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\begin{document}

\title{Host defense or parasite cue: Skin secretions mediate interactions
between amphibians and their parasites}



\author[1]{Martin Mayer}%
\author[2]{Lia Schlippe Justicia}%
\author[3]{Richard Shine}%
\author[3]{Gregory Brown}%
\affil[1]{Aarhus University}%
\affil[2]{University of La Laguna}%
\affil[3]{Macquarie University}%


\vspace{-1em}



  
  \date{June 17, 2024}


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\selectlanguage{english}
\begin{abstract}
Amphibian skin secretions (substances produced by the amphibian plus
microbiota) plausibly act as a first line of defense against pathogen
attack, but may also provide chemical cues for pathogens. To clarify the
role of skin secretions in host-parasite interactions, we conducted
experiments using cane toads (Rhinella marina) and their lungworms
(Rhabdias pseudosphaerocephala) from the range-core and invasion-front
of the introduced anurans' range in Australia. Depending on the
geographic area, toad skin secretions can reduce the longevity and
infection success of parasite larvae, or attract lungworm larvae and
enhance their infection success. These striking differences between the
two regions were due both to differential responses of the larvae, and
differential effects of the skin secretions. Our data suggest that skin
secretions play an important role in host-parasite interactions in
anurans, and that the arms race between a host and parasite can rapidly
generate spatial variation in critical features of that interaction.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Amphibians have suffered immense rates of population declines and
extinctions over recent decades (Houlahan \emph{et al.} 2000; McCallum
2007; Grant \emph{et al.} 2019) and are considered the most threatened
vertebrate class on the planet (Howard \& Bickford 2014). A significant
contributor to that mortality has been the invasive chytrid fungus
(\emph{Batrachochytrium dendrobatidis} ), which attacks the host's
epidermis (L\selectlanguage{ngerman}ötters \emph{et al.} 2009; Fisher \& Garner 2020). The
catastrophic impact of chytrid has drawn attention to the vulnerability
of anurans to a diverse array of pathogens, such as ranaviruses
(Gray\emph{et al.} 2009) and parasites (Hartson \emph{et al.} 2011;
Gustafson\emph{et al.} 2018). In response to challenges induced by
pathogens, anurans exhibit several lines of defense including immune
responses and behavioral avoidance of pathogens (Hossack \emph{et al.}
2013; Koprivnikar \emph{et al.} 2014; McMahon \emph{et al.} 2014).
Moreover, amphibian skin secretions consist of secretions produced by
the amphibian itself (Clarke 1997) and skin microbiota (mostly bacteria
(Federici \emph{et al.} 2015)), hereafter, we refer to this combination
as `skin secretions'. These secretions contain many antimicrobial
properties (Gustavo Tempone \emph{et al.} 2007; Govender \emph{et
al.}2012), which might help to fight off pathogens (Weitzman \emph{et
al.}2019; Christian \emph{et al.} 2021). Simultaneously, pathogens are
under selection to overcome those barriers, generating an `arms race' of
adaptations and counter-adaptations in the host and its adversaries
(Sorci \& Faivre 2008). Those host-parasite interactions may drive the
rapid evolution of spatial variation in the attributes of both
participants, and selection should favor hosts that can either tolerate
infection (limiting the harm caused by a given parasite infection) or
reduce infection probability and burden, i.e., resistance
(Råberg\emph{et al.} 2009).

Although some studies showed that amphibian skin secretions contain
properties to resist chytrid fungus (Rollins-Smith 2009;
Niederle\emph{et al.} 2019) and bacteria (Quintana \emph{et al.} 2017),
little is known about the role of secretions as parasite defense
mechanisms more generally. Similarly, we know little about how parasitic
nematodes find their hosts. Generally, nematodes can utilize olfaction,
gustation, thermosensation, and humidity to locate hosts, which they
seek via strategies ranging from ambushing to actively crawling toward
host-emitted cues (Castelletto \emph{et al.} 2014; Gang \& Hallem 2016).
If such strategies for infection avoidance (in hosts) and host
detection/recognition (in parasites) exist, they are expected to vary
geographically between different populations, due to co-evolved local
adaptations between host and parasite which occur on small spatial
scales (Schmid-Hempel 2011).

Biological invasions provide unparalleled opportunities to investigate
arms races between hosts and their parasites, because they create
spatial heterogeneity in transmission rates and impose novel selective
forces on one or both participants. Recently, we showed that the
invasion of cane toads (\emph{Rhinella marina} , Fig. 1) through
tropical Australia has generated substantial spatial divergence in
host-parasite interactions (Kelehear \emph{et al.} 2012; Brown \emph{et
al.} 2016; Mayer \emph{et al.} 2021). The toads have carried with them a
native-range lungworm (\emph{Rhabdias pseudosphaerocephala} , Fig. 1)
(Dubey \& Shine 2008; Selechnik \emph{et al.} 2017) that can reduce
viability of the host (Kelehear \emph{et al.} 2011; Finnerty \emph{et
al.} 2018). The risk of parasite infection has been modified by the
invasion process, with low population densities of hosts at the
invasion-front reducing opportunities for parasite transfer among toads
(Phillips \emph{et al.} 2010). Apparently as a result, toads at the
western invasion-front have evolved a greater resistance to parasite
infection (Mayer \emph{et al.} 2021), and nematodes close to the
invasion-front have evolved a higher infectiviy (Kelehear \emph{et
al.}2012), continuing the arms race. That situation provides an ideal
opportunity to investigate the biological role of anuran skin secretions
in host-parasite interactions.

Because both parasites and their hosts adapt to local conditions,
geographic variation in the skin secretions of cane toads might have
both positive and negative consequences for host fitness. Skin
secretions might render it more difficult for a parasite to infect their
host, if secretions inhibit the parasite's ability to locate and/or
enter the host's body or find its way to the target organ where it can
grow and mature (here termed the `\emph{host defense} ' hypothesis).
Alternatively, the parasite might evolve to use the host's skin
secretions as a signal for host location, or as a cloak to hide from the
host's immune system during the parasite's migration through the body of
the host (here termed the `\emph{parasite cue} ' hypothesis). Notably,
these two hypotheses are not entirely exclusive; for example, skin
secretions might facilitate host location by the parasite, but still
defend against penetration by the parasite. By studying a system where
host-parasite interactions have diverged rapidly, we have an opportunity
to detect a range of such outcomes. We conducted experiments to (1)
clarify the role of toad skin secretions as a host-finding cue for
lungworms, and (2) test the function of toad skin secretion as defense
mechanism against parasite infection (Table 1).

\textbf{Material and Methods}

\emph{Host-parasite system}

Cane toads were introduced to Australia in 1935, and carried with them
the lung nematode \emph{Rhabdias pseudosphaerocephala} that now occurs
throughout most of the toad's Australian range (Barton 1998;
Phillips\emph{et al.} 2010). Hermaphroditic adult nematodes live inside
the toad's lungs, where they release eggs that hatch into first-stage
male and female free-living forms within the host's alimentary tract and
are defecated. These larvae mate to produce infective third-stage larvae
(L3), which develop for 4-10 days (L2 stage) before breaking out of
their mother's body and entering the soil (Baker 1979). When an L3
locates a host, it pierces the epidermis and migrates through tissue to
reach the lungs of the toad where it matures and feeds on blood from
capillary beds (Kelehear \emph{et al.} 2012). The entire life cycle
takes 5-36 days (Kelehear \emph{et al.} 2012). Infection prevalence of
lungworms varies seasonally and climatically within Australian cane
toads (Barton 1998; Pizzatto \emph{et al.} 2013), but low host densities
mean that the parasites do not occur at the forefront of the toad
invasion (Phillips \emph{et al.} 2010).

\emph{Collection of toads and lungworms}

We obtained cane toads from two geographic areas: (1) Mareeba,
Queensland (17.03° S, 145.43° E), close to the original introduction
site (hereafter `range-core', toads present \textgreater{}80 years), and
(2) Halls Creek, Western Australia (-18.23° S, 127.66° E; hereafter
`invasion-front', toads present \textless{}5 years; Fig. 1). We kept
toads in Middle Point, Northern Territory (-12.56° S, 131.32° E; Fig.
1), individually in 20 L containers. To obtain naive, parasite-free
toads, two pairs of adult toads from each region were induced to spawn
by subcutaneous injection of the gonadotropin-releasing hormone agonist
leuprorelin acetate (Lucrin, Abbott Australasia) (Hudson \emph{et
al.}2016). Although our sample size of parental toads was low for
logistical reasons, low functional genotypic replication is unlikely to
have affected our findings because cane toads have very low genetic
diversity across Australia, with a consequent high similarity in gene
expression among individuals within populations (Rollins \emph{et al.}
2015; Selechnik \emph{et al.} 2019b).

After tadpoles hatched, we raised them in plastic containers (1165 x
1165 x 780 mm; one container per clutch). Emerging metamorphosed toads
were housed in plastic containers (375 x 295 x 195 mm) separated by
clutch prior to the experimental trials. To obtain parasites, we
collected naturally-infected toads from Innisfail, QLD (range-core, n =
61) and Kununurra, WA (close to the invasion-front; toads present \selectlanguage{english}[?]10
years, n = 20), because parasites are absent from the invasion-front
(Phillips \emph{et al.} 2010). The sites where we collected parasites
were about 200 km from the sites where we collected parental toads, to
yield similar spatial differences between collection sites for parasites
and toads at the range-core and invasion-front. We obtained parasites by
collecting toad feces and by sourcing adult \emph{Rhabdias} from toad
lungs, which we then cultured in Petri dishes in a mix of aged tap water
and toad feces to raise first-stage \emph{Rhabdias} larvae to the L3
stage (Langford \& Janovy Jr 2009; Kelehear \emph{et al.} 2012).

\emph{Experimental infections}

To investigate if skin secretions act as a defense mechanism by the host
or as cue for the parasite, or both, we infected toads that had either
intact or reduced skin secretions (Table S1, S2). We used 79
captive-raised toads, comprising 20 individuals (10 from each clutch)
per treatment group: 20 range-core toads that we infected with
range-core L3, 19 range-core toads infected with invasion-front L3, 20
invasion-front toads infected with invasion-front L3, and 20
invasion-front toads infected with range-core L3. Toads were
approximately two months old when we commenced the experiment, and were
housed individually in plastic boxes (15 x 10 x 7 cm). We assigned
individuals to treatment groups such that mean snout-urostyle length
(SUL) was similar among treatment groups (ANOVA: F \textsubscript{(6,
72)} = 0.30, p = 0.94).

Directly before infection, we reduced skin secretions on half the toads
by bathing the animal in water and then swabbing both the dorsal and
ventral surface 15 times with a cotton bud (Fig. 1). This process was
conducted twice per individual to remove most of the skin secretions
(Walker \emph{et al.} 2015). Control toads were also bathed and
mock-swabbed to simulate the same handling time and potential stress as
the other group. That is, we swabbed the toad with a cotton bud covered
with a non-absorptive plastic film (parafilm `M', Pechiney Plastic
Packaging, Chicago, USA), rolling the bud over the body but removing few
if any skin secretions (Christian \emph{et al.} 2021). For infection, we
placed toads individually into round containers (40 mm diameter) lined
with filter paper (Whatman 1, Whatman International Ltd, Maidstone,
England) containing 20 L3 in 300 \selectlanguage{greek}µ\selectlanguage{english}l water. We left the toads in the
infection chamber for 18 h, after which they were placed back in their
housing enclosure, and then rinsed the infection chamber with water onto
a clean Petri dish to count the number of remaining L3 using a
microscope (Leica M60). On the 15\textsuperscript{th} day
post-infection, we euthanized the toads by placing them in a tricaine
methanesulfonate bath (Crossland \& Shine 2011) and dissected them to
count the number of established nematodes in the lungs. We calculated
the proportion of L3 entering the lung by dividing the number of L3 that
established in the lungs by the number of L3 that entered the host
(Mayer \emph{et al.} 2021).

\emph{Performance of parasite larvae}

To investigate if the survival of L3 was affected by exposure to skin
secretions of cane toads, or of a native frog (\emph{Litoria caerulea}
), we placed \textless{}7 day old L3 in a Petri dish containing the
treatment solution for 24 h. To obtain skin secretions of cane toads (n
= 10 range-core toads and 12 invasion-front toads) and frogs (n = 4), we
swabbed individuals as described above. The head of the cotton bud was
then diluted in 1 ml water to obtain our treatment solution. We used
multiple individuals (but not mixing secretions from different
individuals) to avoid pseudoreplication, and used the treatment solution
of individual toads and frogs for L3 originating both from the
range-core and invasion-front to avoid biases induced by individual
variation of the secretions. Aged tap water was used as control. After
keeping L3 in a treatment solution for 24 h, we individually placed them
in 96 well microplates (Greiner Bio-One, Kremsm\selectlanguage{ngerman}ünster, Austria) with 200
\selectlanguage{greek}µ\selectlanguage{english}l water. Twice a week we refilled the water level in the microplates
and checked whether each L3 was alive by touching it with a human
eyelash that was sterilized in 70\% ethanol and then washed in
sterilized water to minimize the risk of introducing pathogens. If the
L3 did not move after 5 attempts, we considered it dead (verified within
3 days as the body began to decompose), and calculated longevity as the
number of days from hatching to death. We used a crossed design, testing
L3 from different geographic regions with toad skin secretion from
different geographic regions as well as frog skin secretion (n = 170 L3;
Table S3). L3 for this experiment were sourced from multiple toads (n =
15 range-core toads and 8 invasion-front toads) to avoid
pseudoreplication.

To investigate if L3 use cane toad skin secretions as a cue, we obtained
skin secretion solutions as described above. The experimental setup
consisted of a 15 mm wide and 50 mm long strip of filter paper, in a
Petri dish. One end of the strip was used as the `control' zone and the
other end as the `treatment' zone. We pipetted 20 \selectlanguage{greek}µ\selectlanguage{english}l of skin secretion
onto the treatment zone and 20 \selectlanguage{greek}µ\selectlanguage{english}l water onto the control zone. For
procedural controls (i.e., to check that L3 randomly move into both
zones), we applied water to both zones. The location of control and
treatment zones was assigned randomly. We then pipetted 10 L3 (also
contained in 20 \selectlanguage{greek}µ\selectlanguage{english}l water) onto the middle of the strip of filter paper.
After 2 h, we cut the filter paper in half, washed out the L3 from the
paper (same method as in experiment 1), and counted the number of L3 in
the control and the treatment zones. We also washed out the Petri dish
to count the remaining L3 to estimate the `recovery rate' (i.e., how
many L3 we were able to find). For each scent trial (procedural control,
range-core toad skin secretions, invasion-front toad skin secretions)
and L3 origin (range-core, invasion-front), we ran 30 replicates,
totaling 180 trials using 1,800 L3 (Table S4).

\emph{Statistical analysis}

To investigate the number of L3 entering the host and the total number
of nematodes established in the lungs (dependent variables in separate
analyses), we used generalized linear mixed models (GLMM) with a
negative binomial response distribution using the R package lme4
(Bates\emph{et al.} 2015) to correct for a non-normal distribution of
the count data (O'hara \& Kotze 2010). To analyze the proportion of L3
entering the lungs (number of parasites in the lungs in relation to the
number that entered the host, dependent variable), we used a GLMM with a
binomial response distribution using the R package lme4 (Bates \emph{et
al.} 2015). For all three analyses we initially ran a full model,
including the initial SUL of the toads, toad origin, L3 origin, and
treatment as fixed effects and clutch ID as random intercept (Table 1,
S1). Moreover, we divided the analyses into two data sets, one for
range-core toads and one for invasion-front toads to avoid higher order
interactions (i.e. toad origin x L3 origin x treatment). For these six
analyses (Table S1), we included the initial SUL of the toads, L3
origin, treatment, and the interaction of L3 origin x treatment (to test
if the reduction of skin secretion had different effects on the
infectiveness of L3 from different origin) as fixed effects and clutch
ID as random intercept. Finally, we analyzed the size of the nematodes
that we had obtained from the toad lungs (dependent variable), using a
LMM with a Gaussian link. We included toad SUL, L3 origin, toad origin,
and the interaction of treatment x L3 origin as fixed effects and toad
ID nested within clutch as random intercept (Table 1, S1).

To investigate differences in L3 survival depending on L3 origin, we
used a Kaplan Maier survival analysis in the R package `survival'
(Therneau \& Lumley 2014). We then analyzed L3 longevity (dependent
variable) using a GLM with a log link, including treatment (separated
between range-core and invasion-front toads), L3 origin and their
interaction (Table 1). To test if L3 use skin secretions to locate their
host, we analyzed the proportion of L3 in the treatment zone (dependent
variable) using a GLM with a binomial distribution and a logit link. We
included the scent type, L3 origin and their interactions as independent
variables (Table 1, S3).

Model selection for all analyses was based on stepwise variable
selection using Akaike's Information Criterion corrected for small
sample size (AIC\textsubscript{c}), selecting the model with the lowest
AIC\textsubscript{c} (Murtaugh 2009), using the R package `MuMIn'(Barton
2016). Parameters that included zero within their 95\% CI were
considered uninformative (Arnold 2010). We validated the most
parsimonious models by plotting the model residuals versus the fitted
values (Zuur \emph{et al.} 2010). All statistical analyses were carried
out in R 4.0.3 (R Core Team 2013).

\textbf{Results}

\emph{Do skin secretions affect the entry and establishment of parasite
larvae into the host?}

On average, 17.4 L3 (\selectlanguage{ngerman}± 2.3 SD) entered the toad. Toad SUL, toad origin,
L3 origin, and treatment were uninformative in explaining the number of
L3 entering the toad, which was best explained by the intercept-only
model (Table S1). Infection prevalence was 92.4\% (73 of 79 toads had
nematodes in their lungs). The proportion of L3 establishing in the
lungs was best explained by toad SUL, toad origin and L3 origin (Table
S1), being higher in toads from the range-core \emph{versus} the
invasion-front, higher for toads infected with L3 from
invasion-front\emph{versus} range-core populations, and higher in larger
toads (Table S2, Fig. S1). When we analyzed the data separately for
range-core and invasion-front toads, we found that the number of L3
establishing in the lungs was increased by intact skin secretions for
range-core L3 but reduced by intact skin secretions for invasion-front
L3 (Table S1, S2, Fig. 2). This effect was stronger in toads from the
invasion-front compared to the range-core (Fig. 2).

Fifteen days post-infection, toads had between 0 and 19 nematodes in
their lungs (mean ± SD: 7.5 ± 4.3), and generally there were more
nematodes in the lungs of larger toads, in range-core toads, and when
infected with invasion-front L3 (Table S1, S2). Range-core toads had
more nematodes in their lungs when the L3 originated from the
invasion-front, but the interaction of treatment and L3 origin was
uninformative (Fig. 2, Table S2). In invasion-front toads, reduced skin
secretions led to a lower number of nematodes in the lungs when the L3
originated from the range-core, but this effect was absent when infected
with invasion-front L3 (Fig. 2).

\emph{Do skin secretions affect the size of parasites in the host lung?}

The size of the adult lungworms that had established in the toads' lungs
ranged between 1.6 to 5.2 mm (mean ± SD: 3.5 ± 0.5 mm). Invasion-front
lungworms were larger than range-core lungworms, and lungworm size
decreased with an increasing number of nematodes in the lungs (Table S2,
Fig. S2). Toad SUL, treatment (skin secretions intact versus reduced),
and toad origin were uninformative in explaining nematode size.

\emph{Effect of skin secretions on the longevity of parasite larvae}

L3 lived between 7 and 59 (mean ± SD: 34 ± 10) days. L3 longevity was
best explained by parasite origin and treatment (Table S4). L3 from
invasion-front populations lived longer than did those from the
range-core (mean ± SD: 40 ± 9 versus 29 ± 7 days; log-rank test: p
\textless{} 0.001). Skin secretions from invasion-front toads reduced
longevity of both groups of L3 (Fig. 3). In contrast, skin secretions
from range-core toads and from frogs did not reduce larval longevity
(Fig. 3, Table S5).

\emph{Are larvae attracted to skin secretions?}

On average, we recovered 90.6\% (± 14.2 SD) of the L3 used for this
experiment (1,630 of 1,800 L3). The proportion of L3 in the treatment
zone was best explained by L3 origin, scent type, and their interaction
(Table S4). More L3 (from both populations) were found in the treatment
zone than the control area (demonstrating attraction to the scent cue)
when skin secretions from range-core toads were used (Fig. 4, Table S5).
Conversely, when skin secretions from invasion-front toads were used as
the treatment scent, L3 from range-core populations were attracted
whereas L3 from invasion-front populations were not (Fig. 4, Table S5).

\textbf{Discussion}

Our data confirm that anuran skin secretions play important and complex
roles in host-parasite interactions. In line with the \emph{host
defense} hypothesis, skin secretions can decrease a toad's vulnerability
to nematodes, by reducing larval longevity and by reducing the ability
of larvae to penetrate to the host's lungs. However, skin secretions
also provide chemical cues that can attract infective larvae
(\emph{parasite cue} hypothesis) as suggested by other studies
(Theodoropoulos \emph{et al.} 2001). Moreover, skin secretions can
enhance rather than decrease the ability of parasite larvae to penetrate
the toad's body and establish an infection in the lungs. Thus, some
effects of skin secretions appear to represent adaptations of the host
that reduce vulnerability to parasite attack (Tempone \emph{et al.}2008;
Christian \emph{et al.} 2021), whereas other effects are more consistent
with adaptations of parasites to exploit the traits of their host (Gang
\& Hallem 2016). The most striking result from our studies is the
magnitude of geographic divergence in host-parasite interactions within
our study system. Although cane toads have been spreading through
tropical Australia for only 85 years (Urban \emph{et al.} 2008), they
appear to have evolved major divergences in the roles that skin
secretions play in host-parasite biology.

Broadly, the skin secretions of cane toads appear to act as a defense
against lungworms in invasion-front populations in Western Australia
(Christian \emph{et al.} 2021), but not in range-core populations in
Queensland. Thus, the presence of skin secretions reduced the rate at
which invasion-front L3 were able to establish infections in
invasion-front hosts, whereas the reverse was true for range-core L3
attacking range-core hosts. In regard to the host, the shift was from
comparatively low protection (or the absence thereof) against parasite
infection by skin secretions at the range-core, to increased protection
at the invasion-front. That geographic divergence fits well with the
hypothesis that cane toads in the range-core have low resistance to
parasite infection (Mayer \emph{et al.} 2021), because parasites are
ubiquitous due to high host densities, and toads thus rely on tolerating
rather than resisting pathogens (Adelman \& Hawley 2017). Conversely, at
the invasion-front, where there is a strong evolutionary pressure for
dispersal ability via both natural selection and spatial sorting
(Brown\emph{et al.} 2014; Phillips \& Perkins 2019), increased host
resistance might be favored if parasite infection reduces dispersal
ability. Moreover, some sets of immune genes are upregulated at the
invasion-front (Selechnik \emph{et al.} 2019a), suggesting that
heightened, possibly non-specific, immune responses arising from
exposure to other pathogens or conditions at the invasion-front may
increase resistance of toads (Brown \emph{et al.} 2018; Mayer \emph{et
al.} 2021). Although mechanisms leading to shifts in host immune
responses are unclear, our study demonstrates that changes in defense
strategies against parasite infection can evolve rapidly.

In regard to the parasite, one surprising result was that in range-core
toads, skin secretions enhanced rather than reduced the rate at which
larvae were able to reach the lungs and establish an infection.
Theodoropoulos \emph{et al.} (2001) suggested that gastro-intestinal
helminth parasites express mucin-like molecules to avoid detection by
the host's immune system (mucin is the main molecule of the intestinal
mucus barrier (Carlisle \emph{et al.} 1991; Sharpe \emph{et al.} 2018)).
If so, larvae entering the host's body may benefit by cloaking
themselves in host-derived skin secretions that enable them to evade
detection by the host's immune system. That tactic apparently does not
work at the invasion-front, perhaps because the immune system of
invasion-front toads is more active against pathogens (Brown \emph{et
al.} 2018) as indicated by the effects of host skin secretion on larval
longevity. Additionally, the larger larvae at invasion-front sites
potentially cannot be cloaked as effectively, leading to increased
susceptibility to the host's immune system. An alternative scenario for
the reduced infection success of range-core L3 when skin secretions were
reduced might be a reduced ability to detect the host. Range-core L3
used skin secretions as a cue and olfaction is important for
host-finding in other species (Gang \& Hallem 2016). However, we found
no significant difference in the number of L3 entering the host as a
function of the presence of skin secretions. Invasion-front L3 also used
skin secretions as a cue -- but only from range-core toads, not
invasion-front toads. This latter finding might have been caused by the
negative effect of skin secretions from invasion-front toads on
longevity of L3. Thus, invasion-front L3 might still be able to use skin
secretions of invasion-front toads as a cue, but avoid exposure to evade
detrimental effects. In contrast, the non-coevolved range-core L3 might
not detect the detrimental effects of the secretions during the short
exposure time of the experiment.

Much remains to be learned about the mechanisms underlying the
interactions between amphibian hosts and the organisms that infect them.
For example, we do not know which components of the skin secretion
function as host defense or parasite cue, respectively. Cane toads from
different regions have different proportions of bacteria with antifungal
properties (Weitzman et al. 2019), suggesting that microbial properties
might also contribute to defense against parasites. It would be of great
interest to tease apart the degree to which the functional attributes of
skin secretions (e.g., in reducing or enhancing parasite success) derive
from molecules produced by the amphibian \emph{versus} the microbiota
that live on its skin. Moreover, the impacts of skin secretions ideally
need to be seen in a wider context, as part of a suite of responses that
also encompass behavior, morphology and physiology (especially, immune
function: Brown \emph{et al.} (2018)). Thus, for example, a highly
effective immune response to pathogens that enter the body might relax
selection for barriers on the skin. Additionally, it is important to
remember that the skin has many other functions, such as respiration,
regulating water flow and as the location of toxin-excreting glands
(Huang \emph{et al.} 2016; Senzano \& Andrade 2018;
Blennerhassett\emph{et al.} 2019; Kosmala \emph{et al.} 2020).

The devastating impacts of diseases (especially chytrid-driven) on
anuran amphibians worldwide places a high priority on the need to
understand factors that render an anuran more or less vulnerable to
infection (Rollins-Smith 2009). Strong phylogenetic and geographic
variation in the magnitude of disease impacts on amphibians (e.g. Savage
\& Zamudio 2016; Fisher \& Garner 2020) suggest that spatially variable
outcomes of host-parasite arms races may be key to understanding -- and
hopefully, ameliorating -- some of those impacts. Our data indicate that
at least part of the diversity in host-parasite interactions can involve
diversity in the role of skin secretions, and that location-specific
selective forces can generate rapid changes in the ways that anuran
hosts interact with their pathogens.

\textbf{Acknowledgements}

We thank Adam Smart for assistance with collecting toads and the
Northern Territory Land Corporation for facilities.

\textbf{Funding}

This study was funded by the Australian Research Council (20190336774,
to R. Shine and L. A. Rollins).

\textbf{Ethics statement}

The study was conducted under the approval of the University of Sydney
Animal Care and Ethics Committee (permit number 2019/1489).

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\textbf{Figures}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Fig. 1: Map (a) showing the collection locations for cane toads
(\emph{Rhinella marina} ) from the range-core (Mareeba, QLD) and
invasion-front (Halls Creek, WA) that were then bred and their offspring
raised in a common garden experiment in Middle Point, NT. Grey shading
depicts the approximate cane toad distribution in 2019. Pictures show a
cane toad (b) that is being swabbed to remove skin secretions, and the
lungworm parasite \emph{Rhabdias pseudosphaerocephala} (c) located
within the toads' lung (red arrow indicates location).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Fig. 2: The effect of the interaction of parasite origin
(range-core\emph{versus} invasion-front) and treatment (skin secretions
intact\emph{versus} reduced) on the proportion of lungworm larvae (L3)
that established in the lungs of cane toads from the range-core (a) and
the invasion-front (b). The lower panels show the effect of the
interaction of parasite origin and treatment on the number of parasites
in the lungs of toads from the range-core (c; note that the interaction
was uninformative) and the invasion-front (d). The 95\% confidence
intervals are given as bars.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Fig. 3: The predicted longevity (black dots) for lungworm larvae (L3)
originating from the range-core (a) and the invasion-front (b)
separately for the different skin secretion treatments. Blue dots
represent raw data for L3 exposed to skin secretions of range-core
toads, orange dots for L3 exposed to secretions of invasion-front toads,
and grey dots for exposure to frog skin secretions. The 95\% confidence
intervals are given as bars and the dashed black line represents the
longevity of the control group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

Fig. 4: The effect of treatment scent (water and cane toad skin
secretions from the invasion-front and range-core, respectively) on the
proportion of lungworm larvae (L3) in the scent treatment area,
separately for L3 from the range-core and the invasion-front. The 95\%
confidence intervals are given as bars and raw data are shown as grey
dots.

\textbf{Tables}

Table 1: Overview of the statistical analyses showing the dependent and
independent variables, the random intercept, model link, and sample
size.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Analysis/Dependent variable & Fixed effects & Random intercept & Link &
n\tabularnewline
\midrule
\endhead
\emph{Infection trial} & & & &\tabularnewline
Number of L3 entering the toad & Snout-urostyle length + L3 origin +
Toad origin + Treatment + L3 origin x Treatment & Clutch & Log -
negative binomial & 79 toads\tabularnewline
Proportion of L3 establishing in the lung & Snout-urostyle length + L3
origin + Toad origin + Treatment + L3 origin x Treatment & Clutch &
Binomial & 79 toads\tabularnewline
Number of nematodes in lungs & Snout-urostyle length + L3 origin +
Treatment + L3 origin x Treatment & Clutch & Log - negative binomial &
79 toads\tabularnewline
Nematode size & Number of nematodes + Snout-urostyle length + Toad
origin + L3 origin + Treatment + Toad origin x Treatment + L3 origin x
Treatment & Toad ID:Clutch & Gaussian & 561 nematodes\tabularnewline
\emph{Nematode performance} & & & &\tabularnewline
L3 longevity & L3 origin + Treatment + L3 origin x Treatment & & Log -
normal & 170 L3\tabularnewline
Scent experiment/ Proportion of L3 in scent treatment area & L3 origin +
Scent + L3 origin x Scent & & Binomial & 180 trials \selectlanguage{ngerman}á 10
L3\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Graphical-abstract/Graphical-abstract}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Cover-image/Cover-image}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

