Download the ecology of chytridiomycosis, an emerging infectious disease of

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.