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Topics in Modern Biology: Population
Collapses: Causes and
Consequences
Global Amphibian Declines
Lecture 2 Part 2
Lemur leaf frog
Hylomantis lemur
Louise A. Rollins-Smith
Amphibian declines: Mitigation
strategies
• Removal of introduced species
• Restoration of habitat
• Survival assurance colonies
• Probiotics or Bioaugmentation
• Immunization
Golden coqui
• The continuing crisis of amphibian declines
and extinctions prompts action.
• However, successful mitigation strategies
are mostly hypothetical.
• Not all amphibian species are declining, and
rescue of any one species will depend on
the unique ecological context of that
species.
Amphibian declines: Mitigation
strategies
• Removal of introduced species • California mountain legged frogs (Rana
muscosa and Rana sierrae) were historically
abundant in high elevation lakes in the Sierra
Nevada mountains.
• Beginning in the 1850s and continuing with the
help of California Department of Fish and Game
from the 1920s, lakes were stocked with
fingerling trout.
• When fish were removed by netting from
experimental lakes, the frog populations
rebounded (Knapp, R. et al. 2007. Biol.
Conserv. 135: 11-20.) Rana muscosa
Amphibian declines: Mitigation
strategies
Amphibian declines: Mitigation
strategies • Restoration of habitat • Kihansi spray toad of Tanzania
(Nectophrynoides asperginis)
was declared extinct in the wild
in 2009.
• Conservationists rescued toads
and brought them to the Bronx
Zoo and Toledo Zoo.
• World bank funded a project to
reproduce a misting system.
• In 2012, offspring of the original
toads held in captive assurance
colonies were returned.
Kihansi spray toad
Amphibian declines: Mitigation
strategies • Survival Assurance Colonies • Amphibian Ark (Kevin Zipple)
• Joint effort of the World Association of Zoos and
Aquariums (WAZA), the International Union for
Conservation of Nature (IUCN) species survival
commission (SSC) (volunteers) Conservation Breeding
Specialist Group (CBSG), and the Amphibian Survival
Alliance (ASA).
• International consortium
– Rescue
– Captive breeding
Kihansi spray toad
Amphibian declines: Mitigation
strategies • Survival Assurance Colonies
• Smithsonian Amphibian Conservation Program
(Brian Gratwicke)
• The Panama Amphibian Rescue and
Conservation Project (part of amphibian ark
effort)
• El Valle amphibian conservation center
(EVACC)
EVACC
Gamboa Amphibian Rescue Center
Amphibian declines: Mitigation
strategies • Survival Assurance Colonies
• The Panama Amphibian Rescue and Conservation
Project (part of amphibian ark effort)
Gamboa Amphibian Rescue Center
Golden frog (Atelopus zeteki)
Amphibian declines: Mitigation
strategies • Survival Assurance Colonies
• Two geographical populations of Boorolong frogs have
been established at Taronga Zoo, Sydney and the
Amphibian Research Centre (part of amphibian ark
effort) in Australia
• A reintroduction program is in place, in addition to
research on immunity to chytrid fungus
•
Booroolong Frog
Litoria booroolongensis
Model of immune defenses in
the skin
G G
M M
Dermis
Mucus:
Antimicrobial
peptides,
lysozyme,
antibodies,
and bacterial
products
Epidermis
Mucus
M
B
Zoospore
B
Granular gland
T DC
Granular gland
Bacteria
Sporangia
Amphibian declines: Mitigation
strategies • Probiotics or Bioaugmentation
• Amphibian skin hosts a rich array of skin
microbes
• Many can be cultured on simple media (R2A)
agar
Amphibian declines: Mitigation
strategies • Probiotics or Bioaugmentation
• Amphibian skin hosts a rich array of skin microbes
• Many species can inhibit growth of Bd
Bd on agar
Inhibitory species
of bacteria
Control bacteria
No inhibition
Amphibian declines: Mitigation
strategies • Probiotics or Bioaugmentation
• Microbial species capable of inhibiting Bd are more
prevalent in Bd-endemic populations than naive and
declining populations of mountain yellow legged
frogs.
Rana muscosa
1
10
100
1000
10000
Sixty Lake (Declining) Conness (Persisting)
Me
dia
n I
nfe
cti
on
In
ten
sit
y
(zo
os
po
re e
qu
iva
len
ts)
Populations of Rana muscosa
*
0
10
20
30
40
50
60
70
80
90
Sixty Lake (Declining) Conness (Persisting)Pe
rce
nt
of
Fro
gs
wit
h A
nti
-Bd
b
ac
teri
a
Populations of Rana muscosa
* Woodhams et al.,
2007. Biol. Conserv.
138: 390-398.
Probiotics or Bioaugmentation
• One naturally
occurring bacterial
species,
Janthinobacterium
lividum, produces an
antifungal metabolite
called violacein.
• Addition of bacteria to
R. muscosa juveniles
protected from Bd
infection. Harris R et al. 2009. ISME J. 3: 818–824
Probiotics or Bioaugmentation
• When J. lividum was used to try to protect
Panama golden frogs, it did not persist and they
were not protected.
Golden frog (Atelopus zeteki)
Becker M. et al. 2011. EcoHealth 8:501-506
R. sphenocephala metamorphs
• Raised from eggs in mesocosms by Shane Hanlon working with Matt
Parris, U. of Memphis.
• Mesocosms had pond water, soil, leaf litter, algae, and insects from the
environment where R. sphenocephala eggs were collected.
• Real time PCR confirms that every metamorph was Bd-negative.
• By mass spectrometry, the juveniles did not consistently express
antimicrobial peptides.
Mesocosm
• Many bacterial isolates from juvenile frogs inhibit growth
of Bd
0%
25%
50%
75%
100%
125%
Ne
g
1 2 3 4 5 6 7 8 9 10
11
12
13
14
15
16
17
18
19
20
21
23
24
25
28
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
% G
row
th n
orm
ali
ze
d t
o P
os
itiv
e
Co
ntr
ol
OTU
Heat-killed Bd
Bd + Supernatant
*
*
*
*
* * *
*
* *
*
*
*
*
*
*
* *
*
*
*
*
*
R. sphenocephala metamorphs
Holden, W. et al.
submitted.
• Microbiome is diverse in these young frogs.
R. sphenocephala metamorphs
B
D
ProteobacteriaActinobacteriaBacteriodetesFirmicutesDeinococcus-Thermus
α-Proteobacteria β-Proteobacteria γ-Proteobacteria ActinobacteriaBacilliFlavobacteriiaDeinococci
Holden, W. et al.
submitted.
0
10
20
30
40
Bacteria-Intact Bacteria-Reduced
cfu
/ s
wa
b
Pre-Treatment Post-Treatment
A
A
A
B
Bacterial reduction experiment
• Reduction of skin bacteria using a cocktail of antibiotics.
• Controls had mesocosm water to maintain bacteria.
• Bd infection of bacteria-reduced frogs resulted in
increased burden of Bd, but survival was not improved.
0
50,000
100,000
150,000
200,000
Bacteria-, Bd+ Bacteria+, Bd+
Zo
osp
ore
Eq
uiv
ale
nts
*
Holden, W. et al.
submitted.
Summary
• Skin microbiome probably plays an
important role in protection of amphibian
skin from pathogens.
– Direct competition
– Production of antifungal metabolites
• The microbial ecology of the skin is poorly
understood.
• Bacterial reduction and augmentation to
protect from Bd is still in the future.
• Immunization to protect valuable threatened
species in captivity is one possible way to
provide a population of resistant breeders for
re-population studies.
• Exposure to Bd followed by clearance with heat
or antifungal drugs might be a way to induce
immunity.
Rana muscosa
Amphibian declines: Mitigation
strategies
Booroolong Frog
Does immunization protect vulnerable
species?
0
.2
.4
.6
.8
1
Cu
m. S
urv
iva
l
Bo
rea
l To
ad
s
0 5 10 15 20 25 30 35 Time
Naïve/Exposed
All controls
Bd/Exposed (106 zsp)
APBS/Exposed
Kaplan-Meier Cum. Survival Plot for Survival
Censor Variable: Censoring
Grouping Variable: Group
Boreal toads were not protected from lethal exposure (105 zsp) by
immunization via dorsal lymph sac route. Likewise R. muscosa were not
protected (Stice, M and Briggs, C, 2010, J. Wildl. Dis. 46:70-77). Why?
Cindy Carey and Lauren Livo
Two opinions about possible success of
vaccine strategy
• Bd infection
followed by
clearance will not
provide protection
from later exposure
• Bd infection
followed by
clearance may
provide protection
Bd infection followed by induced
clearance will not provide protection
from later exposure
• In booroolong frogs (Litoria
booroolongensis) previous exposure and
clearance was not effective (Cashins S.D., et al. 2013. PLoS
ONE 8(2): e56747).
• One exposure (1.5 x 105/ml), clearance by
itraconazole treatment
• No protection of booroolong frogs (L.
booroolongensis) re-infected at 110 days
Bd infection followed by clearance may
provide protection
• In Archey’s frog, some evidence for
protection (Shaw, S.D. et al. Dis. Aquat Organ. 2010. 92: 159–163)
• Leiopelma archeyi infected with Bd “self-
cured”.
• Re-exposed to 2.5 x105 Bd zoospores.
Became infected and then cleared
infections again.
Proposed Skin Immunization protocol
Paint on killed Bd + killed bacteria or expose to
attenuated strain or expose to virulent strain and
clear by antifungal treatment. Keep frogs at
warmest temperature possible.
Repeat 3X
Test for elevated mucosal Abs and elevated AMPs
Expose to known virulent Bd
strain. Expect lower Bd
burden and reduced
morbidity and mortality
How to assess success of vaccine
strategy without sacrificing frogs
• Reinfection will result in reduced fungal
burden assessed by PCR.
– If burden is too high, clear with itraconazole or
another less toxic agent (amphotericin B,
chloramphenicol).
• Assess induction of mucosal antibodies by
ELISA.
Summary Prospects for Vaccine
Strategy • One or more exposures to live Bd or Bd antigens
followed by natural clearance or induced clearance with
antifungal agents may provide time for immune defenses
to amplify and eventually set up defense of the skin.
• Test for induction of mucosal antibodies and AMPs
• Test by reduced fungal burden using PCR
• Final test is exposure to virulent strain