HUFFMANELA HUFFMANI: LIFE CYCLE, NATURAL HISTORY, AND BIOGEOGRAPHY by McLean Worsham, B.S. A thesis submitted to the Graduate Council of Texas State University in partial fulfillment of the requirements for the degree of Master of Science with a Major in Biology May 2015 Committee Members: David Huffman, Chair Chris Nice Randy Gibson COPYRIGHT by McLean Worsham 2015 FAIR USE AND AUTHOR’S PERMISSION STATEMENT Fair Use This work is protected by the Copyright Laws of the United States (Public Law 94-553, section 107). Consistent with fair use as defined in the Copyright Laws, brief quotations from this material are allowed with proper acknowledgment. Use of this material for financial gain without the author’s express written permission is not allowed. Duplication Permission As the copyright holder of this work I, McLean Worsham, authorize duplication of this work, in whole or in part, for educational or scholarly purposes only. ACKNOWLEDGEMENTS I would like to acknowledge Harlan Nicols, Stephen Harding, Eric Julius, Helen Wukasch, and Sungyoung Kim for invaluable help in the field and/or the lab. I would like to acknowledge Dr. David Huffman for incredible and dedicated mentorship. I would like to thank Randy Gibson for his invaluable help in trying to understand the taxonomy and ecology of aquatic invertebrates. I would like to acknowledge Drs. Chris Nice, Weston Nowlin, and Ben Schwartz for invaluable insight and mentorship throughout my research and the graduate student process. I would like to thank my good friend Alex Zalmat for always offering everything he has when a friend is in a time of need. I would like to thank the USFWS for support in conducting this research and The Meadows Center for Water and the Environment for providing access to Spring Lake and boat/diving support. iv TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ........................................................................................................... xi CHAPTER 1. THE AQUATIC ANNELID FAUNA OF THE SAN MARCOS RIVER HEADSPRINGS, HAYS COUNTY, TEXAS.........................................1 Introduction .....................................................................................................1 Materials and methods ....................................................................................3 Results .............................................................................................................3 Taxa collected .....................................................................................3 Dichotomous key to Annelida of San Marcos Springs .......................5 Discussion .....................................................................................................15 2. THE LIFE CYCLE OF HUFFMANELA HUFFMANI (NEMATODA: TRICHOSOMOIDIDAE), A MARINE RELICT PARASITE OF CENTRARCHID FISHES: THE FIRST COMPLETION OF A HUFFMANELA LIFE-CYCLE ......................................................................... 18 Introduction ...................................................................................................18 Methods.........................................................................................................20 Phase 1 Methods – Selection of candidate invertebrate species most likely to be the intermediate host of H. huffmani ......................................................................................20 Determination of probable ecotype of hypothetical intermediate host .............................................................20 Determination of probable taxonomic group of hypothetical intermediate host ........................................23 Phase 2 Methods – Attempted experimental infection of candidate invertebrates................................................................23 Collection of H. huffmani eggs .............................................23 Invertebrate collections .........................................................25 v Preliminary exposure experiment to test susceptibility ...................................................................27 Phase 3 Methods – Titration of optimum exposure intensity and incubation duration of Hyalella exposed to H. huffmani eggs .....................................................................28 Source of H. huffmani eggs ..................................................28 Setup of amphipod exposure titration experiment ................29 Distribution of amphipods to replicate containers ................29 Distribution of H. huffmani eggs to replicate containers ........................................................................30 The titration of amphipod exposure intensity .......................31 The titration of amphipod incubation duration .....................32 Examination and feeding-out of exposed amphipods .......................................................................32 Quantification of infection intensity in amphipods ..............33 Phase 4 Methods – Experimental infection of fish ...........................34 Sources of experimental fish .................................................34 Exposure and incubation of experimental fish ......................35 Examination of experimental fish .........................................35 Phase 5 Methods – Evaluation of relationship of definitive host diet and location of egg deposition for all reported Huffmanela species. .................................................36 Results ...........................................................................................................37 Phase 1 Results – Selection of candidate invertebrate species most likely to be the intermediate host of H. huffmani ......................................................................................37 Clues from naturally infected fishes .....................................37 Phase 2 Results – Attempted experimental infection of candidate invertebrates................................................................40 Phase 3 Results – Titration of optimum exposure intensity and incubation duration of Hyalella exposed to H. huffmani eggs .....................................................................41 Phase 4 Results – Experimental infection of fish .............................44 Phase 5 Results – Evaluation of relationship of definitive host diet and location of egg deposition for all reported Huffmanela species. ......................................................48 Discussion .....................................................................................................53 3. THE BIOGEOGRAPHY AND POPULATION BIOLOGY OF HYALELLA AND HUFFMANELA HUFFMANI .............................................. 58 Introduction ...................................................................................................58 vi Question #1 – Does H. huffmani possess unique adaptations that its marine relatives do not have that allowed it to survive in freshwater? ............................................61 Question #2 – Could H. huffmani have survived droughts which are known to affect spring environments throughout the Edwards Plateau?................................................63 Question #3 – Are headspring Hyalella that co-occur with freshwater Huffmanela unique and reproductively isolated from Hyalella of the H. cf. azteca morphotype? ...............................................................................64 Subquestion 3.1 - Is phenotypic variation between morphologically distinct populations of Hyalella heritable or plastic? ..........................................65 Subquestion 3.2 - If phenotypic differences prove to be heritable, are these morphological races also reproductively isolated?...........................................65 Subquestion 3.3 – Do molecular phylogenies agree with the hypothesized phylogeography and reproductive isolation results? ..................................66 Methods.........................................................................................................66 Question #1 – Salinity tolerance of eggs of H. huffmani ..................66 Question #2 – Desiccation tolerance of eggs of H. huffmani ......................................................................................67 Desiccation Experiment ........................................................67 Comal Fish – 1951 ................................................................67 Question #3 – Are headspring Hyalella that co-occur with freshwater Huffmanela unique and reproductively isolated from Hyalella of the H. cf. azteca morphotype? ...............................................................................67 Source of experimental Hyalella and culture methods ...........................................................................67 Subquestion 3.1 - Common garden methods and design ..............................................................................70 Subquestion 3.2 - Attempted “no-choice” hybridization experiments with Hyalella spp. ................72 Question #4 – COI phylogeny of select Hyalella sequences of North America .......................................................73 Results ...........................................................................................................74 Question #1 .......................................................................................74