Bad neighbours? Dynamics of amphibian chytrid fungus Batrachochytrium dendrobatidis infection in three co-occurring frog species of southern Sydney

Abstract

Wildlife disease is a major cause of global biodiversity loss. Amongst the most devastating is the disease chytridiomycosis, caused by the amphibian chytrid fungus Batrachochytrium dendrobatidis ( Bd ). This disease has contributed to declines and extinctions in hundreds of amphibian species, but not all species are affected equally. Some amphibian hosts are capable of carrying high levels of Bd infection without population declines, acting as reservoir species for the pathogen and driving population declines in sympatric species. In Australia, several species have been proposed as reservoir species; however, our understanding of Bd is derived from studies that are highly geographically and taxonomically biased, and our ability to extrapolate from these systems is unknown. We examined the prevalence and intensity of Bd infection in 3 frog species in a previously unstudied host-pathogen system in temperate eastern Australia: the Blue Mountains tree frog Litoria citropa , a poorly-known species predicted to be susceptible to Bd infection; and the common eastern froglet Crinia signifera and the stony creek frog L. lesueuri , which have both been identified as reservoir species in other regions. We found that L. citropa and L. lesueuri were infected with Bd at a high prevalence and often high intensity, while the reverse was true for C. signifera . All species were detected at moderate abundance and there was no evidence of morbidity and mortality. Our findings do not support C. signifera and L. lesueuri being reservoir species in this system, highlighting the importance of region-specific studies to inform conservation management.

Full text

DISEASES OF AQUATIC ORGANISMS
Dis Aquat Org
Vol. 143: 101–108, 2021
https://doi.org/10.3354/dao03557 Published online February 11
1. INTRODUCTION
One of the greatest global issues at present is biodi-
versity loss (Barnosky et al. 2011), and a significant
driver of this loss is wildlife disease (Murray et al.
2011, Haislip et al. 2012, Aguirre 2017). Disease-
driven population declines and species extinctions
have been recorded across a wide variety of taxa from
geographically disparate regions (Daszak et al. 2000,
Fisher et al. 2009, Whitfield et al. 2012, Scheele et al.
2017b); however, the single most devastating wildlife
disease is chytridiomycosis (Skerratt et al. 2007,
Scheele et al. 2019). Chytridiomycosis is caused by the
amphibian chytrid fungus Batrachochytrium dendro-
batidis (Bd) (Pounds et al. 2006, Collins, 2010, O’Han-
lon et al. 2018, Scheele et al. 2019), which infects the
keratinized skin layers of adult frogs, and mouthparts
of tadpoles (Longcore et al. 1999). It is capable of
causing morbidity and rapid mortality in a wide range
of amphibian taxa (Berger et al. 1998, Scheele et al.
2019), and has been detected on every continent
where amphibians are present (Fisher et al. 2009).
© Inter-Research 2021 · www.int-res.com*Corresponding author:
jordann.crawford-ash@austmus.gov.au
Bad neighbours: amphibian chytrid fungus
Batrachochytrium dendrobatidis infection
dynamics in three co-occurring frog species of
southern Sydney, Australia
Jordann Crawford-Ash1, 2,*, Jodi J. L. Rowley1, 2
1Australian Museum Research Institute, Australian Museum, 1 William St, Sydney, NSW 2010, Australia
2Centre for Ecosystem Science, School of Biological, Earth and Environmental Sciences, University of New South Wales,
Sydney, NSW 2052, Australia
ABSTRACT: Wildlife disease is a major cause of global biodiversity loss. Amongst the most dev-
astating is the disease chytridiomycosis, caused by the amphibian chytrid fungus Batracho -
chytrium dendrobatidis (Bd). This disease has contributed to declines and extinctions in hundreds
of amphibian species, but not all species are affected equally. Some amphibian hosts are capable
of carrying high levels of Bd infection without population declines, acting as reservoir species for
the pathogen and driving population declines in sympatric species. In Australia, several species
have been proposed as reservoir species; however, our understanding of Bd is derived from stud-
ies that are highly geographically and taxonomically biased, and our ability to extrapolate from
these systems is unknown. We examined the prevalence and intensity of Bd infection in 3 frog
species in a previously unstudied host−pathogen system in temperate eastern Australia: the Blue
Mountains tree frog Litoria citropa, a poorly-known species predicted to be susceptible to Bd
infection; and the common eastern froglet Crinia signifera and the stony creek frog L. lesueuri,
which have both been identified as reservoir species in other regions. We found that L. citropa and
L. lesueuri were infected with Bd at a high prevalence and often high intensity, while the reverse
was true for C. signifera. All species were detected at moderate abundance and there was no evi-
dence of morbidity and mortality. Our findings do not support C. signifera and L. lesueuri being
reservoir species in this system, highlighting the importance of region-specific studies to inform
conservation management.
KEY WORDS: Anura · Australia · Infection dynamics · Disease · Management · Chytridiomycosis
Resale or republication not permitted without written consent of the publisher

Dis Aquat Org 143: 101– 108, 2021
Some amphibian species are highly susceptible to
Bd; however, other species, even those in sympatry
with susceptible species, appear to persist with Bd
and not suffer population declines (Retallick et al.
2004, Puschendorf & Bolaños 2006, Woodhams et al.
2007). This interspecific variation in susceptibility to
Bd is multifaceted, with factors such as anti-microbial
peptides in the skin (Brinkworth et al. 2004, Wood-
hams et al. 2007), environmental conditions (Mahony
2006, Kriger & Hero 2007, Rachowicz & Briggs 2007,
Whitfield et al. 2012) and differences in breeding
strategy and microhabitat use playing a large role
(Kriger et al. 2007, Rowley & Alford 2007, Sapsford et
al. 2013).
Despite over 50 yr of research, the geographic
and taxonomic variation of Bd disease dynamics
remains poorly understood in Australia. The vast
majority of research on Bd has been conducted on
the east coast of Australia (Scheele et al. 2017a,b),
particularly in the tropical rainforests of Queens-
land (Laurance et al. 1996, McDonald & Alford 1999,
Retallick et al. 2004) and the alpine areas of far
south-eastern New South Wales (NSW) (Hunter et
al. 2010, Brannelly et al. 2018). Vast areas of Aus-
tralia remain completely unstudied in terms of Bd.
Similarly, many Australian frog species have not
yet been tested for Bd, despite the extinction of ap -
proximately 7 species by the pathogen, with many
others known to be threatened (Hines et al. 1999,
Commonwealth of Australia 2016, Scheele et al.
2017b, 2019). Without an understanding of whether
Bd poses a threat to these unstudied species and
environmental systems, informed conservation deci-
sions cannot be made. It is therefore vital that these
gaps in knowledge are addressed to best manage
the Bd threat in Australia.
Species persisting with Bd infection are capable of
acting as reservoir hosts, increasing or maintaining
Bd infection rates in the environment, while remain-
ing unaffected themselves, either individually or on a
population level (Van Sluys & Hero 2009, Reeder et
al. 2012, Miaud et al. 2016, Stockwell et al. 2016,
Scheele et al. 2017a, Brannelly et al. 2018). In Aus-
tralia, 4 species have been proposed as reservoir spe-
cies for Bd: the common eastern froglet Crinia sig-
nifera, the stony creek frog Litoria lesueuri, the
eastern stony creek frog L. wilcoxii and the brown
tree frog L. ewingii (Van Sluys & Hero 2009, West
2015, Commonwealth of Australia 2016, Stockwell et
al. 2016, Scheele et al. 2017b). These species are
often abundant and have large geographic distribu-
tions, spanning much of eastern Australia, yet have
only been studied in limited geographic areas and
sites. If they are acting as reservoir species across
their range, this could be contributing to population
de clines or inhibiting the recovery of other sym-
patric, more Bd-susceptible species sharing the same
habitats (Hines et al. 1999, Skerratt et al. 2016,
Scheele et al. 2019).
Three species were the focus of our research: 2 of
the previously proposed reservoir species, C. sig-
nifera and L. lesueuri, and the Blue Mountains tree
frog L. citropa. Found east of the Great Dividing
Range in NSW, from the Hunter River to north-east-
ern Victoria (Anstis & Littlejohn 1996), L. citropa
appears to have declined from northern Sydney, with
no recent published sightings from the northern
beaches of Sydney to Ourimbah, a region in which it
was once abundant (Gillespie & Hines 1999, Atlas of
Living Australia 2020). Its closest relatives, Davies’
tree frog L. daviesae and the New England tree frog
L. subglandulosa, are both listed as threatened in
NSW, with Bd thought to be directly involved in their
decline (Mahony et al. 2001, Hines et al. 2002).
Despite these indications that L. citropa is likely to be
susceptible to Bd, the species has never been tested
for the pathogen.
There are large gaps in our knowledge when
investigating Bd dynamics in Australia, both geo-
graphically and taxonomically. We examined the
dynamics of Bd infection in a previously unstudied
host−pathogen system in temperate eastern Aus-
tralia. Our project firstly aimed to understand (1) if C.
signifera and L. lesueuri are likely to be operating as
a reservoir for Bd in the Sydney area and (2) whether
Bd infects L. citropa and, if so, (3) if infection preva-
lence and intensity, and individual infection status
over time are indicative that L. citropa is susceptible
to Bd.
2. MATERIALS AND METHODS
2.1. Study site
The location of our study was Dharawal National
Park, in southern Sydney, NSW, Australia (Fig. 1).
Two sites were selected within the park to investi-
gate the disease dynamics of our 3 study species, one
located on O’Hares Creek itself and the other on a
branching stream, Madden’s Creek; the 2 sites are
~13 km apart, so they are likely to represent distinct
populations of each species. Both sites are open,
rocky streams. Our 3 study species were the most
abundant species at the streams (Atlas of Living Aus-
tralia 2020, J. Crawford-Ash pers. obs.).
102

Crawford-Ash & Rowley: Bd dynamics in Sydney frog species
2.2. Field methods
At each site, a 100 m transect was established
and surveyed 5 times (every 6 wk) from October
2018 to April 2019. This time frame was selected as
all 3 species breed at the study sites in these
months and are therefore detectable. After sunset,
the ambient temperature and elevation was recorded
(Kestrel 2500 Weather Meter, Nielsen-Kellerman),
and the transect was visually and aurally surveyed.
For any Crinia signifera, Litoria lesueuri or L. cit-
ropa encountered, the individual frog was placed
in a clean sandwich bag and swabbed following
protocol as outlined by Hyatt et al. (2007), swabbing
the body surfaces that are most likely to have Bd
present (Waddle et al. 2018), the ventral side of
the frog, the inside of the thighs and each foot 5
times each using a sterile swab (MW110, Medical
Wire & Equipment). The swabs were then immedi-
ately placed in a cooler bag. In order to assess the
health of individuals, the snout− vent length (mm)
and weight (g) were recorded, and any signs of
morbidity such as skin discolouration or lesions
were noted. We photographed each frog to allow
for individual recognition (J. Crawford-Ash & J. J.
L. Rowley unpubl.). Temperature was also re corded
at the end of the survey (Kestrel 2500 Weather
Meter, Nielsen-Kellerman), and mean monthly
temperature was calculated from the temperatures
recorded at the closest weather station (068263
Holsworthy Defence AWS), 15 km away from the
study site. At all times, we abided by the hygiene
protocol for the control of disease in frogs (Hyatt et
al. 2010).
2.3. Laboratory methods
Swabs were stored in a cooler bag with ice blocks
and then transported to the Australian Museum
within 12 h, where they were transferred to −20°C
before testing with diagnostic qPCR using Taqman
chemistry (Boyle et al. 2004, Hyatt et al. 2007). The
tip of each swab was removed and added to a 2 ml
Sarstedt screw-capped tube containing 0.4 g of 0.5 mm
diameter Zirconium/silica beads (Qiagen) and 200 µl
PrepMan™ Ultra Sample Preparation Reagent
(Applied Biosystems). Samples were then vortexed
for 10 s and homogenised in a Qiagen Tissuelyser 2
(2 × 45 s at 30 cycles s−1). Samples were then cen-
trifuged (1 min at 18 407 × g) and incubated at 100°C
for 10 min. Supernatant (20 µl) was then extracted
from each sample and placed into a new sterile 1.5 ml
microcentrifuge tube. Samples were diluted 1:10 and
the aliquots stored at −20°C before thermal cycling.
Each swab was analysed once (no replicates). The
qPCR reagents and reactions followed the methodol-
ogy of Boyle et al. (2004): 1× SensiFAST Probe Lo-
ROX Mix (Bioline), 200 nM ITS-1-3 Forward primer
(CCT TGA TAT AAT ACA GTG TGC CAT ATG TC),
200 nM 5.8S Reverse primer (AGC CAA GAG ATC
CGT TGT CAA A), 1 U of TaqMan™ ChytrMGB2
FAM probe (Applied Biosystems), 1× TaqMan™ exo -
genous internal positive control (IPC) DNA (IPC-VIC
probe) and 1× exogenous IPC reagent mix (Applied
Biosystems). The total reaction volume in each well
of the qPCR plate was 20 µl, comprising 15 µl of the
above master mix and 5 µl of our sample.
The exogenous IPC control was used to monitor
PCR reaction inhibition. Triplicate no-amplification
controls were included using 1× TaqMan™ IPC PCR
blocking reagent. Triplicate no-template controls
were used to monitor probe degradation.
qPCR standards were generated using synthetic
ITS-1 gBlocks®gene fragment from Integrated DNA
Technologies (IDT) (J. Kerby et al. unpubl. data). Five
log10 dilutions ranging from 109to 101copies were set
up in triplicate for generation of the ITS-1 standard
curve used for quantification of ITS-1 copy numbers
(Longo et al. 2013). qPCR reactions were run on an ABI
Quantstudio3 Q-PCR Machine and analysed using
QuantStudio Design and Analysis Software (V. 1.4.1).
2.4. Analysis
A sample was considered Bd positive when the
quantity of ITS-1 copies present was greater than 0
(Vredenburg et al. 2010, DiRenzo et al. 2018).
103
Fig. 1. Location of the study area in Dharawal National Park,
southern Sydney, Australia

Dis Aquat Org 143: 101– 108, 2021
Infection intensity was estimated using zoospore
equivalents (ZSEs) per reaction of 5 µl of our sample
added to 15 µl master mix. ZSE values were ob -
tained from testing cultured zoospore (Australian
GPL Strain, James Cook University) sample volumes
containing 100, 500 and 1250 zoospores in triplicate
alongside the synthetic standards to determine the
average ITS-1 copy numbers per cultured zoospore.
We then divided the ITS-1 copy numbers present in
our samples by the average (58) from the cultured
samples.
In order to determine whether Bd infection af -
fected body condition, we calculated the scaled
mass index (SMI, mean ± SD) (Peig & Green 2009)
for males of each species and used a Wilcoxon test
in JMP (Version 14, SAS Institute) to compare
body condition between infected and uninfected
frogs, as body condition data were not normally
distributed.
3. RESULTS
Ambient temperature during surveys ranged from
15.8 to 28.6°C. We sampled all target species ob -
served, for a total of 152 frogs across both study sites
over a 7 mo period (47 Litoria citropa, 44 L. lesueuri
and 61 Crinia signifera) (Table 1). The 3 study
species were the most abundant species at the site;
however, during surveys, 3 other species were
detected: the Peron’s tree frog L. peronii, the green
stream frog L. phyllochroa and the wallum rocket
frog L. freycineti. The majority of individuals cap-
tured for each study species were males (84 %), with
only 8 female C. signifera, 3 female L. citropa and 7
female L. lesueuri found during the survey period. In
addition, 4 C. signifera and 2 L. lesueuri individuals
could not be sexed in the field (5%).
C. signifera and L. lesueuri were detected at both
sites over the survey period (means: 0.02 ind. m−2 for
C. signifera; 0.01 ind. m−2 for L. lesueuri). C. signifera
was detected most in the later months of surveying
(0.04 ind. m−2 in March and April), while L. lesueuri
was seen more in the earlier months (0.03 ind. m−2 in
October and December).
C. signifera had a relatively low Bd infection
prevalence, while L. lesueuri and L. citropa had a rel-
atively high infection prevalence (Table 1, Fig. 2):
11% of females swabbed were Bd-positive (CI = –3.8
to 26.1%), as compared to 23% of males (CI = 15.5 to
30.2%). The individuals that could not be sexed in
the field were negative for Bd. Due to the relatively
small sample sizes for each sex, species, visit number
and site, these factors could not be compared. Seven
frogs swabbed were juveniles (classified by the
absence of nuptial pads), with 1 L. citropa juvenile
positive for Bd infection.
Infected and uninfected frogs (males only) did not
differ in body condition (SMI: C. signifera, p = 0.8815,
χ2 = 0.0222, 1.24 ± 2.75, df = 1; L. lesueuri, p = 0.4962,
χ2 = 0.4631, 7.16 ± 0.85, df = 1; L. citropa, p = 0.4136,
χ2 = 60.6684, 9.41 ± 2.75, df = 1).
Infection intensity in infected individuals was over-
all highest in L. lesueuri and L. citropa, and lowest in
C. signifera (Table 1, Fig. 2).
We recaptured 2 L. citropa individuals during our
study, the first after 44 d and the second after 88 d.
The second individual was initially found infected
with Bd (1 ZSE), which was unde-
tected when recaptured 88 d later. We
did not detect any Bd on the first indi-
vidual on either capture date.
4. DISCUSSION
For a species to be characterised as a
reservoir species, it should maintain a
high prevalence and high intensity of
infection, while showing no signs of
infection in itself (high host abun-
dance, low to no morbidity or mortal-
ity) (Kriger et al. 2007, Van Sluys
& Hero 2009, Brannelly et al. 2015,
2018, West 2015). We found no evi-
dence to support Crinia signifera and
Litoria lesueuri, both identified as res -
104
N N Prevalence (%) Mean infection
positive sampled (95% CI) intensity (ZSE) (range)
Litoria citropa
Site 1 16 38 42.10 (26.3−59.2) 59 (0−421)
Site 2 3 9 33.30 (7.5−70.1) 26 (0−57)
Total 19 47 40.40 (26.4−55.7) 53 (0−421)
Crinia signifera
Site 1 2 31 6.50 (0.8−21.4) 1 (0−1)
Site 2 2 22 9.10 (1.1−29.2) 5 (0−11)
Total 4 61 6.60 (0.2−15.9)_ 3 (0−11)
Litoria lesueuri
Site 1 4 14 28.60 (8.4−58.1) 122 (0−479)
Site 2 4 30 13.30 (3.8−30.7) 2263 (0−479)
Total 8 44 18.20 (8.2−32.7) 1192 (0−8241)
Table 1. Batrachochytrium dendrobatidis prevalence in Crina signifera, Litoria
lesueuri and L. citropa across Sites 1 and 2, and in total, as well as the range of
zoospore equivalent (ZSE) values and the number of frogs sampled

Crawford-Ash & Rowley: Bd dynamics in Sydney frog species
ervoir species for Bd in other regions, acting as
reservoir species in the system we examined dur-
ing our study.
Both C. signifera and L. lesueuri had relatively low
infection prevalence and intensity of infection across
our study period (Table 1, Fig. 2), much lower than
what was found in past studies in other geographical
areas (Kriger et al. 2007, Van Sluys & Hero 2009,
Brannelly et al. 2015, 2018, West 2015). Although
direct comparisons of Bd infection intensity results
can be misleading due to methodological differences
(i.e. swabbing technique, Clare et al. 2016) and vari-
ation in Bd strains in the number of ITS-1 replicates
per zoospore (Longo et al. 2013), we found lower
infection intensities in C. signifera and in L. lesueuri
than previous studies of the same species in other
areas (Kriger et al. 2007, Brannelly et al. 2015, 2018,
Scheele et al. 2017a). While not conclusive, our
results suggest that C. signifera and L. lesueuri do
not follow the previously stated characteristics of
reservoir species at our sites and during our study
period. However, as Bd infection prevalence and
intensity tend to be higher at cooler temperatures
and at higher elevations (Berger et al. 1998, Kriger &
Hero 2008), it is possible that at cooler times of year
and at nearby higher elevation sites, they do display
such characteristics. It would have been informative
to also have sampled within the winter months
(June−August); however, 2 of the study species (L.
citropa and L. lesueuri) are typically not detectable
during winter months (FrogID 2020). Bd resistance in
frogs at our study site (Woodhams et al. 2007), or mit-
igating environmental factors such as water temper-
ature or canopy cover, are also likely to have
impacted our results (Woodhams et al. 2011, Scheele
et al. 2014).
Lastly, reservoir species are typically more abun-
dant, especially compared to more susceptible spe-
105
0
20
40
60
80
100
120
Bd prevalence (%) Abundance (N swabbed)
Site 1: Madden’s Creek Site 2: O’Hares Creek
0
5
10
15
20
Temp (°C)
5
10
15
20
25
30
35 Crinia signifera
Litoria citropa
Litoria lesueuri
Survey temperature
Average ambient
temperature
KEY
10/18 12/18 01/19 04/1903/19
95% confidence
interval
10/18 12/18 01/19 04/1903/19
10/18 12/18 01/19 04/1903/19
10/18 12/18 01/19 04/1903/19
Fig. 2. Abundance of Crinia signifera, Litoria lesueuri and Litoria citropa over the survey period at Site 1 and Site 2, with start-
ing temperature and mean monthly temperature displayed on the secondary y-axis. Batrachochytrium dendrobatidis (Bd ) in-
fection prevalence present in each species at Site 1 and Site 2 displayed with accompanying 95 % confidence intervals

Dis Aquat Org 143: 101– 108, 2021
cies sharing the same habitat (Kriger et al. 2007,
Scheele et al. 2017a, Brannelly et al. 2018). Both C.
signifera and L. lesueuri were detected at a relatively
low abundance (C. signifera: 0.02 ind. m−2, L. le sueuri:
0.01 ind. m−2) compared to L. citropa (up to 0.07 ind.
m−2 in October). However, the detectability of frogs
during surveys is likely to vary with species and sea-
son, and may not reflect true relative abundance.
L. citropa, a species predicted to be susceptible to
Bd, yet never previously tested, was found to have
high infection prevalence and intensity, higher than
detected in C. signifera at both sites and L. lesueuri
at Site 1. Several factors may suggest some tolerance
to Bd in L. citropa at our sites. Firstly, L. citropa was
present in relatively high abundance (up to 0.07 ind.
m−2, 47 individuals captured), and has been found in
abundance at Site 1 over a 17 yr period (J. J. L. Row-
ley pers. obs.). In addition, when comparing photo-
graphs taken of each individual swabbed during this
study, 2 L. citropa individuals were recaptured after
44 and 88 d, the latter of which was initially Bd-posi-
tive and was found to be negative when recaptured,
suggesting that the species is persisting despite the
presence of the pathogen. Despite this, caution must
be taken before drawing conclusions on the suscepti-
bility of L. citropa based upon our results, and further
research on the susceptibility of this species is needed.
5. CONCLUSION
We found that patterns of Bd infection intensity
and prevalence within our study system did not fol-
low the same trends as previous findings in other sys-
tems and regions. Our results reinforce the problems
in extrapolating patterns and conclusions derived from
studies in other geographic or taxonomic areas when
investigating Bd infection dynamics. Great care needs
to be taken when assuming patterns of Bd infection
dynamics, and efforts need to be made to gather geo-
graphically, taxonomically and temporally diverse
data to understand the true nature of Bd, and more
effectively inform conservation management.
In summary, while we cannot rule out the role of C.
signifera and L. lesueuri as reservoir species in our
study system and at other locations and conditions,
we conclude that a ‘bad neighbour’ in one area may
not be the same ‘bad neighbour’ in another, at least not
across all conditions and sites. We recommend a
stronger ‘neighbourhood watch’, implementing long-
term monitoring of species and ecological systems,
both threatened and non-declining, to develop targeted
conservation strategies to mitigate the impacts of Bd.
Acknowledgements. This study was approved by the AMRI
Animal Ethics Committee at the Australian Museum (project
number: 18-05) and was conducted under an approved Sci-
entific Licence from NSW Parks and Wildlife (SL102135).
We thank Rowena Morris from NSW Parks and Wildlife for
supporting our research and allowing sampling to take
place in Dharawal National Park. Thank you to the Frog and
Tadpole Study Group for awarding J.C.A. a student re -
search grant, and to Chris Portway at the Australian Museum
for his guidance with qPCR procedures. The largest thank
you to all volunteers, Tim Cutajar, Kathy Potter, Stephen
Mahony, Simon Crawford-Ash and Mitchell Hodgson.
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Editorial responsibility: Louise Rollins-Smith,
Nashville, Tennessee, USA
Submitted: March 2, 2020; Accepted: October 28, 2020
Proofs received from author(s): Februar y 2, 2021