Global amphibian declines

and
The evolution of chytridiomycosis resistance in
Lowland Leopard Frogs

Anna Savage, Zamudio Lab, Cornell University

 PART ONE
Global amphibian declines
and chytridiomycosis
(the big picture)

PART TWO
Lowland leopard frogs and
chytridiomycosis
(a little piece of the puzzle)
 PART ONE
Global amphibian declines
and chytridiomycosis
(the big picture)

1. What is chytridiomycosis?

2. Why should you care about it?

3. Where did it come from?

 Where Have All the Froggies Gone?
Marcia Barinaga. 1990. Science 247: 1033-1034

Some factors pose a threat to all organisms, such as habitat

loss, invasive species, pollution, and climate change

Some threats are specific to amphibians:

- amphibians have sensitive skin and use both
terrestrial and aquatic habitats, making them
indicator species = early warning indicators of
environmental change
- Irridoviruses cause disease,
mostly in salamanders
- Parasites cause limb deformities,
mostly in frogs
- Chytridiomycosis is a deadly
fungal disease causing declines
in hundreds of amphibian species
1. What is chytridiomycosis?

“chytrid”

- Chytrids are a large group of fungi

containing over 1,000 species

- Only one species, Batrachochytrium

dendrobatidis (Bd for short), causes
disease in amphibians
 1. What is chytridiomycosis?
Below is a microscope slide of skin
from a frog with chytridiomycosis
The arrow points to one flask-shaped Bd organism
These fungi:
-are single-celled
-reproduce asexually
-consume the keratin in
-amphibian skin cells
-have flagellat zoospores
(spores swim and have tails)

From Bradley et al. 2002

 1. What is chytridiomycosis?

Bd kills frogs by disrupting their osmoregulation

This eventually leads to cardiac arrest

 2. Why should you care?
(even if you don’t like frogs)

Litoria chloris
(Australian tree frog)
skin peptides block
HIV-1 infection of
human T-cells
(J. Virol. 79: 11598)
Photo by D. Woodhams

This species is declining from chytridiomycosis (IUCN 2008)

How many frogs might produce useful skin compounds?

We can never know for the species that have disappeared.
 2. Why should you care?
(even if you don’t like frogs)
Gastric brooding frogs
(The genus
Rheobatrachus,
containing 2 species)

Mom swallows eggs and

incubates them in her
stomach!
A unique form of reproduction that can never be
studied because both species went extinct from
chytridiomycosis in the 1980s (IUCN 2008)
 2. Why should you care?
(even if you don’t like frogs)
Since chytridiomycosis was first noticed in the1970s, fungal
diseases have started to emerge in other groups of animals:
1. White-nose syndrome
in bats: noticed in 2007,
caused by the fungus
Geomyces destructans
(Mycotaxon 2009)

Photo by Al Hicks, NYDEC

2. Sharp increase in the human fungal
disease zygomycosis since the 1990s
(Am J Health-Syst Pharm 2005)

Frogs may have been the first indicator of a larger problem

3. Where did Bd come from?
Global distribution of Bd in 2010

http://www.spatialepidemiology.net/bd-maps
 3. Where did Bd come from?
The First Hypothesis:
Bd is an Emerging Infectious Disease spread from Africa

Weldon et al. 2004. Emerging Infectious Diseases 100: 2100-2105

-Timeline shows the oldest record of Bd infecting a frog

on each continent
-Oldest record is from Africa
-The group of frogs infected (Xenopus) were shipped
around the world for scientific research in the 1960s
-This is when Bd showed up elsewhere, so we think the
fungus may have been transported with these frogs
 3. Where did Bd come from?
The Second Hypothesis:
Chytridiomycosis emerged due to climate change
Average global temperatures over time
Chytridiomycosis starts here (1970s)

Chytridiomycosis starts at
the same time that global
average temperature
increases
(red line vs. green line)

Alford et al. 2007 Nature

Bd may have been present in the environment for a long time,

but has only recently been causing disease because of new
climate patterns that suit the biology of the fungus
 PART ONE
Global amphibian declines
and chytridiomycosis
(the big picture)

PART TWO
Lowland leopard frogs and
chytridiomycosis
(a little piece of the puzzle)
 PART TWO
Lowland leopard frogs and
chytridiomycosis
(a little piece of the puzzle)
1. Anna’s study system

2. Anna’s field methods

3. Chytridiomycosis in Lithobates yavapaiensis

4. Anna’s lab methods

5. Results
 1. Anna’s study system
The Lowland Leopard Frog
(Lithobates yavapaiensis)

Native to Arizona, USA and

Sonora, Mexico
Chytridiomycosis was first documented in the 1980s
1. Anna’s study system

Chytridiomycosis
in Lithobates
yavapaiensis
Other native
Arizona
animals
 1. Anna’s study system
Habitats vary from riparian forest to open desert….
1. Anna’s study system
1. Anna’s study system
1. Anna’s study system
 2. Anna’s field methods
Step 1: Go to study sites in Arizona and catch frogs
 2. Anna’s field methods

Step 2: Collect samples (tissue to look at frog

genes and skin swabs to test for Bd infection)
 2. Anna’s field methods
Step 3: Take lots of photos
and field notes
 2. Anna’s field methods

Step 4: Process samples back in the lab

3. Chytridiomycosis in Lithobates yavapaiensis

Each
black dot
is a study
population
of frogs
3. Chytridiomycosis in Lithobates yavapaiensis

Bd + /
disease +

Bd + /
disease -

Bd - /
disease -

Populations fall into 3 disease categories

3. Chytridiomycosis in Lithobates yavapaiensis

Within each population, only some

individuals die from chytridiomycosis, while
others are infected but survive
3. Chytridiomycosis in Lithobates yavapaiensis

Main research question:

What is the role of frog genetics in
determining chytridiomycosis
susceptibility or resistance?

vs.
4. Anna’s lab methods
Part A: Processing the swabs to
measure Bd infection

-Extract DNA from swabs

using chemicals and
mechanical separation by
weight

-Perform quantitative
Polymerase Chain Reaction
(qPCR) to measure the
amount of Bd DNA in each
sample
 4. Anna’s lab methods
Part A: Processing the swabs to
measure Bd infection
-Each cycle of qPCR
doubles the amount of
DNA in the sample

-Samples with more Bd

DNA will be detectable
at an earlier cycle

-Which sample has

more DNA here: the red
or the yellow?
http://depts.washington.edu/mgcore/services.htm
4. Anna’s lab methods
Part B: Processing the tissue to
look at the genetics of each frog

-Perform Polymerase Chain

Reaction (PCR) on frog DNA
to isolate and amplify (=make
copies of) the gene of interest

-Sequence the gene

(=determine the combination
of nucleotides that make up
the gene in each frog)
 4. Anna’s lab methods
How PCR works:
1. Denature: Separate the
DNA double helix into two
strands by heating it up

2. Anneal: Add short DNA

chunks (primers) that bind
to the gene of interest

3. Extension: Enzymes add

nucleotides onto the
primers to get 2 gene
http://users.ugent.be/~avierstr/principles/pcr.html
copies from one

Repeat 30-40 times to get thousands of gene copies

 4. Anna’s lab methods
Sequencing the genes

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

Run the PCR gene copies through a sequencer to

determine the order of nucleotides (Adenine, Guanine,
Cytosine and Thymine make up all of our DNA)

Different versions of the gene with different functions will

have slightly different combinations of As, Cs, Ts and Gs
 5. Results

A PhD project has many components.

The next few slides will show just a few
of the results so far:

Population genetics

MHC gene sequencing

Experimental infections
5. Results: population genetics
-Shown is a
genetic
representation of
frog populations
-Each color
represents a
different genetic
group
-All populations are
mostly a unique
color, meaning
they are all
genetically different
 5. Results: MHC genetics

What is MHC?
The Major Histocompatibility Complex
(=MHC) is a group of genes that code for the
components of animal immune systems

Humans have hundreds of MHC genes, and

so do frogs (frog MHC gene organization
shown to the left)

These genes determine whether or not an

animal can fight off disease such as
chytridiomycosis

Ohta et al. 2006 J Immunol 176: 3674 –3685.

 5. Results: MHC genetics
A useful analogy:
Gene  Protein
Blueprints  House
MHC genes are instructions for making MHC
proteins that exist in our cells and attack and
destroy diseases


MHC genes might determine whether or
not a frog can fight off Bd infections
 5. Results:
MHC genetics

Phylogenetic tree showing

evolutionary relationships
of MHC genes sequenced
from Lowland Leopard
Frogs

Size of the circles shows

how many times we found
a frog with that MHC
sequence

Big circles = common

sequences
 5. Results:
MHC genetics

Associated with
resistant populations

Associated with
susceptible populations

Chytridiomycosis
susceptibility is related to
which MHC genes are
present in a population!
 5. Results: Experimental infections

Collect egg masses from

Bring them back to the lab
different populations in Arizona

Arizona Bd
?
After 7 months they are
ready for metamorphosis

Hatch the tadpoles and raise

Infect half of the frogs with them in a “common garden” =
Bd and track survival no environmental variation
 5. Results: Experimental infections
Survival across populations (N=149 frogs)

• Three populations have 100% mortality after 30 days

• Two populations have some individuals that survive the experiment!
• Because we controlled for environmental variation, we know the cause
of the differences in survival is genetic
Conclusions
Frog genetics help
determine chytridiomycosis
susceptibility or resistance

This is important because it

means other frog species
may be able to evolve
resistance to this disease
over time
 Conservation implications
Lithobates onca
5 remaining natural populations

Photo by T. Brennan

Lithobates chiricahuensis
Federally listed as endangered
Photo by E. Enderson

Both of these species have declined at least in part

due to chytridiomycosis

Genetic markers identified in L. yavapaiensis can be used in

captive breeding programs for these species
 PEOPLE Acknowledgements
Arizona
Mike Sredl
Mike Booth
Ashley Martin
Dennis Caldwell
Phil Rosen
Phil Hedrick
Jim Collins
Martin Lawrence
Bob Rogers
Mark Haberstitch
Taylor Edwards
Martin Schlaepfer
Betty Davidson

Cornell FUNDING
Kelly Zamudio Cornell Student Environmental Research Grant
Rick Harrison Cornell Presidential Genomics Fellowship
Ton Schat Andrew W. Mellon Student Research Grant
Jon Richmond Amherst College Graduate Fellowship
Steve Bogdanowicz National Science Foundation Graduate Research Fellowship
Abby Duvall National Science Foundation Doctoral Dissertation Improvement Grant
Angela Stevenson National Geographic Society Young Explorers Grant
Melissa Lenker Crossing Boundaries Conservation Scientist fellowship