THE ECOLOGY OF CHYTRIDIOMYCOSIS, AN EMERGING INFECTIOUS DISEASE OF AUSTRALIAN RAINFOREST FROGS

Transcription

1 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 ofdoctor ofphilosophy in Zoology and Tropical Ecology within the School oftropical Biology James Cook University

2 DEDICATION To my friend and coworker, Jesus. "Let everything that has breath praise the LORD..." --Psalm 150:6 11

3 STATEMENT OF ACCESS I, the undersigned, author ofthis 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 properpublic 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 ,' (Name) '), I U 'i.d j."'~ \ I,' \ (Date) III

4 ABSTRACT In the wet tropics ofqueensland, Australia eight species ofstream dwelling frogs have experienced population declines, and individuals have been found with fungal infections ofbatrachochytrium dendrobatidis, an emerging infectious disease ofamphibians. 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 ( m). Tadpoles oflitoria 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 ofsome species, with infection prevalence below 10%. I found no relationships between infection prevalence and body condition, fluctuating asymmetry ofhind limbs, population density, or the presenceof 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 ofinfection. I modelled growth ofchytrid populations using in vitro experiments to estimate the parameters zoospore production, zoospore survival rate, zoospore settlement rate, and time to sporulation ofsporangia. The chytrid lifecycle may be faster at higher temperatures (23 C) than at lower temperatures (8 C), but relatively greater production ofzoospores 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

5 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 ofcaptive Litoria chloris juveniles varied with thermal and hydric environments and time ofday, but not infection status. Litoria genimaculata tadpole behaviour did vary with infection status; tadpoles exposed to chytrid sporangia showed a behavioural fever of6.2 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 ofchytridiomycosis may occur when a particular host-pathogen interaction is achieved involving the complex arrangement of species assemblage structure and environmental conditions. v

6 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 ofat least 13 species ofsavannah frogs! I thank him forproviding 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 ofthis thesis). My fascination with wildlife, and my conservation ethic, although now part ofmy nature, are due primarily to my Dad. My mom is also a source oflove and encouragement to me.,, I think they would be proud ofme no matter what career I chose. Special thanks are due Sara Townsend for her unflinching support ofthis 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

7 John Osborne, Joel Cox, Deb Cochrane, Dan Salkeld, Richard Rowe, Joe Holtum, and Kirsten Heimann. Thank you volunteers and friends ofthe 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 oftropical Biology, subcontracts to L.A. Rollins-Smith and R.A. Alford from an Integrated Research Challenges in Environmental Biology (IRCEB) grant IBN 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

8 CONTENTS Title Page Dedication 11 Statement ofaccess iii Abstract Acknowledgments Table ofcontents List offigures List oftables Statement ofsources IV vi Vll x xii X111 Chapter 1 Introduction: Disease Emergence and Amphibian Population Declines 1.1 Emerging Infectious Disease 1.2 Amphibian Population Declines and Chytridiomycosis 1.3 Factors Implicated in Population Declines 1.4 Mechanisms ofchytridiomycosis Emergence 1.5 Aims Chapter 2 28 The Ecology ofchytridiomycosis in Rainforest Stream Frog Assemblages oftropical Queensland Chapter 3 73 Mechanisms ofchytridiomycosis Emergence: effects ofenvironmental conditions and immune function on disease development in Australian frogs viii

9 ~~4 Amphibian Chytrid in vitro Response to Temperature: Methods ofmeasurement and Hypothesis Regarding Exponential Growth in Cold Climates % Chapter 5 Anti-chytrid Activity ofaustralian Frog Skin Secretions 132 Chapter Behavioural Fever in Tadpoles Infected with Chytrid Fungus and Suggested Research Directions Chapter 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 ofdevelopmental Instability 226 Appendix 2.2 Population surveys ofrainforest stream frog adults and tadpoles 228 Appendix 2.3 Infection or Disease Transmission Risk 229 Appendix 3.1 Emerging disease ofamphibians cured by elevated body 232 temperature IX

10 LIST OF FIGURES Figure Page 1.1 Lifecycle ofbatrachochytrium dendrobatidis Map of stream sites in the Queensland wet tropics Healthy and diseased Litoria genimaculata a Boxplot ofproportion infected sections b Histogram ofdetection efficiency Infection prevalence ofadults and tadpoles Infection prevalence at lowlands and uplands Seasonal infection prevalence ofadults and tadpoles Infection prevalence ofadult Litoria genimaculata Position oflitoria genimaculata along Birthday Creek Morphological characteristics of rainforest stream frogs Condition and infection prevalence oflitoria genimaculata Percent fluctuating asymmetry ofrainforest stream frogs Seasonal population densities ofrainforest stream frogs Infection prevalence ofjuvenile and adult frogs Environmental conditions and infection prevalence Infection prevalence at six stream sites Dry, mist, and rain enclosures Daytime and nighttime behaviours ofjuvenile Litoria chloris Behaviour of infected and uninfected juvenile Litoria chloris Leukocyte populations ofinfected and uninfected Litoria chloris Amphibian chytrids stained with Calcofluor White Protein concentration in chytrid cultures five days after inoculation Chytrid sporangia density on flasks a,b Chytrid colonies after eight days at different temperatures c Chytrid colonies after eight days at different temperatures Amphibian chytrids on host tissues Total chytrid zoospore counts at seven and eleven days after inoculation 124 x

11 Figure 4.7 Proportion active to total zoospores at different temperatures Numbers ofamphibian chytrid colonies growing at different temperatures Protein concentration ofwhole chytrid cultures Zoospore activity through time Zoospore settlement through time Number offree zoospores modelled through time Number ofsporangia modelled through time Minimum inhibitory concentration (MIC) ofaustralian frog skin peptides Percent amphibian chytrid zoospore growth inhibition MIC equivalents per body weight ofaustralian frogs MIC equivalentsper body weight ofadult Australian frogs MIC equivalents per surface area of adult frogs Relationship between infection prevalence and immune function Litoria lesueuri skin peptide recovery by induction technique Skin peptide recovery from five Australian frog species 164 I 5.9 Correlationof frog survival and immune function Skin peptides recovered from wild infected and uninfected tadpoles Skin peptides recovered from adult frogs exposed and unexposed to chytrids Temperature regimes for Litoria genimaculata experiment Correlation ofskin peptide recovery and tadpole water temperature MIC equivalents at different life-history stages Chytrid zoospore growth inhibition assay example chart Thermal gradients Histogram ofpreferred temperatures oftadpoles in thermal gradients Boxplots ofpreferred temperatures oftadpoles in thermal gradients 186 Page Xl

12 LIST OF TABLES Table Page 1.1 Conservation status ofqueensland wet-tropics rainforest stream frogs Stream site descriptions Habitat and frog species occurrence at six stream sites Number ofjuvenile frogs found by season Environmental conditions at stream sites by season ANDVA table ofeffects on infection prevalence Infection risk ofrainforest stream frogs Excel data sheet for developmental instability analysis ANDVA table for developmental instability analysis Population surveys ofrainforest stream frogs Infection risk ofqueensland wet-tropics rainforest stream frogs Ecological and morphological characteristics ofrainforest stream frogs Survival oflitoria chloris in artificial environments Sensitivity and specificity ofchytrid infection diagnostic technique Leukocyte populations ofjuvenile Litoria chloris t\rnphibian chytrid survival and settlement rates Best parameter estimates for chytrid growth model Chytrid colony growth at different temperatures Sensitivity analysis ofchytrid growth model Variation in mean zoospore counts Anti-chytrid skin peptide defences ofaustralian frogs Minimum inhibitory concentration equivalents Skin peptide immune function oflitoria genimaculata 149 xii

13 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 oftertiary education. Information derived from the published or unpublished work ofothers has been acknowledged in the text and a list ofreferences is given. ----~-~-----~---~ (Name) _~}_~~~ ~~L_~~~~:_~ (Date) _ Xlll

14 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 ( 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

15 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.