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Defying decline: Very low chytrid prevalence in tadpoles, yet high infection in adults in a naturally recovering frog species

Animal Conservation

December 2024

DOI:10.1111/acv.13006

Authors:

Jordann Elizabeth Crawford-Ash

Australian National University

Jesse Erens

An Martel

Ghent University

Daniel Noble

Australian National University

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Amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is associated with declines in ~500 amphibian species globally. Decades after initial disease outbreaks, the trajectory of impacted species varies substantially; while some species continue to decline, there are instances of natural recovery, such as the whistling tree frog, Litoria verreauxii, in south‐eastern Australia. The decline and subsequent recovery of this species have been quantified through repeated surveys of historically occupied sites over the past 30 years; however, the underlying mechanisms driving this recovery remain unknown. In this study, we investigate the potential factors facilitating the recovery of L. verreauxii by examining Bd prevalence and intensity in both adults and tadpoles. Specifically, we addressed the following hypotheses: (1) Bd prevalence in tadpoles would be lower compared to adults at the same breeding sites, (2) Bd prevalence in tadpoles would decrease over the spring breeding season due to the increasing availability of warm water microhabitats where tadpoles could potentially avoid or clear Bd infections and (3) there would be a negative correlation between Bd prevalence in tadpoles and the abundance and diversity of microfauna, which may consume Bd zoospores. Our findings indicate that tadpole infection prevalence remained consistently low at 1.36% (95% CI: 0.6–2.47%) throughout our spring sampling period, across different developmental stages. Adults had moderate to high prevalence within the same ponds at 50.53% (95% CI: 43.19–57.84%). No effect of temperature or microfauna diversity and abundance was apparent. While the mechanisms driving the recovery of this species remain unknown, low infection prevalence in tadpoles is likely a key component to the species' recovery. Our results emphasize the need for comprehensive investigations in Bd dynamics across all life history stages within recovering and declining amphibian species.

Location of the 23 study sites in the Australian Capital Territory, Australia. For tadpoles, diurnal sampling was conducted across two time periods to compare earlier and later developmental stages, while nocturnal sampling of adults was undertaken once per site.

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(a) Batrachochytrium dendrobatidis (Bd) infection prevalence for tadpole and adult life stages of Litoria verreauxii, with the first (early) and second (late) tadpole sampling period. Error bars display binomial 95% confidence intervals around the means.

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Bd prevalence and sample sizes for 18 tadpole sampling sites, across two sampling periods (early and late tadpole sampling). In the late tadpole sampling period, no tadpoles were detected at sites 11 and 16.

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(a) Mean daily pond temperatures averaged across a shallow and deep iButton temperature logger at 17 sites during the 4‐week sampling period (one site was excluded due to the loss of iButtons). (b) Water temperatures measured at the time of tadpole sampling, displayed by Bd infection status, with red highlighting Bd‐positive samples. Blue dashed lines on both figures indicate Bd optimal growth temperatures (17–25°C). The red dashed line indicates Bd lethal limit (>28°C).

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Microfauna abundance counts across 18 sites, per 6 L of water filtered, per sampling period (early and late tadpole sampling). In the second sampling (late tadpole sampling), sites 11 and 17 were unable to be processed for identification due to severely eutrophic samples, so are excluded from the analysis. An asterisk indicates that Bd was detected on the tadpoles at that site.

…

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Defying decline: Very low chytrid prevalence in tadpoles,

yet high infection in adults in a naturally recovering frog

species

J. Crawford-Ash

, J. Erens

1,2

, A. Martel

, D.W.A. Noble

, F. Pasmans

& B.C. Scheele

1 Fenner School of Environment and Society, Australian National University, Canberra, ACT, Australia

2 Wildlife Health Ghent, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium

3 Research School of Biology, Australian National University, Canberra, ACT, Australia

Keywords

chytridiomycosis; Batrachochytrium

dendrobatidis; Anura; tadpoles; disease

dynamics; tree frogs; Australia; population

recovery.

Correspondence

Jordann Crawford-Ash, Fenner School of

Environment and Society, Australian National

University, Canberra ACT, Australia.

Email: jordann.crawford-ash@anu.edu.au

Editor: John Ewen

Associate Editor: Francesco Ficetola

Received 16 February 2024; accepted 14

November 2024

doi:10.1111/acv.13006

Abstract

Amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), is associated with

declines in ~500 amphibian species globally. Decades after initial disease out-

breaks, the trajectory of impacted species varies substantially; while some species

continue to decline, there are instances of natural recovery, such as the whistling

tree frog, Litoria verreauxii, in south-eastern Australia. The decline and subsequent

recovery of this species have been quantiﬁed through repeated surveys of histori-

cally occupied sites over the past 30 years; however, the underlying mechanisms

driving this recovery remain unknown. In this study, we investigate the potential

factors facilitating the recovery of L. verreauxii by examining Bd prevalence and

intensity in both adults and tadpoles. Speciﬁcally, we addressed the following

hypotheses: (1) Bd prevalence in tadpoles would be lower compared to adults at

the same breeding sites, (2) Bd prevalence in tadpoles would decrease over the

spring breeding season due to the increasing availability of warm water microhabi-

tats where tadpoles could potentially avoid or clear Bd infections and (3) there

would be a negative correlation between Bd prevalence in tadpoles and the abun-

dance and diversity of microfauna, which may consume Bd zoospores. Our ﬁnd-

ings indicate that tadpole infection prevalence remained consistently low at 1.36%

(95% CI: 0.6–2.47%) throughout our spring sampling period, across different

developmental stages. Adults had moderate to high prevalence within the same

ponds at 50.53% (95% CI: 43.19–57.84%). No effect of temperature or microfauna

diversity and abundance was apparent. While the mechanisms driving the recovery

of this species remain unknown, low infection prevalence in tadpoles is likely a

key component to the species’recovery. Our results emphasize the need for com-

prehensive investigations in Bd dynamics across all life history stages within recov-

ering and declining amphibian species.

Introduction

Amphibian chytrid fungus, Batrachochytrium dendrobatidis

(Bd ), is a pathogen that has caused the decline of amphib-

ians on a global scale, causing the most signiﬁcant

disease-driven losses of vertebrate biodiversity in recorded

history (Skerratt et al., 2007; Scheele et al., 2019a,2019b,

Fisher & Garner, 2020). The fungus, originating in East Asia

(O’Hanlon et al., 2018), has been associated with extensive

mass mortality events across parts of Mesoamerica, South

America and Australia (Berger et al., 1998; Lips

et al., 2006; Carvalho et al., 2017;O’Hanlon et al., 2018;

Scheele et al., 2019a,2019b; Luedtke et al., 2023), with fur-

ther declines reported from North America and Europe

(Briggs et al., 2010; Bosch et al., 2013;V

€

or€

os et al., 2018;

Scheele et al., 2019a,2019b). At a global scale, outbreaks

peaked during the 1980s, cumulatively resulting in the

decline or extinction of approximately 500 amphibian species

globally (Scheele et al., 2019a,2019b). Whilst acute popula-

tion declines have diminished for many species, Bd remains

a pervasive threat to amphibians globally, particularly

coupled with other threats such as habitat loss and climate

change (Luedtke et al., 2023).

Batrachochytrium dendrobatidis infects epithelial cells in

keratinised tissues in amphibians, causing the disease chytri-

diomycosis, which disrupts skin function in susceptible spe-

cies (Berger et al., 1998,1999; Longcore et al., 1999;

Voyles et al., 2011). The location and amount of keratin

Animal Conservation  (2024) – ª2024 Zoological Society of London. 1

Animal Conservation. Print ISSN 1367-9430

layers upon the epithelium varies across amphibian life his-

tory stages. In tadpoles, Bd zoospores are found on the

mouthparts, with a subsequent increase in keratin through

the feet, legs, body and tail during metamorphosis (Maran-

telli et al., 2004). While Bd-induced mortality is not common

in tadpoles (Berger et al., 1998), individuals can experience

sublethal effects such as reduced growth rates and body con-

dition due to damaged mouth parts and reduced feeding efﬁ-

ciency (Rachowicz & Vredenburg, 2004; Cashins, 2009;

Venesky et al., 2009). During metamorphosis, these sublethal

Bd infections can spread throughout the skin resulting in

mortality (Berger et al., 1998; Marantelli et al., 2004;

Humphries et al., 2022,2024). However, research on Bd

infection in amphibian larval stages and during metamorpho-

sis is limited compared to work on adult Bd dynamics. More

broadly, there is little known about how various environmen-

tal factors inﬂuence infection dynamics across amphibian life

history stages.

Approximately 40 years since the initial Bd outbreaks in

Australia, it is apparent that the ongoing impacts of Bd are

highly variable and species-speciﬁc (Woodhams et al., 2007;

Stockwell et al., 2016; Scheele et al., 2019a,2019b;

West, 2015; Crawford-Ash & Rowley 2021). While Australia

has lost four species since the initial outbreak (Scheele

et al., 2019a,2019b), some species are starting to show

signs of recovery (Scheele et al., 2017a,2017b; Luedtke

et al., 2023). An example of a declined, but now recovering

species in south-eastern Australia is the whistling tree frog

(Litoria verreauxii). This species (and the closely related

alpine tree frog, L. verreauxii alpina) experienced major

declines in the southern Highlands bioregion in the 1980s

(Osborne et al., 1999; Scheele et al., 2014). The decline and

subsequent re-expansion of these species have been tracked

through repeated surveys of historically occupied sites over

the past 30 years (Scheele et al., 2014). Across the survey

region, sites in the northern areas have largely recovered

with often abundant populations, however, in the south sites

remain unoccupied, or have repopulated with signiﬁcantly

less abundance than their northern counterparts (Scheele

et al., 2014, JCA pers. obs). The mechanisms inﬂuencing the

recovery of this species remain unknown. Interestingly, Bd is

found across the region and is highly prevalent in the recov-

ering populations. Previous work in L. verreauxii alpina

revealed that adults had high Bd prevalence, with patterns of

increased infection severity over the breeding season

(Scheele et al., 2015,2016). This same study found that

juveniles and tadpoles in the same populations had low Bd

prevalence and intensity (Scheele et al., 2015), however, the

sample size was limited for tadpoles and juveniles, and sam-

pling was undertaken at only three high-elevation sites

(1380–1475 m a.s.l.). Adult L. verreauxii alpina were aged

using skeletochronology and shown to be living to approxi-

mately 2 years of age for males and 3 years of age for

females, suggesting high mortality from Bd post-breeding

(Scheele et al. 2015,2016). Scheele et al. 2015 surmised

that low Bd prevalence in tadpoles was underpinning suc-

cessful recruitment, offsetting ongoing high Bd-associated

mortality in adults. The results from this study contrast with

other studies on susceptible species that report tadpoles to

commonly have a high prevalence of Bd (Cashins, 2009;

Ruggeri et al., 2018; Sapsford et al., 2018).

Many factors shape Bd infection dynamics in frogs and tad-

poles. Some broad examples are behaviour (Rowley &

Alford, 2007; Keiser et al., 2019), susceptibility and resistance

of the host (Blaustein et al., 2005; Bataille et al., 2015; Jim

enez

et al., 2019), ecological dynamics (Blaustein et al., 2012; Ger-

vasi et al., 2013; Scheele et al., 2017a,2017b), virulence of the

pathogen (Berger et al., 2005; Briggs et al., 2010) and geo-

graphical factors such as elevation (Catenazzi et al., 2013;

Sapsford et al., 2013). For this study, we focused on two envi-

ronmental mechanisms that may be central to shaping Bd

dynamics in tadpoles: temperature (Rowley & Alford, 2013;

Blaustein et al., 2018; Sapsford et al., 2018) and the presence of

aquatic microfauna (Schmeller et al., 2014). Bd has a thermal

preference for optimal growth of 17–25°C, with lethal limits

above ~28°C (Piotrowski et al., 2004, Stevenson et al., 2013).

It has therefore been suggested that tadpoles may be able to uti-

lise microhabitats as refugia, such as warmer water, thereby

decreasing infection risk (Woodhams et al., 2003; Piotrowski

et al., 2004; Sapsford et al., 2013). Warm microhabitat use has

been suggested as a possible explanation for the low Bd preva-

lence in L. verreauxii alpina tadpole populations, with tadpoles

found in temperatures between 24 and 35°C during the warmest

part of the day (Scheele et al., 2015), but this mechanism has

not been investigated. Additionally, research in Europe and the

United States has found that common aquatic microfauna spe-

cies can feed on Bd zoospores, inﬂuencing infection dynamics

in tadpoles (Buck et al., 2011; Schmeller et al., 2014; Frenken

et al., 2019; Farthing et al., 2021). The ability to consume zoo-

spores has been found across many microfauna species of vary-

ing sizes and feeding mechanisms such as Cladocerans (Searle

et al., 2013), Ostracods, Copepods, Nematodes, Tardigrades

(Schmeller et al., 2014; Blooi et al., 2017; Deknock

et al., 2021) and Rotifers (Schmeller et al., 2014), highlighting

the potential for a variety of microfauna to shape Bd dynamics

and act as biological controls of the pathogen (Frenken

et al., 2019). However, the role of microfauna in shaping Bd

dynamics in tadpoles has not yet been explored in Australian

systems.

Exploring ﬁne-scale Bd infection dynamics across amphib-

ian life history stages could reveal crucial mechanisms

underlying the recovery and persistence of vulnerable frog

species (Muths et al., 2011). Consistently, low Bd prevalence

in tadpole populations and during metamorphosis, even in

environments with high Bd exposure, could enable suscepti-

ble species to recover and coexist with Bd, by increasing

recruitment in these systems (Scheele et al., 2015). Hence,

research on recovering populations should prioritise examin-

ing Bd dynamics across all life history stages to develop the

most effective management strategies.

Our study explored possible mechanisms facilitating the

re-expansion of L. verreauxii, examining Bd prevalence and

intensity in adult and tadpole populations. Speciﬁcally, we

predicted that (1) Bd prevalence in tadpoles would be lower

than in adults from the same breeding sites, as indicated by

preliminary work on L. verreauxii alpina (Scheele

2Animal Conservation  (2024) – ª2024 Zoological Society of London.

Low Bd prevalence in tadpole of recovering species J. Crawford-Ash et al.

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et al., 2015), and (2) Bd prevalence in tadpoles would

decrease over the spring breeding season as aquatic micro-

habitats warm, providing less preferable conditions for Bd,

and allowing tadpoles to possibly clear or avoid Bd infec-

tions. Furthermore, we hypothesised that (3) there would be

a negative relationship between Bd prevalence in tadpoles

and the abundance and diversity of microfauna, which may

consume Bd zoospores in this system. Our results show that

tadpole infection prevalence remained consistently low

throughout our sampling period, across different develop-

mental stages. We were unable to link this with temperature

or microfauna diversity or abundance due to the exception-

ally low Bd prevalence detected in the tadpoles. Our study

highlights the complexity of Bd infection patterns and sug-

gests that processes other than temperature and microfauna

may regulate Bd dynamics in tadpoles of L. verreauxii, and

in turn other susceptible species.

Materials and methods

Study species and area

Litoria verreauxii is found across a large portion of eastern

Australia, from south-eastern Queensland to eastern Victoria.

For this study, sites were selected in the Australian Capital

Territory (ACT). Our sites aligned with historical and con-

temporary surveys in this region that have documented the

decline and subsequent natural re-expansion of the species

following Bd emergence in the 1980s (Scheele et al., 2014).

This species is a generalist and is found in a range of habi-

tats, across an extensive elevational gradient. To increase

comparability across sites, only closed pond systems were

sampled. Twenty-three sites were selected based on historical

records across an elevational range of 470–1350 m a.s.l.

(Fig. 1; Table S1). Eighteen of these sites were used for tad-

pole sampling (ﬁve sites had no tadpoles present or in very

low abundance, despite evidence of adult calling behaviour).

Infection prevalence

To quantify Bd prevalence in L. verreauxii adults, we sam-

pled 190 individuals across 23 sites, sampling 2–22 frogs

per site in September–October 2022 (Table S1). Each indi-

vidual was swabbed ﬁve times on each side of the abdomen,

hind limb and along the toes of each hind limb, following

Boyle et al. (2004) protocols. Weight was taken using Pesola

scales and snout-vent length (SVL) was measured using digi-

tal callipers. Adults were sampled in the evening, with ambi-

ent temperature, average wind speed and humidity recorded

at each site using a weather meter (Kestrel 3000).

At 18 of the 23 survey sites, up to 40 tadpoles were

swabbed across two sampling periods, resulting in 350 tad-

poles swabbed during the early sampling period, and 314 in

the late sampling period, or 664 in total. This occurred

between September and November 2022 (Table S1). Tad-

poles were held in the hand with washed vinyl gloves (Cash-

ins 2009) and sampled for Bd by brushing a sterile

ﬁne-tipped swab 10 times along the keratinized tooth rows.

We ﬁrst sampled the tadpoles when they developed kerati-

nised mouthparts, which for this species was stage 25–30 on

the Gosner scale (Gosner, 1960). The second sampling

period was approximately 4 weeks later in November, at

Gosner stages 35–40, targeting tadpoles with more developed

hind limbs up until metamorphosis. We occasionally sampled

tadpoles at higher or lower Gosner stages when most of the

detected individuals were before or past the targeted develop-

mental stages. The two surveys yielded distinctly different

body size distributions for all sites (Fig. S2). In cases where

fewer than ﬁve tadpoles could be found in the ﬁrst hour of

searching, we discontinued the survey, which included ﬁve

sites in the ﬁrst sampling round, and two additional sites in

the second sampling round (Table S1). Tadpole sampling

efforts were on average 3 h at each site.

Since Bd infection shifts from the jaw sheaths towards the

increasingly keratinised parts of the hindlimbs and body just

before the completion of metamorphosis (Marantelli

et al., 2004), 11 individuals in our tadpole sampling that

showed tooth row loss (Gosner stage 43 or higher) were

swabbed like adults (see above description). The snout-to-tail

length (STL) of each tadpole was measured, as well as the

approximate depth and distance from the edge of the pond

where the tadpole was located. The surface temperature of

the water at the time and place of capture was recorded,

with roughly equal search effort spent along each side of the

water body, at each site.

All samples were analysed for the presence of Bd using

real-time PCR following Boyle et al. (2004) and were

diluted 1:10 for analyses. Samples were considered positive

when they recovered clear ampliﬁcation curves above a

genomic equivalent (GE) threshold of 0.1.

Microfauna abundance and diversity

At each site, 6 L of pond water was ﬁltered with a 53-micron

ﬁlter, resulting in a 25 mL sample (5 mL of ethanol, 20 mL of

water and microfauna). The 6 L of water was pooled from four

0.5-L shallow scoops and four 1-L deep collections of water,

taken at the four cardinal points around the pond to ensure a

representative microfauna sample. Deep water samples were

taken with a tube extending roughly 1 m down into the water

column to be able to sample zooplankton at variable depths.

The sample was then stored in a 5°C fridge before processing.

A Secchi disk was used to measure the water transparency/tur-

bidity. The disk was slowly submerged in undisturbed water of

the pond until the contrasting colours of the Secchi disk were

unable to be seen, at which point the depth of the Secchi disk

was recorded. Microfauna sampling was conducted during each

of the two tadpole sampling periods. The microfauna was iden-

tiﬁed to different clade groups and abundance was counted

using a compound microscope.

Temperature measurements

Two iButtons (Thermocron) were deployed at each of the 18

sites where tadpoles were sampled. One iButton was placed

in the shallowest point of the pond (within 5 cm of the

Animal Conservation  (2024) – ª2024 Zoological Society of London. 3

J. Crawford-Ash et al. Low Bd prevalence in tadpole of recovering species

surface so that the iButton would remain covered by water

despite ﬂuctuating water levels) and one iButton was placed

in a randomised deep point of the pond, weighted down by

small metal weights. Distance from the edge of the pond

and the depth of each iButton were recorded. The iButtons

recorded water temperature at the two depths every hour for

4 weeks (between the two tadpole sampling periods).

Statistical analysis

All statistical analyses were performed using R version 4.3.1

in R Studio (2021.09.0). To examine variation in Bd infec-

tion prevalence for adult and tadpole L. verreauxii, we used

Generalised Linear Mixed Models (GLMMs) with a binomial

distribution and logit link function using the lme4 package

(Bates et al., 2015). We modelled Bd prevalence (Bernoulli:

‘infected’=1, ‘uninfected’=0) against age group (tadpole

or adult), mean temperature (mean temperature per site for

the 4 weeks between tadpole sampling, averaged from the

shallow and deep iButtons), microfauna abundance, species

richness (Shannon index) and elevation (Model 1). We

additionally modelled tadpoles and adults separately to better

understand how effects varied between the two life history

stages. For our tadpole model (Model 2), we modelled Bd

prevalence by estimating the ﬁxed effects of the survey

period (early or late sampling), mean pond temperature, ele-

vation, microfauna abundance and microfauna species rich-

ness (Shannon index). We modelled Bd prevalence for adults

by estimating the ﬁxed effects of body size (SVL mm),

ambient temperature and elevation (Model 3). Lastly, we

modelled tadpole body size (STL mm) separately against Bd

prevalence (Model 4), incorporating the survey period as a

ﬁxed effect and adhering to the same GLMM model struc-

ture (binomial distribution and logit link function). This

approach was required due to convergence issues encoun-

tered when including all six parameters in Model 2, indicat-

ing over-parameterisation. The site was included as a

random effect in all models.

We additionally modelled Bd intensity for adults against

ambient temperature, body size (SVL mm) and elevation,

using a GLMM with a negative binomial error distribution.

Site was also a random effect to account for variability

Figure 1 Location of the 23 study sites in the Australian Capital Territory, Australia. For tadpoles, diurnal sampling was conducted across

two time periods to compare earlier and later developmental stages, while nocturnal sampling of adults was undertaken once per site.

4Animal Conservation  (2024) – ª2024 Zoological Society of London.

Low Bd prevalence in tadpole of recovering species J. Crawford-Ash et al.

across sites. This model was implemented using the

glmmTMB package and accounted for zero inﬂation (Brooks

et al., 2023).

To assess inﬂuences on microfauna diversity and abun-

dance in our study system, two models were employed to

ﬁrst examine elevation: a Gaussian generalised linear model

(GLM) for species richness (Shannon index), and a negative

binomial model for the abundance. Subsequent analyses were

conducted to investigate the inﬂuence of the trophic state on

microfauna abundance and species richness (Shannon index).

This was done using an averaged Secchi disk measure as a

proxy for water transparency/turbidity. Again, two models

were used, a GLM with Gaussian distribution for the Shan-

non Index and a negative binomial model for the microfauna

abundance.

All model assumptions were veriﬁed by plotting residuals

versus ﬁtted values using the DHARMa package (Hartig &

Lohse, 2020). Data and code are available from https://

github.com/JordannCA/Whistling_tree_frog.

Results

Prevalence of Bd in tadpoles and adults

Batrachochytrium dendrobatidis prevalence was signiﬁcantly

lower in tadpoles compared to adults (Table 1; Estimate

=5.05, SE =0.48, z=10.63, P<0.001). Only ~1% of

tadpoles sampled were found to be Bd-positive, as opposed

to 50.53% of the adult population (Fig. 2). While there was

an increase in Bd prevalence between the two tadpole sam-

pling periods (Table 1; Fig. 2), the increase was not statisti-

cally signiﬁcant (P=0.320). Bd was detected at six sites

where tadpoles were sampled, with two sites (sites 4 and 14)

having Bd present during both the early and late sampling

period (Table S2, Fig. 3). Bd was detected in 20 out of 23

adult sites (Table S2, Fig. S1).

Bd prevalence in tadpoles was not associated with the

sampling period, mean pond temperature, water temperature

at the time of tadpole sampling, elevation, microfauna spe-

cies richness or microfauna abundance (Table 1). Tadpole

body size was modelled separately against Bd prevalence

and was not signiﬁcant (Table 1). Similarly, for adults, body

size and ambient temperature did not have a signiﬁcant

impact on Bd prevalence, however, elevation was nearly sig-

niﬁcant (Table 1).

Due to the low Bd genetic equivalence (GE) detected in

the tadpoles, there was insufﬁcient data to model Bd inten-

sity for the tadpole life group. For adults, elevation, body

size and ambient temperature did not show statistically sig-

niﬁcant effects on Bd intensity (Table 2).

Environmental drivers of Bd across sites

Tadpoles were found at the time of capture mostly within Bd

optimal growth temperatures of between 17 and 25°C

(Woodhams et al. 2008). Mean daily temperatures taken

from iButtons in each pond were largely below the optimal

Bd growth temperatures (Fig. 4). We additionally calculated

the time the tadpoles were exposed to temperatures above

28°C and below 17°C, and within the optimal Bd range of

17–25°C. Generally, temperatures were often below 17°C

with only ~14 h of pond temperature that exceeded the path-

ogen’s lethal limit across all sites (Table 3). Detailed temper-

ature data for each iButton and site can be found in

Table 1 Results of Generalised Linear Mixed Models (GLMM) with binomial distribution and logit link function. The response variable for all

models was Bd presence, with the site as a random effect. Signiﬁcant results are highlighted with an asterisk (*).

Model Fixed effects Estimate SE ZP

Adults and Tadpoles (Model 1) Intercept 0.49 0.23 2.14 0.03*

Age group (tadpole) 5.05 0.48 10.63 <0.01*

Microfauna abundance (raw count) 0.34 0.23 1.49 0.14

Microfauna species richness (Shannon index) 0.28 0.22 1.30 0.19

Mean pond temperature 0.56 0.29 1.90 0.06

Elevation 0.25 0.24 1.01 0.31

Tadpoles (Model 2) Intercept 7.57 66.00 0.12 0.91

Sampling period 0.66 0.74 0.89 0.38

Elevation 1.36 1.78 0.77 0.44

Mean pond temperature 1.95 1.53 1.27 0.20

Water temperature at time and place of tadpole sampling 0.03 0.76 0.04 0.97

Microfauna abundance (raw count) 0.60 0.54 1.12 0.26

Microfauna species richness (Shannon index) 4.15 203.18 0.02 0.98

Adults (Model 3) Intercept 0.11 0.28 0.41 0.68

Ambient Temperature 0.08 0.24 0.33 0.74

Body size (SVL mm) 0.23 0.18 1.24 0.21

Elevation 0.53 0.27 1.96 0.052*

Tadpoles (Model 4) Intercept 5.86 1.57 3.74 <0.01*

Sampling period 0.07 0.63 0.11 0.91

Body size (STL mm) 0.41 0.70 0.59 0.56

Animal Conservation  (2024) – ª2024 Zoological Society of London. 5

J. Crawford-Ash et al. Low Bd prevalence in tadpole of recovering species

Table S4. There was a distinct temperature increase between

sampling periods (Fig. S2).

Aquatic microfauna abundance and diversity varied

between sites and across the elevational gradient. Copepoda

were most abundant, with Ostracoda, Cladocera and Rotifera

similar in abundance across all sites (Fig. 5). We assessed

the impact of elevation and trophic state on microfauna

diversity and abundance. There was a signiﬁcant positive

relationship between the diversity of microfauna species and

elevation, suggesting that there is an increase in diversity at

higher elevations (Estimate =1.171e-04, SE =3.286e-05,

t=3.563, P=0.000394). In contrast, the negative binomial

model for abundance demonstrated that elevation had a sta-

tistically signiﬁcant negative effect on abundance, indicating

a decrease in abundance in higher elevations

(Estimate =0.0008316, SE =0.0004694, t=1.772). Our

averaged Secchi disk measurements were used as a proxy

for water clarity to represent the trophic state of the ponds

sampled. However, for both models, there was no statisti-

cally signiﬁcant relationship between water clarity and spe-

cies diversity (Estimate =0.0001088, SE =0.0013582,

t=0.08, P=0.937) or abundance (Estimate =0.004868,

SE =0.003110, t=1.565, P=0.14).

Discussion

Unique infection patterns support

recruitment

Batrachochytrium dendrobatidis is a key driver of global

amphibian biodiversity loss (Scheele et al., 2019a,2019b;

Luedtke et al., 2023). While general patterns of

disease-driven decline are reasonably well understood, varia-

tion in susceptibility across different life history stages

within species remains poorly known (Venesky et al., 2011;

Sauer et al., 2020;Wu,2023). Furthermore, there is little

known about differences in ecology, behaviour and complex

community interactions (Blaustein et al., 2011). An important

mechanism for persistence, particularly in areas of endemic

Bd, is maintaining or increasing recruitment despite high to

moderate adult mortality (West et al., 2020). It is therefore

crucial to quantify disease dynamics across all amphibian life

stages to better understand mechanisms that could support

high recruitment, and in turn the recovery and coexistence of

susceptible species living with Bd. In this study, we focused

on patterns of Bd prevalence in L. verreauxii across different

life history stages. Our results indicate consistently low Bd

prevalence and intensity in tadpoles, sampled across both

early and late developmental stages. In contrast, we found

that adults had 50 times higher Bd prevalence than tadpoles,

with moderate to high pathogen intensity.

The large difference in Bd prevalence in tadpoles com-

pared to conspeciﬁc adults that we document strongly con-

trasts with infection patterns found in other frog species and

study systems. In populations of species with moderate to

high adult Bd prevalence, tadpoles typically have correspond-

ing moderate to high Bd prevalence and intensity, ranging

from 20 to 90% prevalence across species in North America

(Ouellet et al., 2005; Knapp & Morgan, 2006; Venesky

et al., 2011) and South America (Catenazzi et al., 2013;

Ruggeri et al., 2020; Das Neves-da-Silva et al., 2021; Azat

et al., 2022), Africa (Conradie et al., 2011), Mediterranean

Europe (Clare et al., 2016; Fern

andez-Loras et al., 2019) and

Australia (Stockwell, 2011; Newell et al., 2013; Narayan

et al., 2014; Sapsford et al., 2018). In species where low Bd

prevalence has been documented in tadpoles, adults within

the same populations have correspondingly low infection

levels, suggesting overall lower infection pressure within

these systems (Cashins, 2009; Bataille et al., 2015; Grogan

et al., 2018). These patterns have been summarised with

examples in Table S3. However, our results show consis-

tently low Bd prevalence in tadpoles and moderate to high

Bd prevalence in adults, representing a unique pattern of

infection with opposing infection pressures across life history

stages. This unique ﬁnding suggests high infection pressure

for adults but not for tadpoles. While we do not have

long-term infection data for our adult L. verreauxii popula-

tion, previous studies of the closely related subspecies, L.

verreauxii alpina, show similar prevalence and intensity data,

with infection intensity increasing over the breeding season

and adults only living to approximately 2 years of age

Figure 2 (a) Batrachochytrium dendrobatidis (Bd ) infection preva-

lence for tadpole and adult life stages of Litoria verreauxii, with the

ﬁrst (early) and second (late) tadpole sampling period. Error bars

display binomial 95% conﬁdence intervals around the means.

6Animal Conservation  (2024) – ª2024 Zoological Society of London.

Low Bd prevalence in tadpole of recovering species J. Crawford-Ash et al.

(Scheele et al. 2015,2016). We infer similar infection

dynamics for L. verreauxii, but further investigation is

required. Our results indicate that although adults may be

succumbing to Bd infection shortly after breeding, the

populations can persist due to the low impact of Bd on the

tadpoles. Understanding the drivers of this unique pattern

could be vital for assisting in the recovery of other suscepti-

ble species.

Temperature and microfauna are unlikely

drivers of low Bd prevalence in L.

verreauxii tadpoles

The mechanism(s) underpinning the highly contrasting pat-

terns of Bd prevalence across life history stages in L. ver-

reauxii remain unknown. While temperature is a key element

inﬂuencing Bd infection globally (Woodhams et al., 2003;

Marantelli et al., 2004; Turner et al., 2021), our results sug-

gest that temperature is an unlikely explanation for low tad-

pole Bd prevalence in this system. Despite a seasonal

temperature increase between our two sampling periods

(Fig. S2), and mean pond temperature nearing statistical sig-

niﬁcance as a predictor in Model 1 (Table 1), iButton data

Figure 3 Bd prevalence and sample sizes for 18 tadpole sampling sites, across two sampling periods (early and late tadpole sampling). In

the late tadpole sampling period, no tadpoles were detected at sites 11 and 16.

Table 2 Results of statistical analysis using Generalised Linear

Mixed Models (GLMM) with negative binomial distribution. The

response variable was adult Bd intensity represented by genomic

equivalence, with the site as a random effect. Signifcant results

are highlighted with an asterisk (*).

Model Fixed effects Estimate SE ZP

Adults

(Model 5)

Intercept 6.58 0.30 21.77 <0.01*

Ambient

temperature

0.14 0.23 0.61 0.54

Body size (SVL

mm)

0.01 0.23 0.02 0.99

Elevation 0.16 0.32 0.49 0.62

Animal Conservation  (2024) – ª2024 Zoological Society of London. 7

J. Crawford-Ash et al. Low Bd prevalence in tadpole of recovering species

reveals that water temperatures predominantly stayed within

or below the optimal range for Bd (17–25°C). Notably, tem-

peratures within the ponds only exceeded the lethal threshold

for Bd (~28°C) for approximately 14 h in total across the

sampling period (Table 3). Conversely, Bd can remain alive

and grow slowly at lower temperatures (~4°C), allowing the

pathogen to persist during periods of colder weather (Pio-

trowski et al., 2004). This further suggests that temperature

is not an important determinant of Bd dynamics in tadpoles

in this system, with a temperature regime conducive to high

Bd prevalence. This is supported by the moderate to high Bd

infection prevalence and intensity detected in the adults at

the same ponds.

Studies have found that higher temperatures can reduce

Bd prevalence in tadpoles either by affecting the survival of

Bd or altering host immune defences (Woodhams

et al., 2003; Berger et al., 2004; Rowley, 2006; Sapsford

et al., 2013; Bradley et al., 2016,2019; Goldstein

et al., 2017; Sauer et al., 2020; Bielby et al., 2022). Temper-

ature measurements taken at the time and the place of tad-

pole sampling suggest that L. verreauxii tadpoles typically

inhabited waters within Bd’spreferred thermal range, with

infrequent deviations (Fig. 4). Therefore, while tadpoles

appear to generally select warmer microhabitats (surface of

the pond), the temperatures they experienced during our

sampling are unlikely to be high enough to signiﬁcantly

reduce Bd infection levels. However, it must be noted that a

large proportion of the studies referred to above have been

simulated in laboratory environments and have not accounted

for species’thermal preferences or variable environmental

conditions, and therefore hold little ecological relevance

when applying the outcomes in situ (Sapsford et al., 2018,

Sauer et al., 2020). For example, tadpoles from the midwife

toad, Alytes obstetricans, a highly Bd susceptible species,

were exposed to three temperature treatments (21.4, 26.2 and

~30°C) consistently for 5 days, with infections clearing in

treatments above 26°C (Geiger et al., 2011). In contrast, a

study by Fern

andez-Loras et al. in 2019 established the

CTmax (37°C) of tadpoles from the same species and

exposed them to temperatures between 28 and 37°C for

~20 min. The tadpoles were unable to clear Bd infection

within this time frame, and those infected with Bd had a

lower CTmax threshold (Fern

andez-Loras et al., 2019).

Figure 4 (a) Mean daily pond temperatures averaged across a shallow and deep iButton temperature logger at 17 sites during the 4-week

sampling period (one site was excluded due to the loss of iButtons). (b) Water temperatures measured at the time of tadpole sampling, dis-

played by Bd infection status, with red highlighting Bd-positive samples. Blue dashed lines on both ﬁgures indicate Bd optimal growth tem-

peratures (17–25°C). The red dashed line indicates Bd lethal limit (>28°C).

Table 3 Summary of time spent within and outside of preferred Bd

temperatures according to iButton data collected over a 4-week

sampling period, across 17 sites

iButton

category

(depth)

Hours within optimal

Bd conditions

(17–25°C)

Hours below

17°C

Hours above

lethal limit

for Bd (>28°C)

Shallow 1106 1418 13

Deep 236 2080 14

8Animal Conservation  (2024) – ª2024 Zoological Society of London.

Low Bd prevalence in tadpole of recovering species J. Crawford-Ash et al.

While temperature is acknowledged as a primary factor

inﬂuencing Bd infection dynamics (Woodhams et al., 2003;

Sapsford et al., 2013; Sauer et al., 2020), the intricacies of

its impact across diverse ecological systems, amphibian life

history stages and taxa are not yet fully understood.

Microfauna presence and diversity varied

between sites but were not linked to

disease dynamics

Research from Europe and the United States has found that

common microfauna species feed on Bd zoospores in the

water, particularly inﬂuencing infection dynamics in tadpole

populations (Buck et al., 2011; Schmeller et al., 2014). We

found notable variation in the abundance and diversity of

microfauna in sites across an elevational gradient, with

higher elevations associated with increased diversity, but

reduced abundance of microfauna. Due to consistently low

Bd prevalence and intensity observed in L. verreauxii

tadpoles, we could not establish any correlations between Bd

infection and microfauna presence. This suggests that factors

other than microfauna presence are more likely to be impor-

tant in determining Bd dynamics in our system. However,

our investigation is preliminary and the trophic role of

microfauna in Bd dynamics in Australia remains largely

unknown. Microfauna may be inﬂuencing disease dynamics

across the study region, potentially reducing the pathogen

load generally within the environment. We did not directly

examine the ability of the microfauna species found in this

system to consume Bd zoospores, and we therefore encour-

age further research in this ﬁeld.

Tadpole feeding mechanisms and

characteristics as potential explanations

for low Bd prevalence

The consistently low prevalence in tadpoles and the lack of

associations between temperature, microfauna and Bd

Figure 5 Microfauna abundance counts across 18 sites, per 6 L of water ﬁltered, per sampling period (early and late tadpole sampling). In

the second sampling (late tadpole sampling), sites 11 and 17 were unable to be processed for identiﬁcation due to severely eutrophic sam-

ples, so are excluded from the analysis. An asterisk indicates that Bd was detected on the tadpoles at that site.

Animal Conservation  (2024) – ª2024 Zoological Society of London. 9

J. Crawford-Ash et al. Low Bd prevalence in tadpole of recovering species

prevalence suggest that factors outside of the scope of our

study shape Bd dynamics in L. verreauxii tadpoles. One

potential explanation is tadpole feeding mechanisms. A large

portion of Litoria species including L. verreauxii, inhabit the

mid-water to surface portion of the water column and

employ ﬁltration feeding methods (Anstis, 2017). In contrast,

tadpoles of other Litoria species where higher Bd prevalence

has been documented, dwell at the bottom of streams and

utilise scraping or grazing feeding methods (e.g. Litoria nan-

notis and L. rheocola; Cashins, 2009; Anstis, 2017).

Benthic-dwelling tadpole species have generally higher Bd

infection rates than surface-dwelling species (Skerratt

et al., 2010) (see Table S3). The reasons behind these differ-

ences are unknown, however, it could be due to the larger

specialised mouth parts commonly found in grazing species,

which have more keratin present than ﬁlter-feeding species.

The location of Bd zoospores within the water column may

also drive variation in infection rates between ﬁlter and bot-

tom feeders. While it is poorly researched, zoospore density

within the water column is low, suggesting that exposure

would be low for ﬁlter-feeding species (Farthing

et al., 2021). In contrast, attachment and growth of Bd zoos-

porangia on dead algae and other organic matter has been

observed and may be consumed by benthic tadpoles (John-

son & Speare, 2003). While feeding mechanisms could help

explain the low prevalence we observed in the tadpoles, it is

unlikely to have changed pre- to post-population decline.

Nevertheless, with a greater understanding of the inﬂuence

of feeding mechanisms on disease dynamics in different spe-

cies, disease risk may be able to be predicted and in turn

managed. We are not aware of comparative studies investi-

gating infection risk for tadpoles with different feeding

mechanisms, only observations from individual laboratory

and ﬁeld-based studies. Therefore, we encourage further

investigation comparing tadpole Bd susceptibility based on

their ecologies.

Population density and body size have additionally been

shown to inﬂuence Bd susceptibility of adult frogs and tad-

poles alike (Garner et al., 2009; Briggs et al., 2010; Keiser

et al., 2019). We observed tadpoles across all areas of the

ponds, not clustered in one location, and pond sizes were

generally large, with sufﬁcient habitat (average diameter of

35 m), however, the population density was not directly

measured as part of this study. Tadpole age and resulting

larger body size have been shown to cause higher infection

risk in tadpoles (see Sapsford et al., 2018). We sampled both

early and late stage tadpoles, with distinct body size differ-

ences between sampling periods (Fig. S3), but found no

effect on Bd infection prevalence or intensity. This contrasts

with ﬁndings from Smith et al. (2007) and Cashins (2009),

who reported higher prevalence in larger tadpoles, suggesting

that this is also not a strong predictor in this species.

Environmental inﬂuences on Bd dynamics

Tadpoles that live in lotic environments (streams) have

higher average Bd prevalence than tadpoles from lentic envi-

ronments (ponds), which could also be a possible

explanation for the low tadpole Bd prevalence we observed

(Rowley & Alford, 2007; Cashins, 2009; Valencia-Aguilar

et al., 2016; Ruggeri et al., 2018). We focused on ponds for

this study as they are the predominant habitat used by this

species in this region, and to reduce environmental variation

between sites. Further sampling targeting stream populations

of L. verreauxii would enable a comparison of Bd prevalence

and intensity between the two habitat types and could reveal

that lentic habitat may be associated with the observed low

prevalence. Similarly, tadpoles that overwinter or develop

slowly are more susceptible to Bd infection due to extended

time in the water and often serve as infection reservoirs for

adults and other sympatric species (Venesky et al., 2011;

Narayan et al., 2014; Wetsch et al., 2022). Litoria verreauxii

are fast developers, remaining in the water for an average of

4–6 weeks, unlike other species in this same system

(Table S2), such as Limnodynastes species that develop in 5–

8 months and often overwinter (Anstis 2017), allowing for

longer exposure to Bd than L. verreauxii.

More complex factors to be considered for

future research

Our study does not provide a deﬁnitive explanation for the

low prevalence observed in L. verreauxii tadpoles. Other fac-

tors that were not addressed in this study such as variations

in the virulence of the Bd strain (Berger et al., 2005; Bataille

et al., 2015), or changes in the tadpoles’intrinsic susceptibil-

ity or immunity (Voyles et al., 2011), may be contributing to

the observed infection pattern, however, changes in these

particular factors within the timeframe of the observed

decline and subsequent re-expansion seem unlikely. Addi-

tionally, unexplored environmental shifts including changes

in community composition and density over time, may also

inﬂuence the low infection pressure observed in this ecosys-

tem (Hemingway, 2015; Fern

andez-Beaskoetxea et al., 2016;

Stockwell et al., 2016). Briggs et al. (2010) outlined the pos-

sibility of a shift from epidemic conditions to endemic con-

ditions driving extinction or persistence in amphibian

communities. Briggs et al.’s model proposes that after an ini-

tial outbreak, Bd loads can surge in susceptible individuals,

leading to local extinctions. However, survivors of the initial

epidemic may persist in a new endemic state (Briggs

et al., 2010). Severe declines occurred in L. verreauxii in the

1980s (Osborne et al., 1999; Scheele et al., 2014), corre-

sponding with the arrival and spread of Bd throughout east-

ern Australia (Berger et al., 1998; Skerratt et al., 2007). The

concurrent local disappearance of four sympatric species in

the same region (Osborne, 1990; Osborne, 1992; Osborne &

Hunter, 1998; Hamer et al., 2010) supports this model, how-

ever, without data on broader amphibian Bd dynamics, and

community composition before and during the period of

decline and re-expansion, the impact of such changes on this

system remains unclear.

Studies in other systems indicate that amphibian commu-

nity densities (Rachowicz & Briggs, 2007; Greer

et al., 2008; Ribeiro et al., 2020; Harjoe et al., 2022) and

the presence of reservoir species signiﬁcantly affect Bd

10 Animal Conservation  (2024) – ª2024 Zoological Society of London.

Low Bd prevalence in tadpole of recovering species J. Crawford-Ash et al.

dynamics in susceptible species (Reeder et al., 2012; Scheele

et al., 2017b; Brannelly et al., 2018; Jervis et al., 2020). Ten

species make up the amphibian community utilizing ponds

within our study region, with the most common species

being Crinia signifera, Limnodynastes tasmaniensis, Litoria

peronii and Limnodynastes peronii (ALA 2024). These frogs

are generalist, widespread species with varied Bd susceptibil-

ity, yet all have low mortality from the pathogen (Stockwell

et al., 2010; Abdul-Aziz 2011; Ocock et al., 2013; Besedin

et al., 2022). Crinia signifera is an identiﬁed reservoir spe-

cies in Australia and has inﬂuenced the Bd infection dynam-

ics of other susceptible frog species (See Scheele et al.,

2017b; Brannelly et al., 2018; Burns et al., 2021). We noted

all frog species encountered during the adult surveys, with

Crinia signifera observed at all sites in this study

(Table S2). These observations were supplementary to our

main data and therefore may not represent the amphibian

community in its entirety, however, there are no clear associ-

ations with other species present and the proportion of infec-

tion for either tadpoles or adults in this system (Table S2),

further highlighting the unique nature of very low chytrid

infection in L. verreauxii tadpoles. Overall, community

dynamics have not been a focus in this region and merit

attention in future research.

Conclusions

Understanding mechanisms underpinning population recovery

despite the ongoing presence of Bd can guide the develop-

ment of conservation strategies for at-risk amphibian species.

Our study uncovers a unique pattern of Bd infection dynam-

ics in L. verreauxii, with tadpoles exhibiting consistently

lower Bd infection than adults within the same populations.

However, due to the very low Bd prevalence in tadpoles, the

role that possible drivers such as temperature, elevation,

microfauna abundance and richness and other environmental

factors play in the infection dynamics in this system remain

unclear. This underscores the necessity of a comprehensive

understanding of disease dynamics across life history stages

to inform and enhance conservation strategies.

Acknowledgements

We express our gratitude to Holsworth Wildlife Research

Endowment, Frog and Tadpole Study Group of NSW and

the Peter Rankin Trust Fund for their ﬁnancial support,

which was instrumental in conducting this research. Our

appreciation extends to ACT Parks and Conservation for per-

mitting us to conduct our studies on their managed lands. J.

Erens was supported by the Research Foundation Flanders

(FWO) through PhD fellowships 1173119N-1173121N.

Authors’ contributions

JCA and JE share the ﬁrst authorship on this manuscript. All

authors conceived the ideas and designed the methodology;

JCA and JE collected the data; JCA and JE analysed the

data; JCA and JE visualized the data; JCA led the writing of

the manuscript. All authors contributed critically to the drafts

and gave ﬁnal approval for publication.

References

Abdul-Aziz, A. (2011). Dynamics of chytridiomycosis in a

Tasmanian frog community. Doctoral dissertation, University

of Tasmania.

Anstis, M. (2017). Tadpoles and frogs of Australia. Sydney:

New Holland Publishers Pty Limited.

Atlas of Living Australia. (2024). Occurrence download.

https://biocache.ala.org.au/occurrences/search?&q=qid%

3A1720396471767&disableAllQualityFilters=true

Azat, C., Alvarado-Rybak, M., Solano-Iguaran, J.J., Velasco,

A., Valenzuela-S

anchez, A., Flechas, S.V., Pe~

naﬁel-Ricaurte,

A., Cunningham, A.A. & Bacigalupe, L.D. (2022).

Synthesis of Batrachochytrium dendrobatidis infection in

South America: Amphibian species under risk and areas to

focus research and disease mitigation. Ecography 2022(7),

e05977.

Bataille, A., Cashins, S.D., Grogan, L., Skerratt, L.F., Hunter,

D., McFadden, M., Scheele, B., Brannelly, L.A., Macris, A.,

Harlow, P.S., Bell, S., Berger, L. & Waldman, B. (2015).

Susceptibility of amphibians to chytridiomycosis is

associated with MHC class II conformation. Proc. R. Soc. B

Biol. Sci.,282(1805), 20143127.

Bates, D., Maechler, M., Bolker, B., Walker, S., Christensen,

R. H. B., Singmann, H., Dai, B., Grothendieck, G., Green,

P. & Bolker, M. B. (2015). Package ‘lme4’.convergence,

12(1), 2. I

Berger, L., Marantelli, G., Skerratt, L. F., & Speare, R. (2005).

Virulence of the amphibian chytrid fungus Batrachochytrium

dendrobatidis varies with the strain. Diseases of aquatic

organisms,68(1), 47–50.

Berger, L., Speare, R. & Kent, A. (1999). Diagnosis of

chytridiomycosis in amphibians by histological examination.

Zoos’Print J. 15, 184–190.

Berger, L., Speare, R., Daszak, P., Green, D.E., Cunningham,

A.A., Goggin, C.L., Slocombe, R., Ragan, M.A., Hyatt,

A.D., McDonald, K.R., Hines, H.B., Lips, K.R., Marantelli,

G. & Parkes, H. (1998). Chytridiomycosis causes amphibian

mortality associated with population declines in the rain

forests of Australia and Central America. Proc. Nat. Acad.

Sci. USA 95, 9031–9036.

Berger, L., Speare, R., Hines, H.B., Marantelli, G., Hyatt,

A.D., McDonald, K.R., Skerratt, L.F., Olsen, V., Clarke,

J.M., Gillespie, G. & Mahony, M. (2004). Effect of season

and temperature on mortality in amphibians due to

chytridiomycosis. Australian Veterinary Journal 82(7),

434–439.

Besedin, D., Turner, B.J., Deo, P., Lopes, M.D.B. & Williams,

C.R. (2022). Effect of captivity and water salinity on

culture-dependent frog skin microbiota and

Batrachochytrium dendrobatidis (Bd) infection. Trans. R.

Soc. S. Aust. 146, 273–294.

Animal Conservation  (2024) – ª2024 Zoological Society of London. 11

J. Crawford-Ash et al. Low Bd prevalence in tadpole of recovering species

Bielby, J., Sausor, C., Monsalve-Carcano, C. & Bosch, J.

(2022). Temperature and duration of exposure drive

infection intensity with the amphibian pathogen

Batrachochytrium dendrobatidis.PeerJ 10, e12889.

Blaustein, A.R., Gervasi, S.S., Johnson, P.T., Hoverman, J.T.,

Belden, L.K., Bradley, P.W. & Xie, G.Y. (2012).

Ecophysiology meets conservation: understanding the role of

disease in amphibian population declines. Philosophical

Transactions of the Royal Society of London. Series B,

Biological Sciences 367, 1688–1707.

Blaustein, A.R., Romansic, J.M., Scheessele, E.A., Han, B.A.,

Pessier, A.P. & Longcore, J.E. (2005). Interspeciﬁc variation

in susceptibility of frog tadpoles to the pathogenic fungus

Batrachochytrium dendrobatidis.Conservation Biology 19

(5), 1460–1468.

Blaustein, A.R., Urbina, J., Snyder, P.W., Reynolds, E., Dang,

T., Hoverman, J.T., Han, B., Olson, D.H., Searle, C. &

Hambalek, N.M. (2018). Effects of emerging infectious

diseases on amphibians: A review of experimental studies.

Diversity 10(3), 81.

Blaustein, A.R., Han, B.A., Relyea, R.A., Johnson, P.T.J.,

Buck, J.C., Gervasi, S.S. & Kats, L.B. (2011). The

complexity of amphibian population declines: understanding

the role of cofactors in driving amphibian losses. Ann. N. Y.

Acad. Sci. 1223, 108–119.

Blooi, M., Laking, A.E., Martel, A., Haesebrouck, F., Jocque,

M., Brown, T., Green, S., Vences, M., Bletz, M.C. &

Pasmans, F. (2017). Host niche may determine

disease-driven extinction risk. PLoS One 12,9–12.

Bosch, J., Fern

andez-Beaskoetxea, S., Fisher, M.C. & Garner,

T.W.J. (2013). Evidence for the introduction of lethal

chytridiomycosis affecting wild Betic midwife toads (Alytes

dickhilleni). PLoS One 10,82–89.

Boyle, D.G., Boyle, D.B., Olsen, V., Morgan, J.A.T. & Hyatt,

A.D. (2004). Rapid quantitative detection of

chytridiomycosis (Batrachochytrium dendrobatidis)in

amphibian samples using real-time Taqman PCR assay.

Diseases of Aquatic Organisms 60(2), 141–148.

Bradley, P.W., Brawner, M.D., Raffel, T.R., Rohr, J.R., Olson,

D.H. & Blaustein, A.R. (2016). Effect of temperature on

chytridiomycosis in larval amphibians. Integr. Comp. Biol.

56, E23.

Bradley, P.W., Brawner, M.D., Raffel, T.R., Rohr, J.R., Olson,

D.H. & Blaustein, A.R. (2019). Shifts in temperature

inﬂuence how Batrachochytrium dendrobatidis infects

amphibian larvae. PLoS One 14, e0222237.

Brannelly, L.A., Webb, R.J., Hunter, D.A., Clemann, N.,

Howard, K., Skerratt, L.F., Berger, L. & Scheele, B.C.

(2018). Non-declining amphibians can be important

reservoir hosts for amphibian chytrid fungus. Anim.

Conserv. 21,91–101.

Briggs, C.J., Knapp, R.A. & Vredenburg, V.T. (2010).

Enzootic and epizootic dynamics of the chytrid fungal

pathogen of amphibians. Proc. Natl. Acad. Sci. USA 107,

9695–9700.

Brooks, M., Bolker, B., Kristensen, K., Maechler, M.,

Magnusson, A., McGillycuddy, M. et al. (2023). Package

‘glmmtmb’. R Packag Vers, 1(1), 7.

Buck, J.C., Truong, L. & Blaustein, A.R. (2011). Predation by

zooplankton on Batrachochytrium dendrobatidis: Biological

control of the deadly amphibian chytrid fungus? Biodivers.

Conserv. 20, 3549–3553.

Burns, T.J., Scheele, B.C., Brannelly, L.A., Clemann, N.,

Gilbert, D. & Driscoll, D.A. (2021). Indirect terrestrial

transmission of amphibian chytrid fungus from reservoir to

susceptible host species leads to fatal chytridiomycosis.

Anim. Conserv. 24, 602–612.

Carvalho, T., Becker, C.G. & Toledo, L.F. (2017). Historical

amphibian declines and extinctions in Brazil linked to

chytridiomycosis. Proc. R. Soc. B Biol. Sci. 284(1848),

20162254.

Cashins, S.D. (2009). Epidemiology of chytridiomycosis in

rainforest stream tadpoles. Doctoral dissertation, James Cook

University.

Catenazzi, A., von May, R. & Vredenburg, V.T. (2013). High

prevalence of infection in tadpoles increases vulnerability to

fungal pathogen in high-Andean amphibians. Biol. Conserv.

159, 413–421.

Clare, F., Daniel, O., Garner, T. & Fisher, M. (2016).

Assessing the ability of swab data to determine the true

burden of infection for the amphibian pathogen

Batrachochytrium dendrobatidis.Ecohealth 13, 360–367.

Conradie, W., Weldon, C., Smith, K.G. & Du Preez, L.H.

(2011). Seasonal pattern of chytridiomycosis in common

river frog (Amietia angolensis) tadpoles in the South

African grassland biome. Afr. Zool. 46,95–102.

Crawford-Ash, J. & Rowley, J. (2021). Bad neighbours:

amphibian chytrid fungus Batrachochytrium dendrobatidis

infection dynamics in three co-occurring frog species of

southern Sydney, Australia. Dis. Aquat. Organ. 143,

101–108.

Das Neves-da-Silva, D., Borges, V.N.T., Branco, C.W.C. & De

Carvalho-E-Silva, A. (2021). Effects of intrinsic and

extrinsic factors on the prevalence of the fungus

Batrachochytrium dendrobatidis (Chytridiomycota) in stream

tadpoles in the Atlantic Forest domain. Aquat. Ecol. 55,

891–902.

Deknock, A., Pasmans, F., van Leeuwenberg, R., Van Praet,

S., Bruneel, S., Lens, L., Croubels, S., Martel, A. &

Goethals, P. (2021). Alternative food sources interfere with

removal of a fungal amphibian pathogen by zooplankton. J.

Appl. Ecol. 58, 2914–2923.

Farthing, H.N., Jiang, J., Henwood, A.J., Fenton, A., Garner,

T.W.J., Daversa, D.R., Fisher, M.C. & Montagnes, D.J.S.

(2021). Microbial grazers may aid in controlling infections

caused by the aquatic zoosporic fungus Batrachochytrium

dendrobatidis.Front. Microbiol. 11 , 592286.

Fern

andez-Beaskoetxea, S., Bosch, J. & Bielby, J. (2016).

Infection and transmission heterogeneity of a multi-host

pathogen (Batrachochytrium dendrobatidis) within an

12 Animal Conservation  (2024) – ª2024 Zoological Society of London.

Low Bd prevalence in tadpole of recovering species J. Crawford-Ash et al.

amphibian community. Diseases of Aquatic Organisms

118 (1), 11–20.

Fern

andez-Loras, A., Boyero, L., Correa-Araneda, F., Tejedo,

M., Hettyey, A. & Bosch, J. (2019). Infection with

Batrachochytrium dendrobatidis lowers heat tolerance of

tadpole hosts and cannot be cleared by brief exposure to

CTmax. PLoS One 14(4), e0216090.

Fisher, M.C. & Garner, T.W.J. (2020). Chytrid fungi and

global amphibian declines. Nat. Rev. Microbiol 18, 332–343.

Frenken, T., Agha, R., Schmeller, D.S., van West, P. &

Wolinska, J. (2019). Biological concepts for the control of

aquatic zoosporic diseases. Trends Parasitol. 35, 571–582.

Garner, T.W.J., Garcia, G., Carroll, B. & Fisher, M.C. (2009).

Using itraconazole to clear Batrachochytrium dendrobatidis

infection, and subsequent depigmentation of Alytes

muletensis tadpoles. Dis. Aquat. Organ. 83, 257–260.

Geiger, C.C., Kupfer, E., Schar, S., Wolf, S. & Schmidt, B.R.

(2011). Elevated temperature clears chytrid fungus infections

from tadpoles of the midwife toad, Alytes obstetricans.

Amphib. Reptil. 32, 276–280.

Gervasi, S., Gondhalekar, C., Olson, D.H. & Blaustein, A.R.

(2013). Host identity matters in the amphibian-Batrachochytrium

dendrobatidis system: Fine-scale patterns of variation in

responses to a multi-host pathogen. PLoS One 8(1), e54490.

Goldstein, J.A., von Seckendorr Hoff, K. & Hillyard, S.D.

(2017). The effect of temperature on development and

behaviour of relict leopard frog tadpoles. Conservation.

Phys. Ther. 5(1), cow075.

Gosner, K. (1960). A simpliﬁed table for staging anuran

embryos and larvae with notes on identiﬁcation.

Herpetologica 16(3), 183–190.

Greer, A.L., Briggs, C.J. & Collins, J.P. (2008). Testing a key

assumption of host-pathogen theory: density and disease

transmission. Oikos 117 , 1667–1673.

Grogan, L.F., Cashins, S.D., Skerratt, L.F., Berger, L.,

McFadden, M.S., Harlow, P., Hunter, D.A., Scheele, B.C. &

Mulvenna, J. (2018). Evolution of resistance to

chytridiomycosis is associated with a robust early immune

response. Mol. Ecol. 27, 919–934.

Hamer, A.J., Lane, S.J. & Mahony, M.J. (2010). Using

probabilistic models to investigate the disappearance of a

widespread frog-species complex in high-altitude regions of

south-eastern Australia. Animal Conservation 13(3), 275–

285.

Hartig, F. & Lohse, L. (2020). DHARMa: Residual

Diagnostics for Hierarchical (Multi-Level/Mixed) Regression

Models [Computer software]. https://CRAN.R-project.org/

package=DHARMa

Harjoe, C.C., Buck, J.C., Rohr, J.R., Roberts, C.E., Olson,

D.H. & Blaustein, A.R. (2022). Pathogenic fungus causes

density- and trait-mediated trophic cascades in an aquatic

community. Ecosphere 13(4), e4043.

Hemingway, V. A. (2015). Effects of biotic and abiotic setting

on a host-pathogen relationship: How environmental and

community characteristics inﬂuence infection prevalence and

intensity of amphibian chytrid on California’s central coast.

University of California, Santa Cruz, pp. 6–83.

Humphries, J.E., Lanct^

ot, C.M., McCallum, H.I., Newell, D.A.

& Grogan, L.F. (2024). Chytridiomycosis causes high

amphibian mortality prior to the completion of

metamorphosis. Environ. Res. 247, 118249.

Humphries, J.E., Lanct^

ot, C.M., Robert, J., McCallum, H.I.,

Newell, D.A. & Grogan, L.F. (2022). Do immune system

changes at metamorphosis predict vulnerability to

chytridiomycosis? An update. Dev. Comp. Immunol. 136,

104510.

Jervis, P., Jehle, R., Almeida-Reinoso, D., Almeida-Reinoso, F.,

Ron, S., Fisher, M.C. & Merino-Viteri, A.K.B. (2020). Disease

reservoirs threaten the recently rediscovered Podocarpus stubfoot

toad (Atelopus podocarpus). PLoS One 14(2), 157–164.

Jim

enez, R.R., Alvarado, G., Estrella, J. & Sommer, S. (2019).

Moving beyond the host: unraveling the skin microbiome of

endangered Costa Rican amphibians. Frontiers in

Microbiology 10, 2060.

Johnson, M.L. & Speare, R. (2003). Survival of

Batrachochytrium dendrobatidis in water: quarantine and

disease control implications. Emerg. Infect. Dis. 9, 922.

Keiser, C.N., Wantman, T., Rebollar, E.A. & Harris, R.N.

(2019). Tadpole body size and behaviour alter the social

acquisition of a defensive bacterial symbiont. R. Soc. Open

Sci. 6(9), 191080.

Knapp, R.A. & Morgan, J.A. (2006). Tadpole mouthpart

depigmentation as an accurate indicator of chytridiomycosis,

an emerging disease of amphibians. Copeia 2006, 188–197.

Lips, K.R., Brem, F., Brenes, R., Reeve, J.D., Alford, R.A.,

Voyles, J., Carey, C., Livo, L., Pessier, A.P. & Collins, J.P.

(2006). Emerging infectious disease and the loss of

biodiversity in a Neotropical amphibian community. Proc.

Nat. Acad. Sci. USA 103, 3165–3170.

Longcore, J.E., Pessier, A.P. & Nichols, D.K. (1999).

Batrachochytrium dendrobatidis gen. Et sp. nov., a chytrid

pathogenic to amphibians. Mycologia 91, 219–227.

Luedtke, J.A., Chanson, J., Neam, K., Hobin, L., Maciel,

A.O., Catenazzi, A., Borz

ee, A. et al. (2023). Ongoing

declines for the world’s amphibians in the face of emerging

threats. Nature 622, 308–314.

Marantelli, G., Berger, L., Speare, R. & Keegan, L. (2004).

Distribution of the amphibian chytrid Batrachochytrium

dendrobatidis and keratin during tadpole development. Pac.

Conserv. Biol. 10, 173–179.

Muths, E., Scherer, R.D. & Pilliod, D.S. (2011).

Compensatory effects of recruitment and survival when

amphibian populations are perturbed by disease. J. Appl.

Ecol. 48, 873–879.

Narayan, E.J., Graham, C., McCallum, H. & Hero, J.M.

(2014). Over-wintering tadpoles of Mixophyes fasciolatus

act as reservoir host for Batrachochytrium dendrobatidis.

PLoS One 9, e92499.

Newell, D. A., Goldingay, R. L., & Brooks, L. O. (2013).

Population recovery following decline in an endangered

Animal Conservation  (2024) – ª2024 Zoological Society of London. 13

J. Crawford-Ash et al. Low Bd prevalence in tadpole of recovering species

stream-breeding frog (Mixophyes ﬂeayi) from subtropical

Australia. PLoS ONE,8.

O’Hanlon, S.J., Rieux, A., Farrer, R.A., Rosa, G.M.,

Waldman, B., Bataille, A., Kosch, T.A. et al. (2018). Recent

Asian origin of chytrid fungi causing global amphibian

declines. Science 360, 621–627.

Ocock, J.F., Rowley, J.J.L., Penman, T.D., Rayner, T.S. &

Kingsford, R.T. (2013). Amphibian chytrid prevalence in an

amphibian community in arid Australia. Ecohealth 10,77–81.

Osborne, W. (1990). Declining frog populations and

extinctions in the Canberra region. Bogong 11,4–7.

Osborne, W. (1992). Declines and extinctions in populations

of frogs in the ACT: a discussion paper. Internal Report 92/

8. Canberra: ACT Parks and Conservation Service.

Osborne, W., Hunter, D. & Hollis, G. (1999). Population

declines and range contraction in Australian alpine frogs. In

Declines and disappearances of Australian frogs. Canberra:

Biodiversity Group, Environment Australia, 145–157.

Osborne, W. & Hunter, D. (1998). Frog declines in the

Australian alps: Where are we after ten years? Bogong 19,

3–6.

Ouellet, M., Mikaelian, I., Pauli, B.D., Rodrigue, J. & Green,

D.M. (2005). Historical evidence of widespread chytrid

infection in north American amphibian populations.

Conserv. Biol. 19, 1431–1440.

Piotrowski, J.S., Annis, S.L. & Longcore, J.E. (2004).

Physiology of Batrachochytrium dendrobatidis, a chytrid

pathogen of amphibians. Mycologia 96,9–15.

Rachowicz, L.J. & Briggs, C.J. (2007). Quantifying the

disease transmission function: effects of density on

Batrachochytrium dendrobatidis transmission in the

mountain yellow-legged frog Rana muscosa.Journal of

Animal Ecology 76,711–721.

Rachowicz, L.J. & Vredenburg, V.T. (2004). Transmission of

Batrachochytrium dendrobatidis within and between

amphibian life stages. Dis. Aquat. Organ. 61,75–83.

Reeder, N.M.M., Pessier, A.P. & Vredenburg, V.T. (2012). A

reservoir species for the emerging amphibian pathogen

Batrachochytrium dendrobatidis thrives in a landscape

decimated by disease. PLoS One 7,1–7.

Ribeiro, J.W., Siqueira, T., DiRenzo, G.V., Lambertini, C.,

Lyra, M.L., Toledo, L.F., Haddad, C.F.B. & Becker, C.G.

(2020). Assessing amphibian disease risk across tropical

streams while accounting for imperfect pathogen detection.

Oecologia 193, 237–248.

Rowley, J.J. (2006). Why does Chytridiomycosis drive some

frog populations to extinction and not others?: The effects

of interspeciﬁc variation in host behaviour. (Doctoral

dissertation, James Cook University).

Rowley, J. & Alford, R. (2013). Hot bodies protect

amphibians against chytrid infection in nature. Scientiﬁc

Reports 3, 1515.

Rowley, J.J.L. & Alford, R.A. (2007). Behaviour of Australian

rainforest stream frogs may affect the transmission of

chytridiomycosis. Dis. Aquat. Organ. 77,1–9.

Ruggeri, J., Martins, A.G.D., Domingos, A.H.R., Santos, I.,

Viroomal, I.B. & Toledo, L.F. (2020). Seasonal prevalence

of the amphibian chytrid in a tropical pond-dwelling tadpole

species. Dis. Aquat. Organ. 142, 171–176.

Ruggeri, J., Toledo, L.F. & de Carvalho-e-Silva, S.P. (2018).

Stream tadpoles present high prevalence, but low infection

loads of Batrachochytrium dendrobatidis (Chytridiomycota).

Hydrobiologia 806, 303–311.

Sapsford, S.J., Alford, R.A. & Schwarzkopf, L. (2013).

Elevation, temperature, and aquatic connectivity all inﬂuence

the infection dynamics of the amphibian chytrid fungus in

adult frogs. PLoS One 8, e82425.

Sapsford, S.J., Alford, R.A. & Schwarzkopf, L. (2018).

Disentangling causes of seasonal infection prevalence

patterns: Tropical tadpoles and chytridiomycosis as a model

system. Dis. Aquat. Organ. 130,83–93.

Sauer, E.L., Cohen, J.M., Lajeunesse, M.J., McMahon, T.A.,

Civitello, D.J., Knutie, S.A., Nguyen, K., Roznik, E.A.,

Sears, B.F., Bessler, S., Delius, B.K., Halstead, N.,

Ortega, N., Venesky, M.D., Young, S. & Rohr, J.R. (2020).

A meta-analysis reveals temperature, dose, life stage, and

taxonomy inﬂuence host susceptibility to a fungal parasite.

Ecology 101(4), e02979.

Scheele, B.C., Foster, C.N., Hunter, D.A., Lindenmayer, D.B.,

Schmidt, B.R. & Heard, G.W. (2019b). Living with the

enemy: Facilitating amphibian coexistence with disease.

Biol. Conserv. 236,52–59.

Scheele, B.C., Guarino, F., Osborne, W., Hunter, D.A.,

Skerratt, L.F. & Driscoll, D.A. (2014). Decline and

re-expansion of an amphibian with high prevalence of

chytrid fungus. Biol. Conserv. 170,86–91.

Scheele, B.C., Hunter, D.A., Banks, S.C., Pierson, J.C.,

Skerratt, L.F., Webb, R. & Driscoll, D.A. (2016). High adult

mortality in disease-challenged frog populations increases

vulnerability to drought. J. Anim. Ecol. 85, 1453–1460.

Scheele, B.C., Hunter, D.A., Brannelly, L.A., Skerratt, L.F. &

Driscoll, D.A. (2017b). Reservoir-host ampliﬁcation of

disease impact in an endangered amphibian. Conserv. Biol.

31, 592–600.

Scheele, B.C., Hunter, D.A., Skerratt, L.F., Brannelly, L.A. &

Driscoll, D.A. (2015). Low impact of chytridiomycosis on

frog recruitment enables persistence in refuges despite high

adult mortality. Biol. Conserv. 182,36–43.

Scheele, B.C., Pasmans, F., Skerratt, L.F., Berger, L., Martel,

A., Beukema, W., Acevedo, A.A. et al. (2019a). Amphibian

fungal panzootic causes catastrophic and ongoing loss of

biodiversity. Science 363, 1459–1463.

Scheele, B.C., Skerratt, L.F., Grogan, L.F., Hunter, D.A.,

Clemann, N., McFadden, M., Newell, D., Hoskin, C.J.,

Gillespie, G.R., Heard, G.W., Brannelly, L., Roberts, A.A.

& Berger, L. (2017a). After the epidemic: Ongoing

declines, stabilizations and recoveries in amphibians afﬂicted

by chytridiomycosis. Biol. Conserv. 206,37–46.

Schmeller, D.S., Blooi, M., Martel, A., Garner, T.W.J., Fisher,

M.C., Azemar, F., Clare, F.C., Leclerc, C., J€

ager, L.,

14 Animal Conservation  (2024) – ª2024 Zoological Society of London.

Low Bd prevalence in tadpole of recovering species J. Crawford-Ash et al.

Guevara-Nieto, M., Loyau, A. & Pasmans, F. (2014).

Microscopic aquatic predators strongly affect infection

dynamics of a globally emerged pathogen. Curr. Biol. 24,

176–180.

Searle, C.L., Mendelson, J.R., Green, L.E. & Duffy, M.A.

(2013). Daphnia predation on the amphibian chytrid fungus

and its impacts on disease risk in tadpoles. Ecol. Evol. 3,

4129–4138.

Skerratt, L.F., Berger, L., Speare, R., Cashins, S., McDonald,

K.R., Phillott, A.D., Hines, H.B. & Kenyon, N. (2007).

Spread of chytridiomycosis has caused the rapid global

decline and extinction of frogs. Ecohealth 4(2), 125–134.

Skerratt, L.F., McDonald, K.R., Hines, H.B., Berger, L.,

Mendez, D., Phillott, A.D. et al. (2010). Application of the

survey protocol for chytridiomycosis to Queensland,

Australia. Dis. Aquat. Org. 92,117–129.

Smith, K.G., Weldon, C., Conradie, W. & du Preez, L.H.

(2007). Relationships among size, development, and

Batrachochytrium dendrobatidis infection in African

tadpoles. Diseases of Aquatic Organisms 74(2), 159–164.

Stevenson, L.A., Alford, R.A., Bell, S.C., Roznik, E.A.,

Berger, L. & Pike, D.A. (2013). Variation in thermal

performance of a widespread pathogen, the amphibian

chytrid fungus Batrachochytrium dendrobatidis.PLoS One

8,1–14.

Stockwell, M. (2011). Impact and mitigation of the emerging

infectious disease chytridiomycosis on the green and golden

bell frog. Doctoral dissertation, University of Newcastle.

Stockwell, M.P., Bower, D.S., Clulow, J. & Mahony, M.J.

(2016). The role of non-declining amphibian species as

alternative hosts for Batrachochytrium dendrobatidis in an

amphibian community. Wildl. Res. 43, 341–347.

Stockwell, M.P., Clulow, J. & Mahony, M.J. (2010). Host

species determines whether infection load increases beyond

disease-causing thresholds following exposure to the

amphibian chytrid fungus. Anim. Conserv. 13,62–71.

Turner, A., Wassens, S., Heard, G. & Peters, A. (2021).

Temperature as a driver of the pathogenicity and virulence of

amphibian chytrid fungus Batrachochytrium dendrobatidis:A

systematic review. J. Wildl. Dis. 57, 477–494.

Valencia-Aguilar, A., Toledo, L.F., Vital, M.V.C. & Mott, T.

(2016). Seasonality, environmental factors, and host

behavior linked to disease risk in stream-dwelling tadpoles.

Dis. Aquat. Organ. 72,98–106.

Venesky, M.D., Kerby, J.L., Storfer, A. & Parris, M.J. (2011).

Can differences in host behaviour drive patterns of disease

prevalence in tadpoles? PLoS One 6, e24991.

Venesky, M.D., Parris, M.J. & Storfer, A. (2009). Impacts of

Batrachochytrium dendrobatidis infection on tadpole

foraging performance. Ecohealth 6, 565–575.

Voyles, J., Rosenblum, E.B. & Berger, L. (2011). Interactions

between Batrachochytrium dendrobatidis and its amphibian

hosts: A review of pathogenesis and immunity. Microb.

Infect. 13,25–32.

V€

or€

os, J., Herczeg, D., F€

ul€

op, A., G

al, T.J., D

an,



A., Harmos,

K. & Bosch, J. (2018). Batrachochytrium dendrobatidis in

Hungary: An overview of recent and historical occurrence.

Acta Herpetol. 13, 125–140.

West, M. (2015). Contrasting population responses of

ecologically similar sympatric species to multiple

threatening processes. Doctoral dissertation, University of

Melbourne.

West, M., Todd, C.R., Gillespie, G.R. & McCarthy, M.

(2020). Recruitment is key to understanding amphibian’s

different population-level responses to chytrid fungus

infection. Biological Conservation 241, 108247.

Wetsch, O., Strasburg, M., McQuigg, J. & Boone, M.D.

(2022). Is overwintering mortality driving enigmatic

declines? Evaluating the impacts of trematodes and the

amphibian chytrid fungus on an anuran from hatching

through overwintering. PLoS One 17(1), e0262561.

Woodhams, D.C., Alford, R.A., Briggs, C.J., Johnson, M. &

Rollins-Smith, L.A. (2008). Life-history trade-offs inﬂuence

disease in changing climates: Strategies of an amphibian

pathogen. Ecology 89(6), 1627–1639.

Woodhams, D.C., Alford, R.A. & Marantelli, G. (2003).

Emerging disease of amphibians cured by elevated body

temperature. Dis. Aquat. Organ. 55,65–67.

Woodhams, D.C., Ardipradja, K., Alford, R.A., Reinert, L.K.,

Rollins-Smith, L.A. & Marantelli, G. (2007). Resistance to

chytridiomycosis varies among amphibian species and is

correlated with skin peptide defenses. Anim. Conserv. 10,

409–417.

Wu, N.C. (2023). Pathogen load predicts host functional

disruption: A meta-analysis of an amphibian fungal

panzootic. Funct. Ecol. 37, 900–914.

Supporting information

Additional supporting information may be found online in

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Data S1.

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Systematic assessments of species extinction risk at regular intervals are necessary for informing conservation action1,2. Ongoing developments in taxonomy, threatening processes and research further underscore the need for reassessment3,4. Here we report the findings of the second Global Amphibian Assessment, evaluating 8,011 species for the International Union for Conservation of Nature Red List of Threatened Species. We find that amphibians are the most threatened vertebrate class (40.7% of species are globally threatened). The updated Red List Index shows that the status of amphibians is deteriorating globally, particularly for salamanders and in the Neotropics. Disease and habitat loss drove 91% of status deteriorations between 1980 and 2004. Ongoing and projected climate change effects are now of increasing concern, driving 39% of status deteriorations since 2004, followed by habitat loss (37%). Although signs of species recoveries incentivize immediate conservation action, scaled-up investment is urgently needed to reverse the current trends.

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Article

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Jan 2023
FUNCT ECOL

Nicholas C. Wu

The progression of infectious disease depends on the intensity of and sensitivity to pathogen infection. Understanding commonalities in trait sensitivity to pathogen infection across studies through meta‐analytic approaches could provide insight to the pathogenesis of infectious diseases. The globally devastating amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), offers a good case system due to the widely available dataset on disruption to functional traits across species. Here, I systematically conducted a phylogenetically controlled meta‐analysis to test how infection intensity affects different functional traits (e.g. behaviour, physiology, morphology, reproduction) and the survival in amphibians infected with Bd. There was a consistent effect of Bd infection on energy metabolism, while traits related to body condition, osmoregulation, and behaviour generally decreased with Bd infection. Skin integrity, hormone levels, and osmoregulation were most sensitive to Bd infection (minimum Bd load ln 2.5 zoospore equivalent), while higher minimum Bd loads were required to influence reproduction (ln 10.6 zoospore equivalent). Mortality differed between life stages, where juvenile mortality was dependent on infection intensity and exposure duration, while adult mortality was dependent on infection intensity only. Importantly, there were strong biases for studies on immune response, body condition and survival, while locomotor capacity, energy metabolism and cardiovascular traits were lacking. The influence of pathogen load on functional disruption can help inform pathogen thresholds before the onset of irreversible damage and mortality. Meta‐analytic approaches can provide quantitative assessment across studies to reveal commonalities, differences and biases of panzootic diseases, especially for understanding the ecological relevance of disease impact. Read the free Plain Language Summary for this article on the Journal blog.

Pathogenic fungus causes density‐ and trait‐mediated trophic cascades in an aquatic community

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Full-text available

Apr 2022

Pathogens can alter species composition and ecosystem function by causing direct and indirect effects on communities. Zoospores of the chytrid fungus Batrachochytrium dendrobatidis (hereafter, Bd), a pathogen implicated in worldwide amphibian declines, can be consumed by filter‐feeding zooplankton and can damage mouthparts of infected amphibian larvae. Consequently, we hypothesized that this pathogen would affect the abundance of zooplankton and survival and feeding abilities of larval amphibian hosts. In turn, this could affect the algal food resources of zooplankton and tadpoles, which can include phytoplankton and periphyton. We tested these hypotheses by manipulating the presence of western toad (Anaxyrus boreas) larvae and Bd in outdoor mesocosms and quantifying the densities of amphibians, zooplankton, phytoplankton, and periphyton. Bd infections reduced amphibian larval densities, but this only weakly benefitted periphyton (a density‐mediated indirect effect). After controlling for larval densities, mesocosms with Bd‐exposed larvae had significantly more periphyton biomass than mesocosms with larvae but no Bd, consistent with infection‐induced mouthpart damage reducing larval feeding rates on attached algae (a trait‐mediated indirect effect). In fact, the estimated trait‐mediated effect of Bd on periphyton biomass was larger than the density‐mediated effect. However, we did not find evidence that Bd exposure triggered a switch to filter‐feeding phytoplankton. The presence of Bd was associated with increased copepod abundance, consistent with zooplankton consuming chytrid zoospores. These results suggest that Bd has the potential to cause both trait‐ and density‐mediated indirect effects that can alter community composition and perhaps the primary productivity of freshwater ecosystems.

Synthesis of Batrachochytrium dendrobatidis infection in South America: amphibian species under risk and areas to focus research and disease mitigation

Article

Full-text available

Apr 2022
ECOGRAPHY

Amphibian chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), has been recognized as the infectious disease causing the most catastrophic loss of biodiversity known to science, with South America being the most impacted region. We tested whether Bd prevalence is distributed among host taxonomy, ecoregion, conservation status and habitat preference in South America. Here we provide a synthesis on the extent of Bd infection across South America based on 21 648 molecular diagnostic assays, roles of certain species in the epidemiology of Bd and explore its association with the reported amphibian catastrophic declines in the region. We show that Bd is widespread, with a continental prevalence of 23.2%. Its occurrence in the region shows a phylogenetic signal and the probability of infection is determined by ecoregion, preferred habitat and extinction risk hosts' traits. The taxa exhibiting highest Bd occurrence were mostly aquatic amphibians, including Ranidae, Telmatobiidae, Hylodidae, Calyptocephalellidae and Pipidae. Surprisingly, families exhibiting unusually low Bd prevalence included species in which lethal chytridiomycosis and population declines have been described (genera Atelopus, Rhinoderma and Eleutherodactylus). Higher than expected prevalence of Bd occurred mainly in amphibians living in association with mountain environments in the Andes and Atlantic forests, reflecting highly favourable Bd habitats in these areas. Invasive amphibian species (e.g. Lithobates catesbeianus and Xenopus laevis) exhibited high Bd prevalence; thus we suggest using these as sentinels to understand their potential role as reservoirs, vectors or spreaders of Bd that can be subjected to management. Our results guide on the prioritization of conservation actions to prevent further biodiversity loss due to chytridiomycosis in the world's most amphibian diverse region.

Temperature and duration of exposure drive infection intensity with the amphibian pathogen Batrachochytrium dendrobatidis

Article

Full-text available

Feb 2022

The intensity of a pathogen infection plays a key role in determining how the host responds to infection. Hosts with high infections are more likely to transmit infection to others, and are may be more likely to experience progression from infection to disease symptoms, to being physiologically compromised by disease. Understanding how and why hosts exhibit variation in infection intensity therefore plays a major part in developing and implementing measures aimed at controlling infection spread, its effects, and its chance of persisting and circulating within a population of hosts. To track the relative importance of a number of variables in determining the level of infection intensity, we ran field-surveys at two breeding sites over a 12 month period using marked larvae of the common midwife toad ( Alyes obstetricans ) and their levels of infection with the amphibian pathogen Batrachochytrium dendrobatidis (Bd). At each sampling occasion we measured the density of larvae, the temperature of the water in the 48 h prior to sampling, the period of time the sampled individual had been in the water body, the developmental (Gosner) stage and the intensity of Bd infection of the individual. Overall our data suggest that the temperature and the duration of time spent in the water play a major role in determining the intensity of Bd infection within an individual host. However, although the duration of time spent in the water was clearly associated with infection intensity, the relationship was negative: larvae that had spent less than 3–6 months in the water had significantly higher infection intensities than those that had spent over 12 months, although this infection intensity peaked between 9 and 12 months. This could be due to animals with heavier infections developing more quickly, suffering increased mortality or, more likely, losing their mouthparts (the only part of anuran larvae that can be infected with Bd). Overall, our results identify drivers of infection intensity, and potentially transmissibility and spread, and we attribute these differences to both host and pathogen biology.

Is overwintering mortality driving enigmatic declines? Evaluating the impacts of trematodes and the amphibian chytrid fungus on an anuran from hatching through overwintering

Article

Full-text available

Jan 2022
PLOS ONE

Emerging infectious diseases are increasing globally and are an additional challenge to species dealing with native parasites and pathogens. Therefore, understanding the combined effects of infectious agents on hosts is important for species’ conservation and population management. Amphibians are hosts to many parasites and pathogens, including endemic trematode flatworms (e.g., Echinostoma spp.) and the novel pathogenic amphibian chytrid fungus (Batrachochytrium dendrobatidis [Bd]). Our study examined how exposure to trematodes during larval development influenced the consequences of Bd pathogen exposure through critical life events. We found that prior exposure to trematode parasites negatively impacted metamorphosis but did not influence the effect of Bd infection on terrestrial growth and survival. Bd infection alone, however, resulted in significant mortality during overwintering—an annual occurrence for most temperate amphibians. The results of our study indicated overwintering mortality from Bd could provide an explanation for enigmatic declines and highlights the importance of examining the long-term consequences of novel parasite exposure.

Alternative food sources interfere with removal of a fungal amphibian pathogen by zooplankton

Article

Full-text available

Sep 2021
J APPL ECOL

While the amphibian disease chytridiomycosis is causing ongoing population declines and biodiversity losses around the globe, efficient mitigation strategies are lacking. The free‐living zoospores of the causative agents of this disease, the chytrid pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), are a potential food source for filter‐feeding micropredators as part of the aquatic food web. While consumption of zoospores can lower environmental pathogen loads, alternative food sources may interfere with pathogen removal rates. We compared the ability of three filter‐feeding zooplankton species, the cladoceran Daphnia magna, the rotifer Brachionus calyciflorus and the ostracod Heterocypris incongruens, to remove Bd zoospores in water and investigated the effect of alternative food sources, the green algae Pseudokirchneriella subcapitata and Chlorella vulgaris, on zoospore ingestion by D. magna. Daphnia magna was the only micropredator candidate that effectively removed Bd zoospores from its environment, with an average removal rate of 1,012 ± 542 GE ind.⁻¹ hr⁻¹ within our test system. High concentrations (1 × 10⁵ cells/ml) of large and easily ingestible P. subcapitata reduced pathogen removal rates, whereas the small and less edible C. vulgaris did not interfere with pathogen removal. Synthesis and applications. We showed that Daphnia spp., which are keystone species in all sorts of aquatic habitats worldwide, are promising target agents for biologically mitigating chytridiomycosis infections and how natural food sources may interfere with this strategy. We also suggest potential management actions for biological disease mitigation, aiming to optimize environmental conditions for these target filter feeders, thereby reducing pathogen densities and eventually infection pressure in amphibian hosts. Examples of such management actions include, but are not limited to, removal of planktivorous fish, habitat restoration, nutrient control or agrochemical regulation in the vicinity of amphibian breeding ponds. Further studies, including field trials, are needed to confirm the effects of pathogen consumption on infection dynamics in natural situations and investigate the impact of intervention actions.

Chytridiomycosis causes high amphibian mortality prior to the completion of metamorphosis

Article

Jan 2024
ENVIRON RES

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42-46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay’s barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35-38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35-41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n=5/29) apparently clearing their infection while the majority (n=21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

Do immune system changes at metamorphosis predict vulnerability to chytridiomycosis? An update

Article

Aug 2022
DEV COMP IMMUNOL

Amphibians are among the vertebrate groups suffering great losses of biodiversity due to a variety of causes including diseases, such as chytridiomycosis (caused by the fungal pathogens Batrachochytrium dendrobatidis and B. salamandrivorans). The amphibian metamorphic period has been identified as being particularly vulnerable to chytridiomycosis, with dramatic physiological and immunological reorganisation likely contributing to this vulnerability. Here, we overview the processes behind these changes at metamorphosis and then perform a systematic literature review to capture the breadth of empirical research performed over the last two decades on the metamorphic immune response. We found that few studies focused specifically on the immune response during the peri-metamorphic stages of amphibian development and fewer still on the implications of their findings with respect to chytridiomycosis. We recommend future studies consider components of the immune system that are currently under-represented in the literature on amphibian metamorphosis, particularly pathogen recognition pathways. Although logistically challenging, we suggest varying the timing of exposure to Bd across metamorphosis to examine the relative importance of pathogen evasion, suppression or dysregulation of the immune system. We also suggest elucidating the underlying mechanisms of the increased susceptibility to chytridiomycosis at metamorphosis and the associated implications for population persistence. For species that overlap a distribution where Bd/Bsal are now endemic, we recommend a greater focus on management strategies that consider the important peri-metamorphic period.

Effect of captivity and water salinity on culture-dependent frog skin microbiota and Batrachochytrium dendrobatidis (Bd) infection

Article

Jun 2022

The world is undergoing a sixth mass extinction, and the drastic decline of amphibians in the last several decades is a major contributing factor. The spread of the deadly Batrachochytrium dendrobatidis (Bd) fungus is a major causative agent, and captive breeding programs are under way to try and save endangered populations. However, how captivity affects Bd infection and the skin microbiota is not clear. We identified the skin bacteria of the South Australian frog Crinia signifera and showed that their culture-dependent skin microbiota is moderately stable in cohorts sampled from the same location in subsequent years. Their microbiota was also structurally different to that of a sympatric frog species, Limnodynastes tasmaniensis, indicating a specific microbial signature. Twenty-four C. signifera were placed into captivity for four weeks, exposed to a range of water salinity levels, during which time there was a significant reduction in identifiable skin bacterial species diversity, richness, and a change inmicrobial structure. The infection intensity of Bd was also significantly reduced in captivity, with some frogs becoming completely clear of infection, whilst water salinity level was not a significant determinant of Bd infection or skin microbiota.

Defying decline: Very low chytrid prevalence in tadpoles, yet high infection in adults in a naturally recovering frog species

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