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THE ECOLOGY OF CHYTRIDIOMYCOSIS, AN EMERGING INFECTIOUS DISEASE OF AUSTRALIAN RAINFOREST FROGS Thesis submitted by Douglas Craig WOODHAMS BSc Michigan State University in September 2003 for the degree of Doctor of Philosophy in Zoology and Tropical Ecology within the School of Tropical Biology James Cook University DEDICATION To my friend and coworker, Jesus. "Let everything that has breath praise the LORD ...." --Psalm 150:6 11 STATEMENT OF ACCESS I, the undersigned, author of this thesis, understand that James Cook University will make it available for use within the University Library, and by microfilm or other means, allow access to users in other approved libraries. All users consulting this thesis will have to sign the following statement: In consulting this thesis I agree not to copy or closely paraphrase it in whole or in part without the written consent ofthe author; and to make proper public written acknowledgment for any assistance that I have obtained from it. Beyond this, I do not wish to place any restriction on access to this thesis. ') , ..... ...J --,' ---------------------------------------(Name) ."'~ I U \ (Date) III 'i.D04· I,' \ ABSTRACT In the wet tropics of Queensland, Australia eight species of stream dwelling frogs have experienced population declines, and individuals have been found with fungal infections of Batrachochytrium dendrobatidis, an emerging infectious disease of amphibians. I examined infection prevalence in stream frog assemblages and determined that amphibian chytrid infection is now endemic and persists in adults and tadpoles of declining and non-declining species at all eight streams surveyed. Infection prevalence varied among species, and between adult and tadpole life history stages, seasons, and elevations. Prevalences were higher during the dry season (May to September) and at high elevations (600-800 m). Tadpoles of Litoria nannotis, L. rheocola, and Mixophyes shevilli may be carrier hosts in this system; they have infection prevalences between 48 and 76%, and appear to be resistant to disease. Non-carrier hosts may include adults of all species and tadpoles of some species, with infection prevalence below 10%. I found no relationships between infection prevalence and body condition, fluctuating asymmetry of hind limbs, population density, or the presence of metamorphosing tadpoles and juvenile frogs. I examined how environmental temperature and moisture regimes influenced the chytrid pathogen on frog hosts and in culture. Infected Litoria chloris juveniles survived at the lowest rates in constant mist and constant 20°C conditions, but frogs in constant dry or rain conditions survived longer. All frogs exposed to 37°C on days 12 and 13 after infection survived in good health for 9 mo after infection, and were found to be free of infection. I modelled growth of chytrid populations using in vitro experiments to estimate the parameters zoospore production, zoospore survival rate, zoospore settlement rate, and time to sporulation of sporangia. The chytrid lifecycle may be faster at higher temperatures (23°C) than at lower temperatures (8°C), but relatively greater production of zoospores and better survival rates at lower temperatures may cause chytrid colonies to grow faster under cold conditions. In addition to environmental effects, host immune function and behaviour may also alter host-pathogen dynamics. I found that leukocyte populations in juvenile Litoria chloris responded to infection but not to environmental conditions. Skin peptide immune IV defences were more effective in common species (Litoria lesueuri, Litoria genimaculata, Mixophyes shevilli) and less effective in some endangered species (Litoria rheocola, Nyctimystes dayi). Skin peptide defences against the chytrid were also better in those species that showed greater survival when experimentally infected with B. dendrobatidis. Environmental conditions may also affect skin peptide immune response, with poorer responses under colder conditions. The behaviour of captive Litoria chloris juveniles varied with thermal and hydric environments and time of day, but not infection status. Litoria genimaculata tadpole behaviour did vary with infection status; tadpoles exposed to chytrid sporangia showed a behavioural fever of 6.2 to 7.1"C. The conservation status ofthe rainforest stream frog species examined was correlated with their ranking on a disease risk assessment of behaviour; however, their current infection prevalence is not well correlated with disease risk. This agrees with epidemiological theory, which suggests that a disease involved in population declines should be present in both common and endangered species, but should have higher infection prevalence in species with stable populations. These stable species are also less at risk from chytridiomycosis based on behavioural and immunological assessments. The emergence of chytridiomycosis may occur when a particular host-pathogen interaction is achieved involving the complex arrangement of species assemblage structure and environmental conditions. v ACKNOWLEDGEMENTS I have heaps to be thankful for. God is good to me. What could be better than to study rainforest frogs in Australia? My talented supervisor, Ross A. Alford, is an ideas man with extensive knowledge in areas ranging from electronics and photography to ecology and statistics. I'll never forget my first rainy season in Townsville and the enthusiasm Ross expressed when we visited a chorus of at least 13 species of savannah frogs! I thank him for providing me with great freedom and support to pursue answers to my own questions and allowing me to make this thesis my own. I also thank my undergraduate research advisor, Heather Eisthen, who taught me how to do science (so she should be held at Ieast partly responsible for the contents of this thesis). My fascination with wildlife, and my conservation ethic, although now part of my nature, are due primarily to my Dad. My mom is also a source of love and encouragement to me. , , I think they would be proud of me no matter what career I chose. Special thanks are due Sara Townsend for her unflinching support of this project even when facing countless hours oftedious histology and microscope work. Her ability to catch frogs is a source of wonder for me. And I can't forget her attention to detail! I took great pleasure in collaborating on parts ofthis thesis with Gerry Marantelli and Louise Rollins-Smith. I've never met anyone as passionate about frog conservations as Gerry. After working with Louise, I have decided that a postdoc in immunology and microbial ecology might be fun. Megan Johnson, Diana Mendez, and Rick Speare from the amphibian disease research group at James Cook University were especially helpful with work on the amphibian chytrid fungus, as were the CSIRO amphibian disease research team at Geelong, including: Lee Berger, Alex Hyatt, Veronica Olsen, and Donna Boyle. The following people also deserve my gratitude for their help in sundry ways: Tracy Langkilde, Beth Mott, Ant Baker, Sacha Jelinek, Sue Reilly, Laurie Reilly, Brad Cullen, Jason Doyle, VI John Osborne, Joel Cox, Deb Cochrane, Dan Salkeld, Richard Rowe, Joe Holtum, and Kirsten Heimann. Thank you volunteers and friends of the Amphibian Research Centre, including: C. Steele, K. Ardipradja, R. Hobbs, S. Barber, D. Taylor, T. Dornum, N. Nolas, M. Velik-Lord, A. Crigo, N. Evangelou, P. Oliver, A. Marantelli, and P. Marantelli. My field volunteers were great, and I hope they enjoyed scrambling up and down rainforest streams at night and catching frogs as much as I did. Thanks: Jasmine Cauchi, Michael Bauman, Fiona Marshall, Esther Mitchell, Kathryn Lewthwaite, Katie Mitchell, Fred Sinneger, Brian Brinkman, Thor Sawin, Kendall Trudgen, Mirza Kusrini, Barka Hegadus, John Pedder, and Steven Macey. This research was funded by James Cook University and the School of Tropical Biology, subcontracts to L.A. Rollins-Smith and R.A. Alford from an Integrated Research Challenges in Environmental Biology (IRCEB) grant IBN-9977063 from the U.S. National Science Foundation (James P. Collins, P.L), and a Grant-In-Aid-of-Research from the National Academy of Sciences administered by Sigma Xi, The Scientific Research Society. vii CONTENTS Page Title Dedication 11 Statement of Access iii Abstract IV Acknowledgments vi Table of Contents Vll List of figures x List of tables xii Statement of Sources X111 Chapter 1 1 Introduction: Disease Emergence and Amphibian Population Declines 1.1 Emerging Infectious Disease 1 1.2 Amphibian Population Declines and Chytridiomycosis 2 1.3 Factors Implicated in Population Declines 4 1.4 Mechanisms of Chytridiomycosis Emergence 10 1.5 Aims 24 Chapter 2 28 The Ecology ofChytridiomycosis in Rainforest Stream Frog Assemblages of Tropical Queensland Chapter 3 73 Mechanisms of Chytridiomycosis Emergence: effects of environmental conditions and immune function on disease development in Australian frogs viii ~~4 % Amphibian Chytrid in vitro Response to Temperature: Methods of Measurement and Hypothesis Regarding Exponential Growth in Cold Climates Chapter 5 132 Anti-chytrid Activity of Australian Frog Skin Secretions Chapter 6 172 Behavioural Fever in Tadpoles Infected with Chytrid Fungus and Suggested Research Directions Chapter 7 187 Summary and Conclusion 7.1 Project background and justification 7.2 Aims and objectives 7.3 General discussion 7.4 Synthesis and conclusions Literature Cited 197 Appendix 1.1 Faith Advocating Objectivity in Science 216 Appendix 2.1 Analysis of Developmental Instability 226 Appendix 2.2 Population surveys of rainforest stream frog adults and tadpoles 228 Appendix 2.3 Infection or Disease Transmission Risk 229 Appendix 3.1 Emerging disease of amphibians cured by elevated body 232 , temperature IX LIST OF FIGURES Figure Page 1.1 Lifecycle of Batrachochytrium dendrobatidis. 26 1.2 Map of stream sites in the Queensland wet tropics. 27 2.1 Healthy and diseased Litoria genimaculata 58 2.2a Boxplot of proportion infected sections 59 2.2b Histogram of detection efficiency 60 2.3 Infection prevalence of adults and tadpoles 61 2.4 Infection prevalence at lowlands and uplands 62 2.5 Seasonal infection prevalence of adults and tadpoles 63 2.6 Infection prevalence of adult Litoria genimaculata 64 2.7 Position of Litoria genimaculata along Birthday Creek 65 2.8 Morphological characteristics of rainforest stream frogs 66 2.9 Condition and infection prevalence of Litoria genimaculata 67 2.10 Percent fluctuating asymmetry of rainforest stream frogs 68 2.11. Seasonal population densities of rainforest stream frogs 69 2.14 Infection prevalence ofjuvenile and adult frogs 70 2.13 Environmental conditions and infection prevalence 71 2.14 Infection prevalence at six stream sites 72 3.1 Dry, mist, and rain enclosures 92 3.2Daytime and nighttime behaviours ofjuvenile Litoria chloris 93 3.3 Behaviour of infected and uninfected juvenile Litoria chloris 94 3.4 Leukocyte populations of infected and uninfected Litoria chloris 95 4.1 Amphibian chytrids stained with Calcofluor White 118 4.2 Protein concentration in chytrid cultures five days after inoculation 119 4.3 Chytrid sporangia density on flasks 120 4.4a,b Chytrid colonies after eight days at different temperatures 121 4.4c Chytrid colonies after eight days at different temperatures 122 4.5 Amphibian chytrids on host tissues 123 4.6 Total chytrid zoospore counts at seven and eleven days after inoculation 124 x Figure Page 4.7 Proportion active to total zoospores at different temperatures 125 4.8 Numbers of amphibian chytrid colonies growing at different temperatures 126 4.9 Protein concentration of whole chytrid cultures 127 4.10 Zoospore activity through time 128 4.11 Zoospore settlement through time 129 4.12 Number of free zoospores modelled through time 130 4.13 Number of sporangia modelled through time 131 5.1 Minimum inhibitory concentration (MIC) of Australian frog skin peptides 157 5.2 Percent amphibian chytrid zoospore growth inhibition 158 5.3 MIC equivalents per body weight of Australian frogs 159 5.4 MIC equivalents per body weight of adult Australian frogs 160 5.5 MIC equivalents per surface area of adult frogs 161 5.6 Relationship between infection prevalence and immune function 162 5.7 Litoria lesueuri skin peptide recovery by induction technique 163 5.8 Skin peptide recovery from five Australian frog species 164 I 5.9 Correlation of frog survival and immune function 165 5.10 Skin peptides recovered from wild infected and uninfected tadpoles 166 5.11 Skin peptides recovered from adult frogs exposed and unexposed to chytrids 167 5.12 Temperature regimes for Litoria genimaculata experiment 168 5.13 Correlation of skin peptide recovery and tadpole water temperature 169 5.14 MIC equivalents at different life-history stages 170 5.15 Chytrid zoospore growth inhibition assay example chart 171 6.1 Thermal gradients 176 6.2 Histogram ofpreferred temperatures of tadpoles in thermal gradients 185 6.3 Boxplots of preferred temperatures of tadpoles in thermal gradients 186 Xl LIST OF TABLES Table Page 1.1 Conservation status of Queensland wet-tropics rainforest stream frogs 6 2.1 Stream site descriptions 32 2.2 Habitat and frog species occurrence at six stream sites 32 2.3 Number ofjuvenile frogs found by season 43 2.4 Environmental conditions at stream sites by season 44 2.5 ANDVA table of effects on infection prevalence 46 2.6 Infection risk of rainforest stream frogs 47 2.7 Excel data sheet for developmental instability analysis 219 2.8 ANDVA table for developmental instability analysis 219 2.9 Population surveys of rainforest stream frogs 221 2.10 Infection risk of Queensland wet-tropics rainforest stream frogs 223 2.11 Ecological and morphological characteristics of rainforest stream frogs 224 3.1 Survival of Litoria chloris in artificial environments 81 3.2 Sensitivity and specificity of chytrid infection diagnostic technique 83 3.3 Leukocyte populations ofjuvenile Litoria chloris 85 4.1 t\rnphibian chytrid survival and settlement rates 108 4.2 Best parameter estimates for chytrid growth model 109 4.3 Chytrid colony growth at different temperatures 109 4.4·Sensitivity analysis of chytrid growth model 110 4.5 Variation in mean zoospore counts 111 5.1 Anti-chytrid skin peptide defences ofAustralian frogs 156 5.2 Minimum inhibitory concentration equivalents 145 5.3 Skin peptide immune function of Litoria genimaculata 149 xii STATEMENT OF SOURCES DECLARATION I declare that this thesis is my own work and has not been submitted in any form for another degree or diploma at any university or other institution of tertiary education. Information derived from the published or unpublished work of others has been acknowledged in the text and a list of references is given. ----~-~-----~---~----------------- __ _~}_~~~ ~~L_~~~~:_~ (Name) (Date) Xlll _ Douglas C. Woodhams The Ecology of Chytridiomycosis, an Emerging Infectious Disease of Australian Amphibians Abstract In the wet tropics of Queensland, Australia eight species of stream dwelling frogs have experienced population declines, and individuals have been found with fungal infections of Batrachochytrium dendrobatidis, an emerging infectious disease of amphibians. I examined infection prevalence in stream frog assemblages and determined that amphibian chytrid infection is now endemic and persists in adults and tadpoles of declining and non-declining species at all eight streams surveyed. Infection prevalence varied among species, and between adult and tadpole life history stages, seasons, and elevations. Prevalences were higher during the dry season (May to September) and at high elevations (600-800 m). Tadpoles of Litoria nannotis, L. rheocola, and Mixophyes shevilli may be carrier hosts in this system; they have infection prevalences between 48 and 76%, and appear to be resistant to disease. Non-carrier hosts may include adults of all species and tadpoles of some species, with infection prevalence below 10%. I found no relationships between infection prevalence and body condition, fluctuating asymmetry of hind limbs, population density, or the presence of metamorphosing tadpoles and juvenile frogs. I examined how environmental temperature and moisture regimes influenced the chytrid pathogen on frog hosts and in culture. Infected Litoria chloris juveniles survived at the lowest rates in constant mist and constant 20°C conditions, but frogs in constant dry or rain conditions survived longer. All frogs exposed to 37°C on days 12 and 13 after infection survived in good health for 9 mo after infection, and were found to be free of infection. I modelled growth of chytrid populations using in vitro experiments to estimate the parameters zoospore production, zoospore survival rate, zoospore settlement rate, and time to sporulation of sporangia. The chytrid lifecycle may be faster at higher temperatures (23°C) than at lower temperatures (8°C), but relatively greater production of zoospores and better survival rates at lower temperatures may cause chytrid colonies to grow faster under cold conditions. In addition to environmental effects, host immune function and behaviour may also alter host-pathogen dynamics. I found that leukocyte populations in juvenile Litoria chloris responded to infection but not to environmental conditions. Skin peptide immune defences were more effective in common species (Litoria lesueuri, Litoria genimaculata, Mixophyes shevilli) and less effective in some endangered species (Litoria rheocola, Nyctimystes dayi). Skin peptide defences against the chytrid were also better in those species that showed greater survival when experimentally infected with B. dendrobatidis. Environmental conditions may also affect skin peptide immune response, with poorer responses under colder conditions. The behaviour of captive Litoria chloris juveniles varied with thermal and hydric environments and time of day, but not infection status. Litoria genimaculata tadpole behaviour did vary with infection status; tadpoles exposed to chytrid sporangia showed a behavioural fever of 6.2 to 7.1°C. The conservation status of the rainforest stream frog species examined was correlated with their ranking on a disease risk assessment of behaviour; however, their current infection prevalence is not well correlated with disease risk. This agrees with epidemiological theory, which suggests that a disease involved in population declines should be present in both common and endangered species, but should have higher infection prevalence in species with stable populations. These stable species are also less at risk from chytridiomycosis based on behavioural and immunological assessments. The emergence of chytridiomycosis may occur when a particular host-pathogen interaction is achieved involving the complex arrangement of species assemblage structure and environmental conditions.