Topics in Modern Biology: Population Collapses: Causes and

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