Ecology of the Amphibian Skin Associated Microbiome and Its Role in Pathogen Defense

168 Pages·2017·24.89 MB·English

University of Colorado, Boulder
CU Scholar
Ecology & Evolutionary Biology Graduate Theses &
Ecology & Evolutionary Biology
Dissertations
Spring 1-1-2015
Ecology of the Amphibian Skin Associated
Microbiome and Its Role in Pathogen Defense
Jordan Gehb Kueneman
University of Colorado Boulder, [email protected]
Follow this and additional works at:https://scholar.colorado.edu/ebio_gradetds
Part of theBiology Commons,Ecology and Evolutionary Biology Commons, and theMolecular
Biology Commons
Recommended Citation
Kueneman, Jordan Gehb, "Ecology of the Amphibian Skin Associated Microbiome and Its Role in Pathogen Defense" (2015).Ecology
& Evolutionary Biology Graduate Theses & Dissertations. 76.
https://scholar.colorado.edu/ebio_gradetds/76
This Dissertation is brought to you for free and open access by Ecology & Evolutionary Biology at CU Scholar. It has been accepted for inclusion in
Ecology & Evolutionary Biology Graduate Theses & Dissertations by an authorized administrator of CU Scholar. For more information, please contact
[email protected].
ECOLOGY
 OF
 THE
 AMPHIBIAN
 SKIN-­‐ASSOCIATED

 MICROBIOME
 AND
 ITS
 ROLE
 IN
 PATHOGEN
 DEFENSE

by

Jordan
 Gehb
 Kueneman

B.S.
 University
 of
 Utah,
 2010

A
 thesis
 submitted
 to
 the

Faculty
 of
 the
 Graduate
 School
 of
 the

University
 of
 Colorado
 in
 partial
 fulfillment

of
 the
 requirement
 for
 the
 degree
 of

Doctor
 of
 Philosophy

Department
 of
 Ecology
 and
 Evolutionary
 Biology

2015
This
 thesis
 entitled:

 Ecology
 of
 the
 Amphibian
 Skin-­‐associated

Microbiome
 and
 Its
 Role
 in
 Pathogen
 Defense

written
 by
 Jordan
 G.
 Kueneman

has
 been
 approved
 by
 the
 Department
 of
 Ecology
 and
 Evolutionary
 Biology

_________________________________________

Valerie
 McKenzie

_________________________________________

Andrew
 Martin

_________________________________________

Sharon
 Collinge

_________________________________________

Reid
 Harris

_________________________________________

Steve
 Schmitt

Date__________________

The
 final
 copy
 of
 this
 thesis
 has
 been
 examined
 by
 the
 signatories
 and
 we

find
 that
 both
 the
 content
 and
 the
 form
 meet
 acceptable
 presentation
 standards

of
 scholarly
 work
 in
 the
 above
 mentioned
 discipline.

IACUC
 protocol
 #
 __
 1112.01_&_
 1104.05_______________
iii

Kueneman,
 Jordan
 Gehb
 (Ph.D.,
 Ecology
 and
 Evolutionary
 Biology)

Thesis
 directed
 by
 Associate
 Professor
 Valerie
 J.
 McKenzie

ECOLOGY
 OF
 THE
 AMPHIBIAN

SKIN-­‐ASSOCIATED
 MICROBIOME
 AND
 ITS
 ROLE
 IN
 PATHOGEN
 DEFENSE

ABSTRACT

Ecologists
 have
 long
 been
 challenged
 to
 understand
 the
 role
 of
 host-­‐associated
 microbial

communities
 in
 host
 health
 and
 pathogen
 defense.
 Recent
 technological
 advances
 in
 high

throughput
 sequencing
 have
 allowed
 scientists
 to
 observe
 the
 immense
 microbial
 diversity

of
 host-­‐associated
 habitats.

 However,
 we
 are
 only
 beginning
 to
 appreciate
 evolutionary

interactions
 of
 the
 host
 and
 its
 microbiome.
 The
 ability
 to
 mitigate
 diseases
 of
 host

organisms
 through
 biological
 manipulations
 of
 the
 host’s
 biotic
 community
 is
 a
 new

research
 area
 for
 wildlife
 disease
 managers.
 One
 understudied
 area
 is
 how
 amphibian
 skin-­‐
associated
 microbial
 communities
 interact
 with
 a
 known
 fungal
 pathogen
 and
 how
 they

may
 function
 to
 protect
 the
 host
 from
 developing
 disease.
 In
 order
 to
 address
 this

understudied
 area,
 the
 first
 objective
 is
 to
 characterize
 the
 patterns
 of
 natural
 variation

within
 species,
 across
 populations,
 and
 through
 developmental
 life
 stages
 of
 amphibians.

This
 will
 elucidate
 the
 ecological
 drivers
 of
 host-­‐associated
 microbiota
 and
 how
 pathogen-­‐
inhibiting
 microbes
 are
 distributed
 across
 species,
 space,
 and
 life
 stages.
 The
 second
 goal
 is

to
 test
 whether
 an
 amphibian
 skin
 microbiome
 can
 be
 manipulated
 to
 provide
 a
 protective

effect
 against
 the
 fungal
 pathogen.
 How
 amphibian
 skin-­‐associated
 microbial
 communities

respond
 to
 pathogen
 invasion
 and
 probiotic
 applications
 may
 provide
 unique
 insight
 into
iv

invasion
 ecology
 and
 disturbance
 ecology
 of
 the
 host-­‐associated
 microbiota.

 Amphibians

and
 diverse
 bacteria
 and
 fungi
 that
 inhabit
 their
 skin
 provide
 an
 excellent
 model
 system
 for

studying
 interactions
 between
 hosts,
 their
 microbial
 communities,
 and
 pathogens.

Amphibian
 skin
 can
 be
 repeatedly
 and
 nondestructively
 sampled,
 and
 can
 be
 manipulated

through
 antibiotics
 or
 bioaugmentation.
 This
 system
 also
 offers
 an
 opportunity
 to
 develop

therapeutic
 tools
 for
 one
 of
 the
 world’s
 most
 threatened
 taxa.
v

DEDICATION

This
 thesis
 is
 dedicated
 to
 my
 parents
 Caroline
 Kueneman
 and
 Eric
 Kueneman
 for
 their

support
 in
 my
 pursuit
 of
 advanced
 training
 in
 Biology.

And
 to
 Alba
 Diana
 Vilar
 and
 Fabiola
 Vilar
 for
 their
 consistent
 support
 accommodating
 the

logistics
 of
 this
 research
vi

ACKNOWLEDGEMENTS

First
 and
 foremost
 I
 have
 to
 thank
 my
 advisor
 Valerie
 McKenzie,
 who
 has
 trained
 me
 to

become
 a
 professional
 ecologist.
 Her
 mentoring
 strategy
 provided
 an
 appropriate
 balance

of
 breath
 and
 depth.
 I
 also
 thank
 my
 dissertation
 committee,
 Sharon
 Collinge,
 Andrew

Martin,
 Steve
 Schmitt
 and
 Reid
 Harris
 who
 provided
 valuable
 guidance,
 advice
 and

resources
 throughout
 my
 dissertation.

 Several
 post-­‐docs
 and
 graduate
 students
 in
 the

Mckenzie
 Lab
 including,
 Douglas
 Woodhams,
 Lissette
 Arelleno,
 Christine
 Avena,
 Anna

Perteson,
 Se
 Jin
 Song,
 are
 great
 colleagues
 and
 friends.
 I
 hope
 we
 can
 work
 and
 play

together
 in
 the
 future.
 A
 special
 thanks
 is
 given
 to
 Holly
 Archer
 for
 her
 help
 with
 labwork,

molecular
 knowledge
 and
 assistance.
 Additional
 graduate
 students
 of
 substantial
 support

include
 Max
 Joseph,
 Antonio
 Gonzalez
 and
 Matt
 Wilkins.

 I
 would
 like
 to
 acknowledge

Colorado
 Parks
 and
 Wildlife
 for
 assistance
 in
 sample
 collection
 and
 Bd
 prevalence
 data
 at

sample
 sites.
 In
 particular,
 Kevin
 Thompson,
 Boyd
 Wright,
 Jennifer
 Logan,
 and
 Paul
 Jones

assisted
 in
 sample
 collection
 and
 site
 access.
 Alba
 Diana
 Vilar
 provided
 extraordinary
 field

help.
 We
 also
 thank
 Joseph
 Grant
 State
 Park,
 and
 California
 Fish
 and
 Game
 for
 access
 to

sites.
 Katie
 Richgels
 thank
 you
 for
 guidance
 on
 amphibian
 sampling
 in
 the
 Central
 Valley

region
 in
 CA
 and
 Jonah
 Piovia-­‐Scott
 and
 Karen
 Pope
 in
 providing
 expertise
 regarding

amphibian
 populations
 in
 northern
 California.
 I
 especially
 would
 like
 to
 thank
 the
 Ecology

and
 Evolutionary
 Biology
 Department,
 of
 the
 University
 of
 Colorado
 in
 Boulder,
 and
 in

particular
 Jill
 Skarstad.

 Thank
 you
 for
 all,
 for
 your
 expertise
 and
 kindness,
 I
 appreciate

your
 efforts.
vii

CONTENTS

CHAPTER

I.

  Introduction
 ..................................................................................................................................................
 1

Organization
 of
 the
 dissertation

 .........................................................................................................
 1

Context:
 Applying
 metagenomic
 tools
 to
 amphibian
 conservation
 .....................................
 4

II.

  The
 amphibian
 skin-­‐associated
 microbiome
 across
 species,
 space
 and
 life
 history

stages

 ..............................................................................................................................................................
 9

ABSTRACT
 ...........................................................................................................................................
 9

Introduction
 .................................................................................................................................
 10

Methods
 .........................................................................................................................................
 14

Results
 ............................................................................................................................................
 23

Discussion
 .....................................................................................................................................
 37

Appendix
 A
 ...................................................................................................................................
 42

III.

  Inhibitory
 bacteria
 reduce
 fungi
 on
 early
 life
 stages
 of
 endangered
 Colorado
 boreal

toads
 (Anaxyrus
 boreas)
 .......................................................................................................................
 43

ABSTRACT
 ........................................................................................................................................
 43

Introduction
 .................................................................................................................................
 44

Methods
 .........................................................................................................................................
 47

Results
 ............................................................................................................................................
 53

Discussion
 .....................................................................................................................................
 64

Appendix
 B
 ...................................................................................................................................
 72

IV.

  Captivity
 leads
 to
 the
 loss
 of
 bacterial
 symbiont
 diversity
 and
 increased
 fungal

infection
 .......................................................................................................................................................
 87

ABSTRACT
 ........................................................................................................................................
 87

Introduction
 .................................................................................................................................
 88

Results
 ............................................................................................................................................
 91

Methods
 .........................................................................................................................................
 96

Discussion
 ...................................................................................................................................
 108

Appendix
 C
 ..................................................................................................................................
 115

V.

  Summary
 and
 future
 of
 amphibian
 microbiome
 research

 ...................................................
 118

REFERENCES
 ..............................................................................................................................................................
 127
viii

TABLES

Table

2.1

  Samples
 included
 from
 each
 region,
 site,
 amphibian
 species,

and
 life-­‐history
 stage.
 ............................................................................................................................
 17

4.1

  Probiotics
 used
 and
 their
 origin

 ......................................................................................................
 93

 SUPPLEMENTARY
 TABLES

B.1

  For
 supplemental
 tables
 B.1-­‐5,
 please
 see
 published
 data
 (To
 large
 for
 thesis)
 ...............

C.1

  Metadata
 of
 amphibian
 toad
 sampling
 including
 species,
 location
 and
 number
 of

samples
 collected
 ....................................................................................................................................
 85

C.2

  Summary
 statistics
 for
 PERMANOVA
 analysis
 including
 factors
 Life
 stage,
 Sample

Site
 and
 Bd
 presence
 or
 absence

 .....................................................................................................
 86

FIGURES

2.1

  Map
 of
 amphibian
 sampling
 areas:
 Joseph
 Grant
 Park,

California’s
 Central
 Valley
 and
 the
 Trinity
 Alps,
 in
 Northern
 California.

 .......................
 16

2.2

  Alpha
 diversity
 of
 amphibian
 species,

life-­‐history
 stages,
 and
 environmental
 samples

 .......................................................................
 25

2.3

 Microbial
 communities
 associated
 with
 amphibian

skin
 are
 differentiated
 across
 host
 species
 in
 the
 Central
 Valley

 ......................................
 26

2.4

  Heatmap
 comparing
 the
 relative
 abundances
 of
 selected
 OTUs

across
 life-­‐history
 stages
 of
 R.
 cascadae,
 other

amphibian
 species,
 and
 environmental
 samples

 .....................................................................
 28

2.5

  Amphibian
 skin
 bacterial
 communities
 is
 structured

by
 geographic
 locations
 within
 host
 species

 ..............................................................................
 29

2.6

  Bacterial
 communities
 associated
 with
 amphibian
 skin
ix

vary
 according
 to
 life
 cycle
 stage
 within
 R.
 cascadae

 .............................................................
 31

3.1

  Life
 stage
 is
 the
 best
 predictor
 of
 community
 composition
 of
 skin
 associated
 bacteria

found
 on
 Anaxyrus
 boreas.
 ...................................................................................................................
 55

3.2

  Proportional
 abundance
 of
 bacterial
 and
 micro-­‐eukaryotic
 OTUs
 found
 across

developmental
 life
 stages
 of
 Anaxyrus
 boreas
 ............................................................................
 57

3.3

  Shannon
 diversity
 of
 bacterial
 and
 micro-­‐eukaryotic
 organisms
 through

developmental
 life
 stages
 ....................................................................................................................
 59

3.4

  Network
 interactions
 between
 bacteria
 and
 fungal
 taxa
 found
 on
 Anaxyrus
 boreas

summarized
 by
 significant
 negative
 interactions
 between
 bacterial
 and
 Fungal
 OTUs

 .........................................................................................................................................................................
 61

3.5

  Percent
 Bd-­‐inhibitory
 bacteria
 found
 across
 life
 stages
 and
 environment
 of
 Anaxyrus

boreas
 summarized
 by
 abundant
 taxa
 ...........................................................................................
 63

4.1

  Comparing
 alpha
 and
 beta
 diversity
 of
 captive
 and
 wild
 Anaxyrus
 boreas
 with

environmental
 samples,
 summarized
 by
 abundant
 taxa
 found
 on
 sample
 types.
 ......
 98

4.2

  Shannon
 diversity
 of
 Bd-­‐inhibitory
 bacteria
 found
 on
 captive
 and
 wild
 Anaxyrus

boreas,
 summarized
 by
 abundant
 Bd-­‐inhibitory
 taxa
 found
 on
 sample
 types
 ...........
 100

4.3

  Proportion
 and
 composition
 of
 Bd-­‐inhibitory
 bacteria
 found
 on
 exposed
 individuals

summarized
 through
 time
 .................................................................................................................
 102

4.4

  Proportion
 of
 Bd-­‐inhibitory
 bacteria
 found
 at
 the
 time
 of
 exposure
 to
 fungal

pathogen
 Bd,
 subsequent
 infection
 incidence
 is
 summarized
 by
 exposure
 trial.
 .....
 105

4.5
  Anaxyrus
 boreas
 response
 to
 probiotic
 treatment
 reveals
 decreased
 morbidity
 and

morality
 effects,
 microbial
 communities
 are
 in
 part
 explained
 by
 Bd
 infection

intensity
 .....................................................................................................................................................
 106

5.1
  Immune
 functions
 of
 amphibians
 through
 development
 ....................................................
 122

SUPPLEMENTAL
 FIGURES

B.1

  Proportional
 abundance
 of
 bacterial
 OTUs
 by
 individual
 amphibian
 sampled
 ...........
 35