The Structure and Function of Amphibian Skin Bacterial Communities and Their Role in ...

129 Pages·2014·1.87 MB·English

by  Jeni Walke

The Structure and Function of Amphibian Skin Bacterial Communities and Their Role in
Susceptibility to a Fungal Pathogen

Jenifer Banning Walke

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in
partial fulfillment of the requirements for the degree of

Doctor of Philosophy
In
Biological Sciences

Lisa K. Belden, Chair
Reid N. Harris
Dana M. Hawley
Ann M. Stevens

July 23, 2014
Blacksburg, Virginia

Keywords: amphibian, microbiota, transmission, symbiosis, structure-function, disease, fungus
The Structure and Function of Amphibian Skin Bacterial Communities and Their Role in
Susceptibility to a Fungal Pathogen

Jenifer B. Walke

ABSTRACT

As part of the ongoing loss of global biodiversity, amphibian populations are experiencing
declines and extinctions. A primary factor in these declines is the skin disease chytridiomycosis,
which is caused by the fungus Batrachochytrium dendrobatidis (Bd). Recent research suggests
that the amphibian skin microbiota has anti-Bd activity and may be an important factor in host
disease resistance. However, little is known about the basic ecology of this host-microbe
symbiosis, such as how much variation there is in microbial symbionts among host species and
populations, and the nature of symbiont transmission, culturability, and function. My dissertation
research addressed these basic questions in microbial ecology, as well as used a novel system to
examine the long-standing ecological theory of community structure-function relationships.
First, host-specificity, population-level variation and potential environmental transmission of the
microbiota were examined by conducting a field survey of bacterial communities from bullfrogs,
newts, pond water, and pond substrate at a single pond, and newts from multiple ponds. There
was variation among amphibian host species and populations in their skin symbionts, and, in a
host species-specific manner, amphibian skin may select for microbes that are generally in low
abundance in the environment. Second, the culturability of amphibian skin bacteria was assessed
by directly comparing culture-dependent and -independent bacterial sequences from the same
individuals. Although less than 7% of the amphibian skin microbes were captured using R2A
medium, most of the dominant bacteria were represented in our cultures, and similar patterns of
diversity among four amphibian species were captured with both approaches. Third, the
relationship between microbial community structure and function and selective forces shaping
structure and function were examined in bullfrogs by tracking microbial community structure
and function following experimental manipulation of the skin microbiota and pathogen exposure.
Results of this study demonstrated that Bd is a selective force on cutaneous bacterial community
structure and function, and suggest that beneficial states of bacterial structure and function may
ii
serve to limit infection and negative fitness consequences of Bd exposure. Using a combination
of observational and experimental approaches, my dissertation contributes to understanding
structure-function relationships of these complex symbiotic communities of vertebrates.

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Acknowledgements

I am thankful for the support and mentorship of my advisor, Lisa Belden. In addition to
investing time and effort into my development as a scientist, Lisa has provided me with
wonderful life opportunities and guidance, and for that I am thankful. I value the opportunity to
have Lisa as a mentor and a friend. I would also like to thank my advisory committee, Reid
Harris, Dana Hawley, and Ann Stevens, for their invaluable guidance throughout my graduate
program. I would like to especially thank Reid, who sparked my interest in amphibian skin
bacteria a decade ago at James Madison University and opened my eyes to the amazing
microbial world.
I thank the members of the Belden Lab (Matthew Becker, Sally Zemmer, Daniel Medina,
Myra Hughey, and Skylar Hopkins) for their friendship, support, and valuable discussions about
science. I would especially like to thank Matt Becker for the numerous insightful conversations
we’ve had while travelling to field sites and conferences, swabbing in the field, or pipetting
endlessly in the lab. I am also thankful for Laila Kirkpatrick’s invaluable advice regarding
molecular laboratory procedures, as well as the numerous undergraduate researchers who have
helped with my dissertation research.
I am grateful to the agencies, awards, and programs that have made this work possible,
including the Cunningham Doctoral Scholar Award, Robert and Marion Patterson Scholarship,
Perry Holt Scholarship, Virginia Tech Graduate Research and Development Program, The Fralin
Life Sciences Institute at Virginia Tech, Morris Animal Foundation, and the National Science
Foundation. Also, thanks to Virginia Tech’s Department of Biological Sciences and the
University of Virginia’s Mountain Lake Biological Station, whose people and scenery made
working very pleasant.
Finally, I thank my family. The unconditional love and support provided by my parents in
so many ways made this possible. I also thank my husband, Kirby, for helping with field work,
providing constant and reassuring motivation, and always offering a fun and unique perspective
on life. I am forever thankful for my daughter, Claire, who motivates and inspires me every day.
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Attributions

Chapter 2: Amphibian skin may select for rare environmental microbes

This chapter has been reproduced legally from The ISME Journal, 2014, online ahead of print,
doi:10.1038/ismej.2014.77

The published article included six additional authors:

Matthew H. Becker, Virginia Tech, Blacksburg, VA: contributed to the conceptual design of the
study, field and lab work, analysis, and manuscript development

Stephen C. Loftus, Virginia Tech, Blacksburg, VA: contributed to statistical analysis

Leanna L. House, Virginia Tech, Blacksburg, VA: contributed to statistical analysis

Guy Cormier, Virginia Tech, Blacksburg, VA: contributed to bioinformatics

Roderick V. Jensen, Virginia Tech, Blacksburg, VA: contributed to bioinformatics

Lisa K. Belden, Virginia Tech, Blacksburg, VA: contributed to the conceptual design of the
study, analysis, and manuscript development

Chapter 3: Linking culture-dependent and -independent characterizations of amphibian
skin microbial communities: Important insights into the use of probiotics in amphibian
conservation

The chapter included five additional authors:

Matthew H. Becker, Virginia Tech, Blacksburg, VA: contributed to the conceptual design of the
study, field and lab work, analysis, and manuscript development

Myra C. Hughey, Virginia Tech, Blacksburg, VA: contributed to field and lab work, analysis,
and manuscript development

Meredith C. Swartwout, Virginia Tech, Blacksburg, VA: contributed to field and lab work

Roderick V. Jensen, Virginia Tech, Blacksburg, VA: contributed to bioinformatics

Lisa K. Belden, Virginia Tech, Blacksburg, VA: contributed to the conceptual design of the
study, analysis, and manuscript development

v
Chapter 4: Community structure and function of amphibian skin microbes: an
experimental test with bullfrogs exposed to chytrid fungus

The chapter included six additional authors:

Matthew H. Becker, Virginia Tech, Blacksburg, VA: contributed to the conceptual design of the
study, field and lab work, analysis

Thais L. Teotonio, Temple University, Philadelphia, PA: contributed to metabolite profile
generation and analysis

Stephen C. Loftus, Virginia Tech, Blacksburg, VA: contributed to statistical analysis of
metabolite profiles

Leanna L. House, Virginia Tech, Blacksburg, VA: contributed to statistical analysis of
metabolite profiles

Kevin P. C. Minbiole, Villanova University, Villanova, PA: contributed to metabolite profile
generation and analysis

Lisa K. Belden, Virginia Tech, Blacksburg, VA: contributed to the conceptual design of the
study, analysis, and manuscript development

vi
Table of Contents
Abstract .................................................................................................................................ii

Acknowledgements ..............................................................................................................iv

Attributions ..........................................................................................................................v

Table of Contents .................................................................................................................vii

List of Tables ........................................................................................................................viii

List of Figures .......................................................................................................................ix

Chapter 1: General Introduction ......................................................................................1
Dissertation Research Overview ................................................................................6
References ..................................................................................................................6

Chapter 2: Amphibian skin may select for rare environmental microbes ..................... 16
Abstract ......................................................................................................................16
Introduction ................................................................................................................16
Materials and Methods ...............................................................................................19
Results ........................................................................................................................22
Discussion ........................................................................................................................... 25
References ..................................................................................................................29

Chapter 3: Linking culture-dependent and -independent characterizations of amphibian
skin microbial communities: Important insights into the use of probiotics in amphibian
conservation ..........................................................................................................................44
Abstract ......................................................................................................................44
Introduction ................................................................................................................44
Materials and Methods ...............................................................................................46
Results ........................................................................................................................53
Discussion ..................................................................................................................55
References ..................................................................................................................60

Chapter 4: Community structure and function of amphibian skin microbes: an
experimental test with bullfrogs exposed to chytrid fungus ............................................78
Abstract ......................................................................................................................78
Introduction ................................................................................................................79
Materials and Methods ...............................................................................................81
Results ........................................................................................................................90
Discussion ..................................................................................................................94
References ..................................................................................................................98

Chapter 5: Synthesis ...........................................................................................................115
References ..................................................................................................................118
vii
List of Tables

Chapter 2: Amphibian skin may select for rare environmental microbes

Table 1. List of amphibian core OTUs at all three sites, and the mean relative abundances of
those OTUs on amphibian skin and in the environment at Mountain Lake .............37

Chapter 3: Linking culture-dependent and -independent characterizations of amphibian
skin microbial communities: Important insights into the use of probiotics in amphibian
conservation

Table 1. List of the most abundant OTUs on each amphibian species, with the mean relative
abundances, prevalence, and culturability of these OTUs .......................................69

Table 2. List of the most abundant bacterial families on each amphibian species based on the
culture-independent characterization and whether there were cultured representatives
within the families across any species ......................................................................71

Chapter 4: Community structure and function of amphibian skin microbes: an
experimental test with bullfrogs exposed to chytrid fungus

Table 1. Experimental timeline showing field collection, bacterial treatment applications, Bd
exposures, and swabbing regime ..............................................................................109

Table 2. List of abundant OTUs across all samples and time points and whether they changed in
abundance significantly over time ............................................................................109
viii
List of Figures

Chapter 2: Amphibian skin may select for rare environmental microbes

Figure 1. Within-site variation in microbial communities .....................................................38

Figure 2. Venn diagram summarizing the overlap of environmental (pond water and substrate)
and amphibian (newts and bullfrog) OTUs at Mountain Lake ...............................39

Figure 3. Venn diagram showing overlap of newt OTUs across three sites ..........................40

Figure 4. Relative abundances of OTUs shared between amphibians and environmental samples
(pond water and pond substrate) .............................................................................41

Figure 5. Across-site variation in newt microbial communities ............................................42

Figure 6. Mean relative abundances of bacterial phyla across amphibian populations .........43

Chapter 3: Linking culture-dependent and -independent characterizations of amphibian
skin microbial communities: Important insights into the use of probiotics in amphibian
conservation

Figure 1. Phylogenetic tree of 259 culture-dependent OTUs (clustered at 97% sequence
similarity) ................................................................................................................72
Figure 2. Amphibian skin bacterial OTU richness (number of OTUs) and phylogenetic diversity
based on culture-independent and culture-dependent characterizations .................73
Figure 3. NMDS ordinations of Sorensen similarity and unweighted UniFrac distance matrices
for culture-independent and culture-dependent microbial communities associated with
four amphibian species ...........................................................................................74
Figure 4. Mean percentage of total OTUs “individually matched” and “species matched” to
cultured OTUs for each amphibian species ............................................................75
Figure 5. Phylogenetic tree of culture-independent OTUs, with culturability and mean relative
abundances of OTUs on each amphibian species shown ........................................76
Figure 6. Relative abundances of culture-independent bacterial phyla associated with bullfrogs,
newts, spring peepers, and toads .............................................................................77
Chapter 4: Community structure and function of amphibian skin microbes: an
experimental test with bullfrogs exposed to chytrid fungus
Figure 1. Conceptual model representing potential responses and interpretations of microbial
community structure and function in the presence of a pathogen ..........................110
ix
Figure 2. Effects of treatment on bullfrogs’ Bd infection intensity at day 7 and proportional
growth at day 42, and the relationship between individual bullfrogs’ Bd infection
intensity at day 7 and proportional growth at day 42..............................................111
Figure 3. NMDS ordinations based on weighted UniFrac distance matrices and Sorensen
dissimilarity matrices representing differences among bacterial treatments in microbial
community structure and metabolite profiles of frogs exposed and unexposed to Bd one
week after initial exposure to Bd ............................................................................112
Figure 4. Effects of Bd exposure and microbiota manipulation treatment (normal, antibiotics,
and augmented with J. lividum) on OTU richness, phylogenetic diversity, and relative
abundance of the probiotic J. lividum on bullfrog skin one week following exposure to
Bd ............................................................................................................................113
Figure 5. Effects of microbiota manipulation treatment (normal, antibiotics, and augmented with
J. lividum) on changes in OTU richness and phylogenetic diversity of bullfrogs’ skin
microbiota from the field to day 0 ..........................................................................114
x