Marq uesas Keys region August and December 2007 and March and June 2008. A total of 45 fecal samples were collected from the area. Results of fecal examinations are given in Table 4-6. Some of these samples may be from the same turtles given the collection technique, thus these results should be regarded as indication of the species of pa rasites present and do not suppor t any extrapolation regarding

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217 frequency or prevalence. Ov a of two spirorchiid genera, Learedius and Neospirorchis were detected as were ova of the non-spirorchiid digenean genera Rhytidodes Deuterobaris Polyangium and Schizamphistomoides Some unidentified immature trematodes and ova also were observed. The abundance of gastropods was relatively low in the grazing areas, which had a depth of 3 to 4 meters and periods of intense tidal currents. As a re sult of these conditions and lower abundance, proportionately more effort was expe nded in the collections from grazing plots despite the lower number of gast ropods collected relative to M ooney Harbor and shallower near shore zones. Both C. mydas and C. caretta were frequently observed during collections. Of the 295 gastropods examined by microdissection (T able 4-7), developing trematodes were only observed in one snail ( Cerithium species). These microdissections included the four individuals of Patelloidea pustulata the only limpet species observed. A list of gastropods collected and screened by PCR is shown in Table 4-8. None of the samples tested were positive by PCR for the pr esence of spirorchiids. Select amplicons produced by the first round PCR (consensus pr imers) were sequenced when present and interpretable sequence was obtained for 29 samples, which included pooled tissues of the species Modulus modulus Columbella mercatoria Columbella rusticoides Lithopoma americanum Cerithium atratum and Crepidula species (Figure 4-11). Th ree different samples of M. modulus collected from Mooney Harbor yielded ITS2 sequence that was 100% homologous with Angiodictyum parallelum an alimentary trematode found in C. mydas In addition, another ITS2 sequence was obtained from a sample of M. modulus which included individuals collected from a grazing plot. This second sequence shared 92 % identity with an unidentified pronocephalid

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218 species collected from the stomach of a C. mydas None of the remaining sequences could be matched to specific digenean ITS sequen ces available in the public databases. Florida Bay Gastropods were collected from seagrass beds in Florida Bay in October 2007. A list of species collected and screened by PCR is given in Table 4-9. No evidence of spirorchiid infection was detected by PCR. As for the Marq uesas samples, select amplicons were sequenced for bands obtained using consensus trematode prim ers. Interpretable sequence was obtained for twelve sample batches, that included Columbella mercatoria Columbella rusticoides Tegula fasciata Cerithium eburneum, and Lithopoma americanum None of the sequences matched or were similar to available digenean sequences fr om sea turtles. Of the specimens collected, 295 also were examined by microdissection (Table 4-7). Only one Cerithium species had cercarial development, the morphology of which was not cons istent with a spirorchiid and ITS2 sequence matched that of trematodes detected in other Cerithium including ceracaria recovered from a single specimen from the Marquesas region. Discussion The utility of the DNA extraction and PCR method developed for high throughput screening of gastropods was evidenced by successful detection of digenean infections in many of the species examined. Ideally, gastropods e xposed to infectious miracidia with known time points of infection would used to define sensi tivity; however, this was not possible given that none of the life cycles had been discovered for any digenean parasites of sea turtles. The use of a non-target gastropod species, a cap tive-propagated freshwater species in this study, spiked with parasite material was a useful surrogate for vali dating the technique. The threshold of detection was found to be equivalent to the earliest possi ble prepatent infection based on the ability to detect a single egg equivalent in 1.5 grams of host tissue. Although this measure of sensitivity is

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219 only an approximation, it is more than adequate for applications in life cycle discovery. Naturally infected gastropods likely will include i ndividuals in which host organs are extensively effaced by developing parasites; therefore, the am ount of parasite materi al present will exceed that of an embryonated egg by many orders of magnitude. Furthermore, the ribosomal gene, including the ITS targeted by the PCR technique, is present in the genome as numerous tandem repeats. Thus, each parasite cell contai ns many copies for detection by PCR. The detection L. learedi in the limpet F. nodosa is the first evidence of the identity of an intermediate host of a marine spirorchiid; however confirmatory studies are necessary. Efforts to observe spirorchiid cercarial development in additional specimens to support the molecular findings were unsuccessful, despite the gross examination of over 400 individual F. nodosa. A total of 137 F. nodosa were screened in the initial collection that included the positive sample, thus prevalence in the collected limpets was as low as less than 1% (assuming only one limpet was positive in the pooled sample). This finding is not inconsistent with expected prevalence in intermediate host populations. Pr evalence can be very low in in termediate host populations and result in high prevalence in definitive hosts due to high fecundity of asexual stages, i.e. abundant production of cercaria, as well as prolonged survival of infected intermediate hosts. Holliman (1971) reported a prevalence of 0.11% for the freshwater spirorchiid Spirorchis parvus and hypothesized that high cercarial out put rather than abundance of in fected snails maintains this spirorchiid life-cycle. Gastropod hosts of fres hwater spirorchiids have been documented to produce as many as 100 cercariae per day and li ve as long as several months (Goodchild and Fried, 1963; Holliman, 1971; Peiper, 1953). Daily cercaria output in the hundreds has been documented in the intermediate hosts of some Schistosoma (Marquardt et al., 2000). Other investigators have documented seasonal variation in the prevalence of spirorchiids, including

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220 periods where parasites were undetectable (Fer nandez, 1991; Rosen et al., 1994). In addition, although the prevalence of L. learedi infection appears to be high in harvest-age C. mydas at the CTFL, which are between 3 and 4 years old, fecal egg numbers were observed to be very low in all turtles examined. Only ten percent of infected turtles had eggs detected in the feces, and eggs were consistently rare and difficult to find in positive samples. Therefore, L. learedi may be cycling at a relatively low level of abundance in terms of fecal output of eggs into in the system. Another consideration is that the distribution of infected F. nodosa extends or is more concentrated outside of the study area, which was limited to boundaries of the CTFL under the collection permit. The flow of the effluent discharge is highly variable and depends on the prevailing current. Thus, eggs of L. learedi may be broadly distributed within the coastal habitat outside of the CTFL. The possibility of laboratory contamination as an explanation for the positive result is remote. No addition positive PCR results for L. learedi were detected in the other 13,729 gastropods (570 pooled samples) screened duri ng this study. Furthermore, dissection of gastropods and DNA extraction were performe d in a laboratory separate from the PCR laboratory, and H. mistroides DNA rather than L. leraedi was used as a positive PCR control to facilitate recognition of any contamination. Another possible, although improbable, scenario other than infection is that the positive F. nodosa ingested the eggs, but was not actually infected. Fissurella nodosa is an intertidal species and feeds on algae. It is plausible that eggs adhered to the algae may be ingested. The intensity of the positive PCR result, however, was much stronger than observed in the detection mo del studies when small quantities, such as egg equivalents, were introduced. Al so, none of the other limpet sp ecies or other gastropod species that also feed on algae yielded a positive result Our conclusion is that the positive PCR result

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221 supports that F. nodosa is an intermediate host for L. learedi, although confirmatory studies are needed, especially given that our findings were limited to a single positive sample. Furthermore, limpet species should be included among the species examined in future life cycle discovery efforts. Although no evidence of spirorchiid infection wa s found in any of the other study sites, we were able to demonstrate that Modulus modulus one of the most common gastropod species found in seagrass beds, is the intermediate host for Angiodictyum parallelum (Loss 1901), a digenean trematode of th e alimentary tract of C. mydas This parasite was detected in three separate M. modulus samples from two different collecting trips and all originated from Mooney Harbor, which is frequented by C. mydas In addition, M. modulus also may be a host for an as of yet genetically uncharacterized pronocephalid species. The digenean ITS2 sequence obtained from a sample of M. modulus collected from a grazing plot was highly similar (92%) to that of an unidentified pronocephalid colle cted from the stomach of a C. mydas This degree of similarity suggests that the digenean detected in M. modulus is a closely rela ted organism and very likely is a sea turtle parasite. The identity of the digenean may be discovered as the library of comparative genetic sequences available for sea turtle parasites is expanded. Examination of fecal samples opportunistically collected from the Marquesas grazing area provided useful insight into the parasite species pres ent in turtles foraging at the study site. Although none of the parasite genera detected co uld be specifically linked with those found in the gastropod samples, we were able to demonstrate that C. mydas foraging in the area were infected by at least four genera of alimentary trematodes and two spirorchiid genera. This parasite surveillance data may be useful to future life cy cle studies in the Marquesas region.

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222 Conclusions Digenean trematodes are the most diverse and numerous endoparasites of sea turtles; however, there has been very l ittle progress toward discovery of their life cycles. The implications of this missing information are that critical aspects of the host-parasite relationship, including epidemiology and factors relevant to tu rtle health and disease, are poorly understood. The results of this study provide a useful mol ecular tool for life cycle discovery studies with proven field application and the fi rst evidence of the id entity of the gastro pod intermediate hosts of at least two trem atode parasites of C. mydas In addition, the discovery that M. modulus serves as an intermediate host for A. parallelum and possibly a pronocephalid species, supports the hypothesis that seagrass beds are a suitable ha bitat for transmission of trematode parasites of sea turtles. These findings may be used to guide the necessary follow-up studies and to overcome many of the seemingly overwhelming difficulties of life cycle discovery in the marine environment.

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223 Figure 4-1. Marine system adjoining The Turtle Hospital, Marathon, Florida. The predominant gastropod habitat at this site is seagrass beds ( Thalassia testudinum ) (inset).

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224 Figure 4-2. Satellite image of the Cayman Tu rtle Farm, Limited, Grand Cayman Island. The intake and effluent channels are labeled a nd are approximately 215 meters apart. The side-by-side round and square struct ures are tanks used for rearing C. mydas

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225 Figure 4-3. The limestone shore (ironshore) ad joining the Cayman Turtle Farm, Limited. Several large tide pools are visible. Figure 4-4. Burrows of rock-boring sea urchin ( Echinometra sp.) pocket the subtidal surface of the ironshore

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226 Figure 4-5. The Marquesas Keys and surrounding region. Globa l positional system waypoints of areas from which gastropods were collect ed included a large gr azing area west of the islands, a small near-shore site on th e outer shore of th e southwestern-most islands, and Mooney Harbor lagoon. Th e scale bar equals 1 kilometer. Figure 4-6. Grazing plot created by foraging C. mydas in seagrass beds west of the Marquesas Keys. The Thalassia and Syringodium have been cropped by grazing turtles.

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227 Figure 4-7. The collection areas w ithin Florida Bay are shown here as three shaded areas, which include Twin Key Basin, Rabbit Key Basin, and Arsniker Basin.

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228 Figure 4-8. Gel electrophoreses of products produced by PCR amp lification of the partial ITS2 of Neospirorchis species using specific primers. Each numbered sample lane pair represents 1.5 grams of Pomacea bridgesii tissue spiked with the following egg equivalents of Neospirorchis DNA: samples 1 and 2 (1 egg); samples 3, 4, and 5 (3 eggs); samples 6 and 7 (5 eggs), samples 8 and 9 (10 eggs). Sample 10 only contains snail tissue and sample 11 was extracted with a complete adult Neospirorchis species. The positive control (+) is Neospirorchis DNA and the negative control (-) is PCR reagents only. Lanes labeled A have been processed using a QIAquick spin column, whereas lanes B are have been simply diluted in TE buffer to equivalent template concentrations. The ITS2 is detected in one of the samples spiked with a single egg equivalent and all of the samples containing 3 or more egg equivalents. Brighter amplicons are observed in samples processed using the QIA quick column as compared the TE dilutions.

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229 1 2 3 4 5 6 7 8 9 10 11 12 + Figure 4-9. Gel electrophoresis of products obtained from PCR am plification of the trematode ITS2 from DNA extracted from the limpet Fissurella nodosa. The brightest band in the ladder lanes is 500 base pairs. The bright bands in lane six reflect the detection of the 300 base pair complete ITS2 using consensus primers and the smaller 175 base pair indicates specific amplification of the 5 region of the ITS2. Products of similar size are present in the positive control lane (+), which is Hapalotrema mistroides DNA. Direct sequencing of the products excised from lane six confirmed the sequence to be that of Learedius learedi ITS2. Figure 4-10. Two Fissurella nodosa are adhered to the intertidal zone of the ironshore at the Cayman Turtle Farm, Limited.

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230 Figure 4-11. Gel electrophoresis of products re sulting from hemi-nested PCR amplification of the trematode ITS2 region from the gastropod Modulus modulus. The R1 gel reflects amplification of trematode ITS2 using cons ensus primers in the first reaction. The two hemi-nested reactions R2A and R2B use spirorchiid-specific reverse primers and the bright smaller bands (black circles) in the positive controls reflect specific amplification of the 5 region of the Neospirorchis ITS2 (R2A) and Hapalotrema/Learedius ITS2 (R2B). The positive controls are Neospirorchis sp. (+A) and Hapalotrema mistroides (+B). Note that the specific primers do not amplify the non-target control.

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231 Table 4-1. Gastropod DNA extraction protocol. Gastropod preparation and tissue lysis 1. Remove the hepatopancreas (digestive gland) and gonad 2. Pool these tissues to a combined wet weight of 1.5 grams 3. Place wet tissue into an aluminum foil pouc h and freeze in liquid nitrogen for 20 seconds 4. Crush tissues and place in 10 ml s of CTAB lysis buffer with 75 l of proteinase K (Qiagen) 5. Incubate overnight at 55 C DNA extraction 1. Add 12 mls of chloroform:isamyl alcohol (24:1) and mix by gentle inversion 2. Centrifuge at 7,700 G for 30 minutes 3. Transfer aqueous phase to clean tube and repeat chloroform extraction Precipitation 1. Add Na acetate (pH 5.4) to aqueous phase to obtain a final concentration of 0.3M 2. Add 2 volumes of 100% ethanol and mix by swirling 3. Centrifuge at 17,000 G for 1 hour 4. Discard supernatant and wash with 70% ethanol 5. Centrifuge at 17,000 G for 30 minutes 6. Dry pellet and resuspend in 1 ml of TE (pH 8.0) DNA clean-up (pigment removal) 1. Add aliquot of DNA to QIAquick spin column according to manufacturer protocol 2. Dilute eluted DNA in TE for PCR Table 4-2. Recipe for CTAB tissue lysis buffera. Recipe for a 0.5 liter volume 2% w/v CTAB 10.0 g 1.4M NaCl 41.0 g 20mM EDTA 2.9 g 100mM TrisCl 7.9 g Fill to a volume of 0.5 L and buffer to pH 8.0 aModified from Winnepenninckx et al, 1993.

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232 Table 4-3. PCR reaction conditions for trematode detection primers. Parameter Consensus trematode PCR Spirorchiid specific PCR Primers SPIR1 & SPIR2 SPIR1 & NSC2 / SPIR1 & HLC4 Denaturation 95 C 5 min 95 C 5 min 45 cycles of: Denaturation Annealing Extension 95 C 60 sec 50 C 45 sec 72 C 120 sec 95 C 60 sec 56 C 45 sec 72 C 120 sec Final extension 72 C 10 min 72 C 10 min Table 4-4. Marine gastropods collected and sc reened for spirorchiid trematode infection from The Turtle Hospital, Mara thon, Florida and surrounding area. Species Number Lithopoma americanum 455 Collumbella mercatoria 596 Fasciolaria tulipa 2 Ceritheum algicola/eburneum 114 Ceritheum muscarum 115 Cerithium littoratum 8 Modulus modulus 335 Tegula fasciata 204 Turbo castanea 154 Astraea phoebia 48 Crepidula sp. 69 Total 2,100

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233 Table 4-5. Marine gastropods collected and sc reened for spirorchiid trematode infection from the Cayman Turtle Farm, Limited, Grand Cayman Island, British West Indies. Species Number Leucozonia nassa 292 Columbella mercatoria 310 Hemitoma octoradiata 38 Lithopoma caelatum 56 Tegula lividomaculata 11 Tegula fasciata 16 Coralliophila abreviata 10 Thais deltoidea 66 Pupurita pupa 276 Nodolittorina ziczac 360 Cenchritas muricatus 203 Nodolittorina angustior 247 Nodolittorina dilatata 273 Engina turbinella 115 Nodolittorina mespillum 317 Cerithiopsis sp. 146 Cerithium littoratum 75 Cerithium eburneum 22 Nerita versicolor 81 Nerita peloranta 46 Fissurella nodosa 550 Fissurella fascicularis 106 Fissurella barbadensis 40 Diodora listeri 43 Limpet sp. 184 Total 3,883

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234 Table 4-6. Digenean genera detected by f ecal floatation and sedimentation for samples collected from wild C. mydas in the Marquesa s Keys region. Sample number Results 8/2007 8.07.1 No parasites observed 8.07.2 Rhytidodes sp., unidentified immature fluke 8.07.3 Rhytidodes sp., presumptive Deuterobaris sp., Neospirorchis sp. 8.07.4 Learedius sp., Rhytidodes sp., unidentified immature fluke 12/2007 12.07.1 Rhytidodes sp., Rhytidodes-like sp. 12.07.2 Unidentified trematode eggs 12.07.3 Rhytidodes sp., Rhytidodes-like sp., Learedius sp. 12.07.4 Rhytidodes sp. 12.07.5 Rhytidodes sp. 12.07.6 No parasites observed 12.07.7 Rhytidodes -like sp., Learedius sp. 12.07.8 Learedius sp. 12.07.9 Rhytidodes -like sp. 12.07.10 Unidentified trematode eggs 12.07.11 Rhytidodes sp. 12.07.12 Rhytidodes sp. 12.07.13 Rhytidodes sp. 12.07.14 Rhytidodes sp. 12.07.15 Presumptive Rhytidodes sp. 12.07.16 Rhytidodes sp. 12.07.17 Rhytidodes sp. 12.07.18 Presumptive Rhytidodes sp. 12.07.19 No parasites observed 12.07.20 Learedius sp., Rhytidodes sp. 3/2008 3.08.01 Polyangium sp. 3.08.02 Rhytidodes sp., Polyangium sp. 3.08.03 Polyangium sp., Rhytidodes sp. 3.08.04 Rhytidodes sp, Polyangium sp. and Schizamphistomoides sp. 3.08.05 Rhytidodes sp., Polyangium sp. 3.08.06 Rhytidodes sp., Polyangium sp. 3.08.07 Deuterobaris sp., Polyangium sp. 3.08.08 Schizamphistomoides sp. 3.08.09 Polyangium sp. 3.08.10 Deuterobaris sp., Polyangium sp, and Schizamphistomoides sp. 3.08.11 Deuterobaris sp., Polyangium sp. 3.08.12 Polyangium sp. 3.08.13 Polyangium sp., Schizamphistomoides sp. 3.08.14 Polyangium sp., Deuterobaris sp., Schizamphistomoides sp. 6/2007 6.07.01 Polyangium sp., Schizamphistomoides sp. 6.07.02 No parasites observed 6.07.03 Learedius sp., Polyangium sp. 6.07.04 No parasites observed 6.07.05 Polyangium sp., Rhytidodes sp. 6.07.06 Polyangium sp. 6.07.07 Polyangium Schizamphistomoides sp.

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235 Table 4-7. Gastropods colle cted from the Marquesas Keys region and Florida Bay examined by microdissection. Species Number examined Marquesas Keys region Astraea pheobia 13 Tegula fasciata 39 Lithopoma americanum 53 Collumbella mercatoria 25 Modulus modulus 16 Cerithium atratum 69 Crepidula sp. 9 Fasciolaria tulipa* 1 Horse conch* 3 Cerithium littoratum 8 Turbo castanea 50 Conus sp.* 3 Patelloida pustulata 4 Emerald nerite* 2 Total 295 Florida Bay Modulus modulus 92 Turbo castanea 48 Cerithium sp. 21 Lithopoma americanum 50 Tegula fasciata 67 Astraea pheobia 17 Total 295

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236 Table 4-8. Marine gastropods collected and sc reened for spirorchiid trematode infection from Marquesas Keys region, Florida. Species Number Grazing plots (west of Marquesas) Modulus modulus 263 Astraea phoebia 71 Cerithium atratum 33 Columbella mercatoria 74 Tegula fasciata 44 Lithopoma americanum 2 Crepidula sp. 85 Murex sp. 4 Fasciolaria tulipa 2 Pleuroploca gigantea 1 Conus sp. 1 Total 580 Mooney Harbor and near shore zones Modulus modulus 3573 Cerithium atratum 916 Lithopoma americanum 501 Turbo castanea 396 Columbella rusticoides 146 Tegula fasciata 59 Cerithium sp. 50 Crepidula sp. 38 Cerithium littoratum 27 Astraea phoebia 10 Total 5,716

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237 Table 4-9. Marine gastropods collected and sc reened for spirorchiid trematode infection from Florida Bay (Twin Key Basin, Rabb it Key Basin), Everglades National Park. Species Number Modulus modulus 478 Cerithium eburneum 270 Cerithium sp. 20 Turbo castanea 32 Columbella rusticoides 58 Zafrona taylorae 105 Tegula fasciata 50 Lithopoma americanum 450 Total 1,463

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238 LIST OF REFERENCES Altschul, S. F., W. Gish et al 1990. Basic local alignment search tool. Journal of Molecular Biology 215: 403. Balazs, G. H. 1986. Fibropapillomas in Hawaiia n green turtles. Marine Turtle Newsletter 39: 13. Ballard, J. W. and M. C. Whitlock. 2004. The incomplete natural history of mitochondria. Molecular Ecology 13: 729-744. Barker, S. C. and D. Blair. 1996. Molecular phylogeny of Schistosoma species supports traditional groupings within the genus. Journal of Parasitology 82: 292-298. Birky, C. W. 2001. The inheritance of gene s in mitochondria and chloroplasts: laws, mechanisms, and models. Annual Review of Genetics 35: 125-148. Bjorndal, K. A., A. B. Bolten, et al. 2000. Gr een turtle somatic grow th model: Evidence for density dependence. Ecological Applications 10: 269-282. Bjorndal, K.A., A. B. Bolten, et al. 2003. Compensatory growth in oceanic loggerhead sea turtles: response to a stochastic environment. Ecology 84: 1237-1249. Blair, D., J. Waikagul, et al. 1998. Phylogene tic relationships among the Thai species of Paragonimus inferred from DNA sequences. Pro ceedings of the 9th International Congress of Parasitology, Chiba, Japan. Blair, D., B. Wu, et al. 1999. A molecular prospective on the genera Paragonimus Braun, Euparagonimus Chen and Pagugonimus Chen. Journal of Helminthology 73: 295-299. Blair, J. E. and S. B. Hedges. 2005. Molecula r phylogeny and divergence times of deuterostome animals. Molecular Biology and Evolution 22: 2275-84. Blazov K, Hork P. 2005. Trichobilharzia regenti : the developmental differences in natural and abnormal hosts. Parasitology International 54: 167-72. Bolten, A. B. 2003. Variation in sea turtle life history patterns: neritic vs. oceanic developmental stages. In Biology of Sea Turtles, Volume II, P. L. Lutz, J. A. Musick, and J. Wyneken (eds.). CRC Press, Boca Raton, U.S.A., p. 243-258. Bolten, A. B. 2003. Active Swimmers-Passive Drifters. In Loggerhead sea turtles, A. B. Bolten and B. E. Witherington (eds.). Smith sonian Books, Washington D.C., p. 63-78. Bowen, B. W. and S. A. Karl 2007. Populati on genetics and phylogeography of sea turtles. Molecular Ecology 16: 4886-4907. Bowen, B. W., A. B. Meylan, et al. 1992. Global population structure and natural history of the green turtle ( Chelonia mydas ) in trems of matriarchal phylogeny. Evolution 86: 573-576.

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240 Dyer, W. G., E. H. J. Williams, et al. 1991. Some digeneans trematods of the green turtle Chelonia mydas testudines Cheloniidae fr om Puerto Rico West Indies. Journal of the Helminthological Society of Washington 58: 176-180. Edgar, R. C. 2004. MUSCLE: multiple seque nce alignment with high accuracy and high throughput. Nucleic Acids Research 32: 1792-7. Erhart, L. M. 1991. Fibropapillomas in green turtles of the Indian River Lagoon, Florida: Distribution over time and area. In Research plan for marine turtle fibropapilloma, G. H. Balazs and S. G. Pooley (eds.). U.S. Department of Co mmerce (NOAA-TM-NMFSSWFSC-156), 156), Washington, D.C., p. 59. Felsenstein, J. 1985. Confidence limits on phylogenies: an appro ach using bootstrap. Evolution 39: 783-791. Felsenstein, J. 1989. PHYLIP-phylogeny inference package. Cladistics 5: 164-166. Fischthal, J. H. and A. D. Acholonu. 1976. So me digenetic trematodes from the Atlantic hawksbill turtle Eretmochelys imbricata imbicata from Puerto Rico. Proceedings of the Helminthological Society of Washington 43: 174-185. Foley, A. M., K. S. Minch, et al. 2007. Characteristics of a Green Turtle ( Chelonia mydas) Assemblage in Northwestern Florida Dete rmined During a Hypothermic Stunning Event. Gulf of Mexico Science 2: 131-143. Foley, A. M., K. S. Minch, et al. 2007. Distribu tions relative abundances, and mortality factors for sea turtles in Florida from 1980 th rough 2005 as determined from stranding (unpublished). Jacksonville, Fl orida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute: 284. Foley, A. M., B. A. Schroeder, et al. 2005. Fibropapillomatosis in stranded green turtles ( Chelonia mydas ) from the eastern United States (198098): trends and associations with environmental factors. Journal of Wildlife Diseases 41: 29-41. Frankovich, T. A. and J.C. Zieman. 2005. A temporal investigati on of grazer dynamics, nutrients, seagrass leaf productivity, a nd epiphyte standing stock. Estuaries 28: 39-50. Glazebrook, J. S., R. S. Campbell, et al. 1981. Pathological changes associated with cardiovascular trematodes (Digenea: Sp irorchidae) in a green sea turtle Chelonia mydas (L.). Journal of Comparative Pathology 91: 361-8. Glazebrook, J. S., R. S. Campbell, et al. 1989. Studies on cardiovascular fluke (Digenea: Spirorchiidae) infections in s ea turtles from the Great Barri er Reef, Queensland, Australia. Journal of Comparative Pathology 101: 231-50. Glazebrook, J. S. and R. S. F. Campbell 1990. A survey of the diseases of marine turtles in northern Australia II. Oceanarium-reared and wi ld turtles. Diseases of Aquatic Organisms 9: 97-104.

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241 Gohar, N. 1934. Liste des trematodes parasites et de leurs hotes vertebres signales dans la Vallee du Nil. Annales de Parasitologie Humaine et Compare 12: 322-331. Gohar, N. 1935. Liste des trematodes parasites et de leurs hotes vertebres signales dans la Vallee du Nil. (Suite et fin). Annale s de Parasitologie Humaine et Compare 13: 80-90. Goodchild, G. Chouncey, et al. 1967. Compara tive egg counts and histopathology in turtles infected with Spirorchis (Trematoda: Spirorchiidae) Journal of Parasitology 53: 38-45. Goodchild, C. G. and B. Fried. 1963. Expe rimental infection of the planorbid snail Menetus dilatatus buchanensis (Lea) with Spirorchis sp. (Tremat oda). Journal of Parasitology 49: 586-592. Goodchild, C. G. and D. E. Ki rk. 1960. The life history of Spirorchis elegans Stunkard, 1923 (Trematoda: Spirorchiidae) from the pain ted turtle. Journal of Parasitology 46: 219-228. Goodman, J. D. 1987. A new blood fluke, Hapalorhynchus beadlei n. sp. ( Spirorchiidae), and a note on Allossostomoides [sic] (Paramphistomistomidae), in Pelusios williamsi lutescens from Uganda. Transactions of the American Microscopical Society 106: 80-84. Gordon, A. N., W. R. Kelly, et al. 1998. Le sions caused by cardiovascular flukes (Digenea: Spirorchidae) in stranded green turtles ( Chelonia mydas). Veterinary Pathology 35: 21-30. Graczyk, T. K., A. A. Aguirre, et al. 1995. Detection by ELISA of circulating anti-blood fluke ( Carettacola Hapalotrema and Learedius) immunoglobulins in Ha waiian green turtles ( Chelonia mydas ). Journal of Parasitology 81: 416-21. Greiner, E. C., D. J. Forrest er, et al. 1980. Helminths of mariculture-reared green turtles ( Chelonia mydas mydas ) from Grand Cayman, British West Indes. Proceedings of the Helminthological Society of Washington 47: 142-144. Gupta, N. K. and V. Mehrotra. 1975. On th ree blood flukes Spirorchiid ae Spirorchiinae from the freshwater chelonians and discussion on the synonymy of Plasmiorchis pellucidus Rivista di Parassitologia 362: 165-170. Gupta, N. K. and V. Mehrotra 1981. Two blood flukes trematoda of the family Spirorchidae from Indian marine turtles. Acta Parasitologica Polonica 281: 11-20. Herbst, L. 1994. Fibropapillomatosis of marine turtles. Annual Review of Fish Diseases 4: 389425. Herbst, L. H., A. Ene, et al. 2004. T umor outbreaks in marine turtles are not due to recent herpesvirus mutations. Current Biology 14: R697-R699. Herbst, L. H., E. C. Greiner, et al. 1998. Serological association be tween spirorchidiasis, herpesvirus infection, and fibropapillomatosis in green turtles from Florida. Journal of Wildlife Diseases 34: 496-507.

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248 BIOGRAPHICAL SKETCH Brian Stacy was born in Stone Mountain, Georgia in 1975. He developed a love of the outdoors and wildlife at an early age growing up in north Georgia and periodically spending time at the family farm in rural Montgomery county. A special interest in rept iles developed early on. He attended undergraduate school at the University of Georgia during which time he worked as a farm hand at the University dairy and on a priv ate farm working with a large collection of psittacines. He completed an externship w ith the Puerto Rican Parrot Project under the Department of Natural Resources. After deciding to pursue a career in wildlife health and conservation, he was admitted into veterinary school at UGA where he received his Doctor of Vete rinary Medicine degree in 2001. Dr. Stacy worked with captive and free-ra nging wildlife throughout ve terinary school and completed two projects abroad at the Madras Cr ocodile Bank Trust, Institute for Herpetology. He also worked as research assistant with the Southeastern W ildlife Disease Study and completed externships with the Armed Forces Institute of Pathology, Wildlife Conservation Society, and Western College of Veterinary Me dicine, Saskatoon. Following veterinary school, Dr. Stacy completed a residency program in anat omic pathology at the Un iversity of California at Davis, Veterinary Medical Teaching Hospita l and the Zoological Society of San Diego, and became a board certified pathologist in 2004. His in terest in health and disease in free-ranging wildlife led him to pursue the study of marine sp irorchiids and their effects on sea turtles. Currently, Dr. Stacy is a vete rinary pathologist with the UF Aquatic Animal Health Program. He studies and works as a diagnostician and consultant on health and disease related issues affecting sea turtles, marine mammals, and other aquatic species. He also is involved in a number of other wildlife health pr ojects within the US and abroad.