RESEARCH ARTICLE

Epidermal epidemic: unravelling the pathogenesis ofchytridiomycosisNicholas C. Wu1, Rebecca L. Cramp1, Michel E. B. Ohmer2 and Craig E. Franklin1,*

ABSTRACTChytridiomycosis, a lethal fungal skin disease of amphibians, fatallydisrupts ionic and osmotic homeostasis. Infected amphibiansincrease their skin shedding rate (sloughing) to slow pathogengrowth, but the sloughing process also increases skin permeability.Healthy amphibians increase active ion uptake during sloughing byincreasing ion transporter abundance to offset the increased skinpermeability. How chytridiomycosis affects the skin function duringand between sloughing events remains unknown. Here, we show thatnon-sloughing frogs with chytridiomycosis have impaired cutaneoussodium uptake, in part because they have fewer sodium transportersin their skin. Interestingly, sloughing was associated with a transientincrease in sodium transporter activity and abundance, suggestingthat the newly exposed skin layer is initially fully functional until therecolonization of the skin by the fungus again impedes cutaneousfunction. However, the temporary restoration of skin function duringsloughing does not restore ionic homeostasis, and the underlying lossof ion uptake capacity is ultimately detrimental for amphibians withchytridiomycosis.

KEYWORDS: Anuran, Amphibian, Batrachochytrium dendrobatidis,Electrophysiology, Epithelial disruption, Pathogen, Skin sloughing

INTRODUCTIONAmphibians are the most threatened class of vertebrates, withapproximately 30% globally classified as threatened with extinction(IUCN, 2017). Although anthropogenic disturbances are the maincause of amphibian declines (Stuart et al., 2004), roughly 27% ofthese declines have occurred in pristine habitats such as protectednational parks (Pimm et al., 2014). Many of these non-anthropogenic declines have been attributed to a cutaneouspathogen, Batrachochytrium dendrobatidis (Bd), which infects thekeratinised layers of amphibian skin and can cause the diseasechytridiomycosis (Berger et al., 1998; Pessier et al., 1999).Amphibian skin is unique amongst tetrapods given its highpermeability, which allows it to serve a variety of physiologicalroles such as cutaneous gas exchange and osmotic and ionicregulation, but this necessitates a moist epidermis and so the activesecretion of aqueous substances from the dermal glands rendersamphibians more susceptible to relatively high rates of evaporativewater loss (Boutilier et al., 1992; Larsen et al., 2014). Given theimportance of physiological regulation via the skin, a disruption in

cutaneous function tends to have serious consequences foramphibian health (Pessier, 2002).

Amphibians with chytridiomycosis display altered behaviour(lethargy, lack of appetite) and suffer physical damage to theskin (cutaneous erythema, hyperkeratosis), leading to the loss ofphysiological homeostasis (low electrolyte levels) (Berger et al., 2005;Voyles et al., 2012; Peterson et al., 2013). In healthy frogs, electrolytesare exchanged through paracellular spaces or transcellularly via iontransport proteins (channels, co-transporters, exchangers, ATPase) incutaneous epithelial cells (Hillyard et al., 2008). In frogs withchytridiomycosis, low levels of circulating electrolytes (hyponatremiaand hypochloremia) correlate with a loss of cutaneous ion uptakecapacity (Voyles et al., 2009). Bd produces a complex mixture ofproteins (proteases, biofilm-associated proteins and a carotenoid esterlipase) that can disrupt epidermal intercellular junctions (Brutyn et al.,2012) and suppress genes related to the production of keratin andcollagen (Rosenblum et al., 2012). In addition to its effects on skinintegrity, Campbell et al. (2012) suggested that cutaneousBd infectionsmay directly inhibit the epithelial sodium channels (ENaC), which areprimarily responsible for the cutaneous re-uptake of Na+ fromcutaneous secretions and/or from the environment (Schild, 2010;Larsen and Ramløv, 2013). This hypothesis was based on theirobservation that the amiloride-sensitive short-circuit current of the skinof infected frogs was lower than that of healthy frogs (Voyles et al.,2009). Dysfunction of ENaC transport is often associated withdisorders of Na+ and fluid homeostasis, and blood pressure (Schild,2004). HowBd influences cutaneous ion uptake pathways, specificallythose relating to Na+ movement, including ENaC and Na+/K+-ATPase(NKA), which is responsible for generating electrochemical gradientsin the epidermis (Lingrel and Kuntzweiler, 1994), remains unknown.

Given the vital role of the skin in sustaining amphibianhomeostasis, maintaining skin integrity and function is ofconsiderable importance. To this end, the outer keratinised layer isperiodically removed and replaced by ‘sloughing’. Sloughing alsoplays an important role in regulating cutaneous microbe abundances(Meyer et al., 2012; Cramp et al., 2014), and in frogs infected withBd, sloughing helps remove Bd from the keratinised layer (Ohmeret al., 2017). Indeed, Ohmer et al. (2017) found that sloughingreduced Bd load in five anuran species, with less susceptible speciesclearing infection. However, susceptible species continued todevelop chytridiomycosis despite an increase in sloughingfrequency, and in spite of a temporary reduction in Bd load aftersloughing (Ohmer et al., 2017). An increase in sloughing frequencymight act as an immune response to remove the pathogen before theonset of disease (Ohmer et al., 2015). Importantly though, increasedsloughing frequency may be a double-edged sword, as sloughingitself causes transient osmoregulatory disruption in amphibians(Jørgensen, 1949; Wu et al., 2017). In healthy amphibians (Rhinellamarina), sloughing is accompanied by an increase in cutaneouspermeability (Wu et al., 2017). However, an increase in theexpression and activity of epithelial Na+ channels offsets theReceived 5 September 2018; Accepted 5 December 2018

1School of Biological Sciences, The University of Queensland, Brisbane,Queensland 4072, Australia. 2Department of Biological Sciences, University ofPittsburgh, Pittsburgh, PA 15260, USA.

*Author for correspondence (c.franklin@uq.edu.au)

N.C.W., 0000-0002-7130-1279; R.L.C., 0000-0001-9798-2271; M.E.B.O., 0000-0002-5937-6585; C.E.F., 0000-0003-1315-3797

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temporary increase in skin permeability such that animals suffer noloss of physiological homeostasis (Wu et al., 2017). Conversely, ingreen tree frogs (Litoria caerulea) with high Bd loads, cutaneousion loss is substantially elevated during non-sloughing periods andincreases further during sloughing (Wu et al., 2018). Given thatanimals slough more frequently, the cumulative impact of sloughingand increased ion loss during non-sloughing periods leads to theloss of physiological homeostasis. Although it is clear that Bdaffects cutaneous ion transport processes in infected frogs, themechanistic basis for the disruption of ion transport, and the effectsof sloughing on this, remains unclear.Understanding how the disruption of skin function leads to the

loss of physiological homeostasis in frogs with chytridiomycosis,and whether sloughing worsens cutaneous regulation, is importantfrom a management standpoint. A greater understanding of themechanistic basis for loss of homeostasis in infected frogs couldlead to better treatment options, particularly for criticallyendangered species and captive insurance populations. Thus, theaim of this study was to investigate the effects of Bd on cutaneousion transport processes, focusing on the abundance, activity, andexpression of ENaC and NKA in a susceptible species, theAustralian green tree frog. We hypothesised that both increasedskin permeability and the reduction or inhibition of regulatory iontransporters resulting from Bd infection would contribute to thedisruption of cutaneous ion flow, and sloughing would furtherexacerbate electrolyte permeability in infected frogs. The resultingdisruption of both chytridiomycosis and sloughing could prolongelectrolyte imbalance, causing conditions of low ion concentrationsin the blood plasma such as hyponatremia.

MATERIALS AND METHODSAnimal collection and maintenanceLitoria caerulea (White 1790) spawn was collected from BribieIsland, southeast Queensland, Australia, in March 2015, andraised in the laboratory at The University of Queensland untilmetamorphosis. The resulting 20 juveniles (10–20 g) were used forexperimentation. An additional 17 L. caerulea (15–70 g) adults andsubadults were collected from wet roads in non-protected areas nearFernvale, southeast Queensland, Australia, in January 2015. Frogswere housed individually in either small (235×170×120 mm) orlarge (265×235×12 mm) ventilated clear plastic containers, withpaper towels saturated with chemically aged water (dilution 1:4000;VitaPet, NSW, Australia) as substrate, and a half PVC pipe forshelter. The lighting conditions were set at a 12 h:12 h light:darkphotoperiod cycle, and temperature was set at a constant 20.5±0.5°C. Frogs were checked daily and fed once a week on vitamin-dusted crickets (Acheta domesticus), and enclosures were cleanedweekly. Prior to experiments, all frogs were swabbed to confirm theabsence of Bd infection given its widespread distribution in naturalL. caerulea populations (Berger et al., 1998). A quantitativepolymerase chain reaction (qPCR; details below) assay was used tomeasure Bd loads following protocols by Ohmer et al. (2015)before experiments began. All animals were collected under theQueensland Department of Environment and Heritage ProtectionScientific Purposes Permit (WISP15102214), and procedures in thisstudy were conducted with approval of The University ofQueensland’s Animal Ethics Welfare Unit (SBS/316/14/URG).

Monitoring sloughing frequencyThe intermoult interval (IMI; time between two successfulsloughing events) was monitored continuously using infraredsurveillance cameras (Eonboom Electronics Limited, and K

Guard Security, New Taipei City, Taiwan), and a generic 16-channel H.264 Digital Video Recorder (DVR) system, mounted to amoveable metal frame in front of enclosures, with two cameras perrow. Each camera monitored two frog enclosures at a time, at asample rate of 1.56 frames s−1.

Bd culture and experimental exposureBd strain EPS4 from Ohmer et al. (2015) was used for experimentalinfections. Cultures were maintained at 4°C until 4–5 days beforeexposure. Isolate EPS4 was passaged to sterile 1% agar, 0.5%tryptone, 0.5% tryptone-soy plates and maintained at 20°C. After4–5 days, zoospores were harvested by flooding plates with aged tapwater for 30 min. The zoospore suspension was collected, and theconcentration was measured using a haemocytometer (Boyle et al.,2004). A randomised subset of frogs (n=21) was exposed to∼500,000 zoospores. Frogs were exposed to Bd for 5 h in 300 mlplastic containers containing zoospores suspended in 100 ml agedwater. Control animals (n=16) were exposed to aged water only. At14 days post-exposure, each frog was swabbed with a sterile fine-tipped cotton swab (MW100-100; Medical Wire & Equipment,Wiltshire, UK) three times over the frog’s ventral surface, thighs,armpit, forelimb feet and hindlimb feet (Kriger et al., 2006; Ohmeret al., 2015) to assess infection status. To determine infection load aszoospore equivalents (ZE), swabs were extracted in 50 µl PrepManUltra (Applied Biosystems) and analysed in duplicate with qPCRin a thermal cycler (MiniOpticon™ Real-Time PCR DetectionSystem, Bio-Rad Laboratories) with a modified 15 µl Taqmanreaction following Ohmer et al. (2015). After 1 month, exposedfrogs with no Bd-positive swabs were re-exposed with ∼500,000zoospores following the same procedure detailed above. Theinfection load or number of zoospore equivalent (ZE) wascalculated from the mean value of each triplicate assay and log+1transformed [log(ZE+1)].

Measuring infection load and samplingAll frogs were checked daily for clinical signs of disease progressionincluding a lack of appetite (monitoring food intake), abnormalposture, excessive or irregular sloughing, lethargy, cutaneouserythema and discolouration, and loss of righting reflex (Voyleset al., 2009). Once the IMI was determined for each frog (bothuninfected and infected animals), frogs from the uninfected (n=8intermoult, n=8 sloughing) and infected (n=11 intermoult, n=7sloughing) groups were anaesthetised with an intracoelomicinjection of 60 mg kg−1 body mass thiopentone sodium (ThiobarbPowder, Jurox Pty Limited, NSW, Australia) and then euthanisedvia double pithing. Animals were euthanised at one of two timepoints: (1) sloughing (no more than 1 h after the onset of sloughingbehaviours) or (2) intermoult (at a point halfway through the IMI).Sloughing behaviour takes approximately 5–10 min to complete.Snout–vent length (SVL; mm) and body mass (g) were measuredimmediately. A final skin swab was also taken to determine Bdinfection load.

Blood plasma electrolytesWhole blood was collected from euthanised animals via cardiacpuncture into a lithium heparinised syringe. Samples were thencentrifuged at 5000 g for 5 min and the plasma collected and storedat −20°C for subsequent electrolyte analysis. Plasma Na+ and K+

levels (mmol l−1) were measured using flame photometry (BWB,Berkshire, UK), and plasma Cl− levels (mmol l−1) were determinedspectrophotometrically (DTX 880 Multimode Detector; BeckmanCoulter) with a chloride assay kit (MAK023; Sigma-Aldrich).

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Electrophysiology of the ventral skinFollowing collection of blood samples, isolated ventral skinsamples (<1 cm2) were collected from the lower abdominal pelvicpatch region and mounted in a self-contained, single-channelUssing chamber (model U-9926;Warner Instruments, Hamden, CT,USA) with a single-channel epithelial voltage clamp amplifier(model EC-800;Warner Instruments), and connected to a PowerLab4/35 interface (Mod n: PL3504, ADInstruments). Apical andbasolateral surfaces of the skin were perfused with an oxygenated(95% O2 and 5% CO2) modified frog Ringer’s solution based onVoyles et al. (2009) [in mmol l−1: NaCl (112), KCl (2.5), D-glucose(10), Na2HPO4 (2), CaCl2 (1), MgCl2 (1), Hepes sodium salt (5),Hepes (5) at pH 7.3–7.4 with osmolality of 230±20 mosmol l−1]maintained at 20.5±0.5°C. The Ringer’s solution on the basolateralside represents the internal fluid composition, and on the apical siderepresents the cutaneous surface fluid composition (Larsen andRamløv, 2013). Electrophysiological parameters were measured asfollows: (1) transepithelial potential (TEP or VT; mV) under open-circuit conditions (clamp current to 0 μA) and the resulting voltagein reference to the basolateral side, (2) active ion transport viaclamping voltage to 0 mV and the instantaneous short-circuitcurrent (ISC) response as μA cm−2, and (3) transepithelial arearesistance (Ω cm2) by applying 3 s of 1 µA pulses across theepithelium every 60 s, or under voltage clamp conditions byapplying 3 s of 1 mV at 60 s intervals (Wu et al., 2017). Theinflections from the resulting change in ISC and VT during pulsingwere used to calculate resistance using Ohm’s law. Sodium flux(mol cm−2 s−1) was calculated by dividing the ISC (µA) by Faraday’sconstant (96,000 C mol−1). ISC and VT readings were recorded inLabChart (ADInstruments).

Pharmacological inhibitor experimentsAmiloride hydrochloride hydrate (Sigma-Aldrich) was applied onthe apical side of the chamber to calculate the amiloride-sensitiveshort circuit current (ISC). Although amiloride is a non-specific Na+

channel inhibitor, ENaC is normally inhibited by very low(<1 µmol) concentrations of amiloride compared with othertransporters such as Na+/H+ exchanger (>100 µmol), Na+/Ca2+

exchanger (1000 µmol) and NKA (>3000 µmol) (Kleyman andCragoe, 1988). Amiloride was dissolved in dimethyl sulfoxide(DMSO) and added to the chamber to achieve final concentrationsof 0.1, 0.5, 1, 2, 5, 10, 20 and 50 µmol l−1. ISC readings at eachconcentration were obtained 5 min post-application. The half-maximal inhibitory concentration (IC50) of amiloride was calculatedfor each individual. Preliminary trials with DMSO only (vehiclecontrol) did not significantly affect the overall voltage and currentreadings. After amiloride treatment, skin preparations were washedout with Ringer’s solution until readings returned to starting values(or close to it), then 100 µmol l−1 ouabain octahydrate (Sigma-Aldrich) dissolved in distilled H2O was applied to the basolateralskin side chamber, and measurements were recorded until 15 minpost-application of ouabain.

Skin histologyVentral skin samples (<1 cm2) from the lower abdominal pelvicpatch region were collected and placed into aqueous buffered zincformalin fixative (Z-fix; Anatech, MI, USA) for 24 h, thentransferred to 70% ethanol, and stored at 4°C. Tissues sampleswere then dehydrated through an ascending ethanol series, clearedin xylene and embedded in paraffin wax (Histoplast Paraffin;ThermoFisher Scientific). Tissue samples were then transverselysectioned into approximately 6 µm thick sections, (Leica RM2245;

Leica Microsystems). A sub-sample of sections was stained withMayer’s hematoxylin and 1% eosin in 70% ethanol, andphotographed with an iPhone 6s (Apple Inc.) and NIS-Elementssoftware (v. 4.10, Nikon Instruments Inc.) under a bright-fieldillumination microscope (Nikon Eclipse E200 MV, NikonInstruments Inc.).

ENaC and NKA abundanceVentral skin samples (<1 cm2) from the lower abdominal pelvicpatch region were collected and stored at −80°C. Frozen tissueswere then homogenised in RIPA buffer [150 nmol l−1 NaCl, 1%Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and50 nmol l−1 Tris-HCl with general protease inhibitors (Sigma-Aldrich General protease inhibitor cocktail P2714 with 1 mmol l−1

PMSF)] using an IKA Ultra-Turrax. The protein content wasquantified via a Qubit fluorometer (ThermoFisher Scientific).Protein (30 µg for NKA-α, 50 µg for ENaC-α) was dissolved inNuPAGE LDS sample buffer (Invitrogen) and denatured at 70°C for10 min, then loaded in duplicate into Bolt 4-12% Bis-Tris Plus gels(ThermoFisher Scientific) and electrophoresed at 120 V for 40 min.Each membrane included a pre-stained protein ladder(ThermoFisher Scientific). Gels were subsequently transferredonto 0.45 µm Immun-Blot LF PVDF membranes (Bio-Rad) at20 V for 60 min. Total protein staining was utilised to normaliseprotein loading (internal loading control) via the REVERT TotalProtein Stain protocol (926-11010; LI-COR Biosciences). Aftertotal protein staining, membranes were then blocked in Odysseyblocking buffer (TBS) at pH 7.6 (LI-COR Biosciences) for 1 h atroom temperature before incubation overnight at 4°C with theirrespective primary antibody [NKA-α5 primary antibody (1:1000dilution) and ENaC-α primary antibody (1:1000 dilution; Anti-SCNN1A antibody; HPA012939, Sigma-Aldrich)] in blockingbuffer with 0.1% Tween-20 (TBST). NKA α5 was deposited in theDevelopmental Studies Hybridoma Bank, University of Iowa, byD. M. Fambrough (DSHB Hybridoma Product a5). Membraneswere then incubated in IRDye 800CW goat anti-mouse secondaryantibody (1:20,000; 926-32210, LI-COR BioSciences) and IRDye800CW donkey anti-rabbit secondary antibody (1:20,000;926-32213, LI-COR BioSciences), respectively, for 1 h at roomtemperature in the dark. Membranes were dried and visualised withan Odyssey CLx imaging system (LI-COR BioSciences). Targetproteins were normalised to loaded protein content usingImageStudio software (version 5.2; LI-COR BioSciences). Proteinlevels are expressed as abundance relative to the non-infectedintermoult group.

ENaC and NKA mRNA expressionRNA extraction and cDNA synthesisVentral skin samples (<1 cm2) from the lower abdominal pelvicpatch region were collected, and stored in RNA-later (Ambion Inc.)at −20°C. The skin samples were then homogenised with stainlesssteel beads using a TissueLyser II (Qiagen). Total RNAwas isolated(RNeasy Mini kit; Qiagen), and the resulting RNA yield wasmeasured using a Qubit fluorometer (ThermoFisher Scientific).Genomic DNA contamination in RNA samples was removed, thenthe RNAwas then reversed transcribed into cDNA, with appropriateno reverse transcriptase controls (QuantiTech Reverse TranscriptionKit; Qiagen).

Primer design and quantification of mRNA by qPCRTranscripts for each gene of interest were located in an in-house L.caerulea larval and adult tissue transcriptome using homologous

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sequences from other amphibians as the reference query. Referencesequences were blasted against the transcriptome using the ‘blastn’tool in Galaxy/GVL 4.0.1 (Afgan et al., 2016). Putative L. caeruleasequences were then compared against the entire National Centre forBiotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/) using ‘blastn’ with acceptance of the default parameters.qPCR primers against the target genes (Table S1) were designedusing OligoPerfect™ Designer (Thermofisher Scientific) withacceptance of the default parameters. qPCR was performed usingPower SYBR Green PCR Master Mix (ThermoFisher Scientific)using recommended cycling parameters. Each assay (in triplicate)included a no-template control and a no reverse transcriptasecontrol. All PCR efficiencies were >90% and all the assaysproduced unique dissociation curves. Bio-Rad CXF Managersoftware (version 3.1, Bio-Rad) results were exported and eachgene was quantified relative to the expression of the housekeepinggene, β-actin. Changes in expression levels were presented as fold-change relative to the uninfected intermoult group.

Statistical analysisAll analyses were performed in R 3.5.1 (https://www.r-project.org/).Data are either presented as means±s.e.m. or individual data points,and α was set at 0.05 for all statistical tests. Data that were notnormally distributed were log-transformed prior to analysis.Percentage data were logit transformed prior to analysis. Allmodels used a Gaussian error structure, and the confidence intervalsfor each model are presented in Tables S2–S6.

Blood plasma levels and electrophysiologyBlood plasma ion measurements (Na+, Cl−, K+) andelectrophysiological measurements of the skin [TEP, amiloride-sensitive ISC (50 µmol l−1 concentration), skin resistance and percentouabain inhibition of ISC] between each group were initially analysedusing linear models from the R default ‘stats’ package, with Bd loadas an interactive effect and bodymass as an additive effect. To test theeffects of body mass and exposure period (exposed once or twice),models with body mass and exposure as additive effects werecompared with models with no additive effects. The final model waschosen based on Akaike’s information criterion (AIC) with the‘anova’ function. To test for interactions between treatments withingroups while adjusting for Bd slope, a Holm-adjusted F-test wasperformed (package ‘phia’, function ‘testInteractions’; https://cran.r-project.org/web/packages/phia/index.html).The effect of amiloride dose and Bd load on the ISC between

intermoult and sloughing animals was analysed using a quadraticmodel (package ‘nlme’, function ‘lme’; https://cran.r-project.org/web/packages/nlme/index.html), with log(ISC) as the dependentvariable, log(amiloride concentration) as a fixed effect, Bd load asan interactive effect and frog ID as a random effect to account forrepeated measurements on the same individuals at different dosages.The IC50 was calculated for each individual, and the differencesbetween the uninfected and infected individuals for the intermoultand sloughing groups were analysed using a linear model with Bdload as an interactive effect.

ENaC and NKA subunit abundance and expressionRelative abundance of ENaC-α and NKA-α proteins in the ventralskin between each group was analysed using linear models, with Bdload as the interactive effect. To test the effects of body mass andexposure period (exposed once or twice), models with body massand exposure as additive effects were compared with models withno additive effects. The final model was chosen based on the AIC

with the ‘anova’ function. To test for factor interactions betweentreatments within groups and adjusting for Bd slope, a Holm-adjusted F-test was performed (package ‘phia’, function‘testInteractions’).

Relative mRNA expression of ENaC subunits (α, β, γ) and NKAsubunits (α1, β1, β3) in the ventral skin between each treatment andsloughing group was analysed with the following equation fromYuan et al. (2006):

E�DDCT ¼b0 þ btreatment þ bgene þ bgroup þ btreatment�gene

þ btreatment�group þ bgroup�gene þ btreatment�gene�group

þ bID þ e;

ð1Þwhere E�DDCT is the ΔΔ threshold cycle (CT); β is the fixed-effectsparameter where 0 is the intercept; × is the interaction term; bID is therandom intercept for frog ID expressed as bID∼N(o,δ2), where δ2 isthe variance of random intercept; and ε is the Gaussian error termexpressed as ɛ∼N(o,σ2), where σ2 is the variance of the residual. Themodel examines relative CT values of the target gene normalised tothe CT values of the reference gene β-actin, and the three-wayinteraction between treatment, group and gene with respect to thereference gene gives the ΔΔCT, where uninfected intermoult frogswere set as the control. To test the effects of body mass and exposureperiod (exposed once or twice), models with body mass andexposure as additive effects were compared with models with noadditive effects. The final model was chosen based on the AIC. Totest for factor interactions between treatments within groups,controlling for gene, a Holm-adjusted Chi-square test wasperformed (package ‘phia’, function ‘testInteractions’).

Data availabilityThe L. caerulea specific primer set sequence is available in Table S1.All datasets generated during and/or analysed during the study areavailable from the corresponding author on reasonable request.

RESULTSBd prevalence in the skinPrevalence of infection with Bd was high: in the first set ofexposures, 60.8% of exposed frogs developed an infection, and afterre-exposure, 73.9% of originally exposed frogs had developed aninfection. The three frogs that did not develop infection after re-exposure were excluded from further analysis.

Gross pathology and histopathology of the ventral skinThe histopathology of the skin of infected L. caerulea aftersloughing was the focus of this study, as infected intermoult (non-sloughing) animals have been detailed in past studies (Berger et al.,1999; Pessier et al., 1999; Nichols et al., 2001). In general, heavilyinfected frogs displayed cutaneous erythema, histological lesionsand thinning of the skin (Fig. 1A,B). However, the skin of recentlysloughed animals with low Bd zoospore loads (<10,000 ZE) wassimilar to that of uninfected animals (Fig. 1C,D). In heavily infectedanimals (>10,000 ZE) after sloughing, Bd sporangia were observedattached to the sloughed skin that had been fully removed (Fig. 1E,F,G), and also remained within the deeper layers of the epidermis(stratum granulosum; Fig. 1E,F).

Blood plasma electrolytesFrogs infected with Bd (both during the intermoult and sloughingperiods) in general showed a significant decrease in plasma sodium

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[Na+] and chloride [Cl−] levels with increasing Bd load (Table 1).Plasma potassium [K+] did not differ significantly between infectedand uninfected animals (t25=1, P=0.3). For intermoult animals only,there was a decrease in both plasma [Na+] and [Cl−] as Bd loadsincreased (Table 1). For sloughing animals, only plasma [Na+]levels remained significantly lower in infected frogs relative touninfected frogs (F1,25=11, P=0.003), while [Cl−] levels were notsignificantly different between infected and uninfected treatments(F1,25=1.6, P=0.2; Table S2).

Electrophysiology of the ventral skinThe TEP of the ventral skin across infection load was dependent onthe sloughing phase (t28=4.9, P<0.001; Table S3). As infection loadincreased in intermoult animals, the TEP decreased by 66.7%(−32.5±5.2 to −10.8±1.7 mV; F1,28=16.9, P<0.001) comparedwith uninfected animals. However, in sloughing animals, the TEPincreased by 58% (−8.8±0.85 to −21±5.5 mV) as infection loadincreased (F1,28=8.9, P=0.006; Fig. 2A).

Similarly, skin resistance decreased with increasing Bd load(Fig. 2B; Table S3). After sloughing (both infected and uninfectedanimals), the skin resistance was the lowest (301±79 Ω cm2).However, there was no significant difference in skin resistancebetween infected and uninfected animals after sloughing (Fig. 2B).There was also a borderline significant difference between theintermoult and sloughing animals (F1,28=5.6, P=0.05), with thesloughing animals showing lower skin resistance (321±51 Ω cm2)compared with the intermoult animals (478±82 Ω cm2; Fig. 2B).

Changes in active sodium transport, represented by amiloride-sensitive instantaneous short-circuit current (ISC) across infectionload, was dependent on the sloughing phase (Table S3). Forintermoult animals, as infection load increased, the ISC decreased byapproximately 40% (59.2±9.8 to 35.6±4.5 µA cm−2; F1,27=7.3,P=0.01). When converted to sodium flux, the rate of sodiumtransport decreased from 6.1×10−4±1×10−4 mol cm−2 s−1 inuninfected animals to 3.7×10−4±4.6×10−5 mol cm−2 s−1 ininfected animals. Recently sloughed animals increased ISC by 66%

C D

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50 μm 10 μm

10 μm

Fig. 1. Gross pathology and histopathology[transverse haematoxylin and eosin (H&E)-stained section 6 µm] of the ventral skin fromLitoria caerulea with chytridiomycosis. Ventralview of (A) an uninfected [0 zoospore equivalents(ZE)] and (B) an infected (∼44,000 ZE) L. caeruleaduring the intermoult (non-sloughing) period.Infected individuals showed various grossmorphological abnormalities such as cutaneouserythema with visible capillary vessels. Transversesection through the ventral skin of (C) an uninfected(intermoult) animal with no visible abnormalities.(D) Transverse section through the ventral skin of alightly infected animal (∼1600 ZE) collectedimmediately after sloughing occurred. The newlyexposed stratum corneum was similar tothat of uninfected animals, and had no visibleabnormalities. (E) Transverse section through theventral skin of a heavily infected, sloughing animal(20,000 ZE). Batrachochytrium dendrobatidis (Bd)sporangia below the shed s. coreneum (arrow) areclearly visible. (F,G) High-power views of the ventralskin from two infected sloughing animals showingBd sporangia below and within (arrows) the olds. corneum. EXIF data: f/2.2, ISO 80, exposure time1/33 s, focal length 4 mm.

Table 1. Blood plasma ion levels of infected and uninfected (control) Litoria caerulea during the intermoult and sloughing periods

Intermoult Sloughing

Uninfected Infected Uninfected Infectedn Mean±s.e.m. n Mean±s.e.m. n Mean±s.e.m. n Mean±s.e.m.

Sodium (mmol l−1) 7 147.6±3.9 9 84.5±8 5 145±8.7 4 90.5±5.2Chloride (mmol l−1) 7 57±2.2 9 40±5.3 5 58.9±2 4 53.2±9.6Potassium (mmol l−1) 7 3.4±0.3 9 2.6±0.3 5 2.9±0.4 4 3.3±0.6

n, sample size. Summary statistics are provided in Table S2.

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(28.8±4.2 to 84.7±14.3 µA cm−2; F1,27=27, P<0.001; Fig. 2C) asinfection load increased. This represents an increase in the rate of sodiumtransport during sloughing from 3×10−4±4.4×10−4 mol cm−2 s−1 inuninfected animals to 8.8×10−4±1.5×10−4 mol cm−2 s−1 in infectedanimals.For ouabain inhibition of ISC, there was an overall significant

difference between groups, where post-sloughing animals hadgreater inhibition from 100 µmol l−1 ouabain compared withintermoult animals (t15=2.3, P=0.04; Fig. 2D). Infection did nothave a significant effect on the ISC inhibition (Table S4).For amiloride dosage experiment, therewas a significant decrease

in ISC as amiloride dose increased in both intermoult and sloughinggroups, independent of infection load (Table S4). In the intermoultgroup, amiloride concentration on the ISC was dependent on theinfection load, where at a high dosage of amiloride the ISC slopedecreases to a plateau with increasing infection load (Fig. 2E). This

indicates the isolated ventral skin during the intermoult phase ofinfected animals was more sensitive to amiloride dosage, requiring0.7±0.14 µmol l−1 amiloride to inhibit 50% of the ISC, comparedwith uninfected frogs (IC50=2.35±0.6 µmol l−1; t12=2.6, P=0.02).

For animals after sloughing, there was a significant effect of Bdinfection alone (t11=3.5, P=0.004), with a decrease in ISC asinfection load increased. No interactions between infection load andamiloride concentration were observed (Table S4), suggesting nodifference in the sensitivity to amiloride in the newly exposed skinbetween uninfected (IC50=1.4±0.26 µmol l−1) and infected frogs(IC50=0.84±0.2 µmol l−1; t12=0.7, P=0.5; Fig. 2F).

ENaC and NKA protein abundance in the ventral skinThere was a decrease in ENaC-α subunit (SCNN1A) proteinabundancewith increasing infection load (Table S5); however, therewas no significant difference in SCNN1A abundance between the

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Fig. 2. Electrophysiological parameters of isolated ventral skins of Litoria caerulea. (A) Transepithelial potential (mV), (B) transepithelial resistance (Ω cm2)and (C) amiloride-sensitive short-circuit current (ISC; µA cm2) of isolated ventral skins, either during the intermoult period (solid line) or after sloughing (dashedline) in relation to infection intensity [log(ZE+1)]. Shaded area around regression lines represents 95% confidence intervals, and all data are presented [intermoult:uninfected (grey circles) n=6, infected (orange circles) n=11; sloughing: uninfected (grey triangles) n=9, infected (orange triangles) n=7]. (D) The percentageinhibition of ISC between infected and uninfected animals after application of 100 µmol l−1 ouabain. All data points are presented [intermoult: uninfected (greycircles) n=6, infected (orange circles) n=4; sloughing: uninfected (grey triangles) n=7, infected (orange triangles) n=4]. Different letters indicate significantdifferences at P<0.05. ISC (µA cm−2) response of isolated ventral skin from (E) Intermoult and (F) sloughing L. caerulea to different amiloride concentrations (0.1–50 µmol l−1) in the apical reservoir. All data are presented (intermoult: uninfected n=5, infected n=10, sloughing: uninfected n=7, infected n=7), and solid colouredlines for each Bd load range [0–5 log(ZE+1)] represent model predictions. Summary statistics are provided in Tables S3 and S4.

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intermoult and sloughing groups (t23=0.9, P=0.3). Within theintermoult group, as infection load increased, the relative abundanceof SCNN1A protein decreased (F1,23=6.7, P=0.03; Fig. 3A). Forsloughing animals, there was no significant difference in SCNN1Aprotein abundance as infection load increased (F1,23=1, P=0.3).The abundance of the NKA-α subunit (ATP1A1) was

significantly greater in infected animals compared with uninfectedanimals in the sloughing group (F1,23=0.65, P<0.001; Fig. 3B),whereas there was no difference between infected and uninfectedanimals in the intermoult group (F1,23=0.65, P=0.42).

ENaC and NKA mRNA expression in the ventral skinWithin the ENaC family, therewere significant differences in α- andβ-subunit mRNA expression between infected and uninfected frogs(Fig. 4). For ENaC-α (SCNN1A) there was a significant increase inexpression in the infected group compared with the uninfectedgroup (t22=3.8,P=0.001), whereas for ENaC-β (SCNN1B) therewasa significant decrease in expression (t22=2.7, P=0.01). No effect ofsloughing phase was observed for either gene (Fig. 4A; Table S6).There was no effect of Bd infection or sloughing phase on theexpression of the ENaC-γ (SCNN1G) subunit (Fig. 4A).Within the NKA family, there were significant increases in the

expression of both α1 (ATP1A1) and β1 (ATP1B1) subunit mRNAin the infected group relative to the uninfected group (Fig. 4B;Table S6). NKA-β3 (ATP1B3) subunit expression did not differbetween infected and uninfected animals (t22=2, P=0.06). However,there was a significant effect of sloughing phase on ATP1B3expression (t22=3.2, P=0.003), with expression increasing in thesloughing group relative to the intermoult group (Fig. 4B).

DISCUSSIONWith the recent discoveries that sloughing can regulate cutaneousBd loads (Ohmer et al., 2017) and that increasing Bd loadscontribute to the severity of disease symptoms in infected animals(Wu et al., 2018), it is important to understand the mechanistic basisfor these relationships. The present study confirms that reduction inENaC-associated cutaneous sodium transport contributes to the lossof ionic homeostasis in frogs with chytridiomycosis. We showedthat elevated cutaneous sodium efflux in Bd-infected frogs is due in

part to reduced Na+ uptake capacity, attributable to a reducedabundance of ENaC transporters. Although ENaC mRNAexpression was elevated in non-sloughing frogs withchytridiomycosis, the abundance of ENaC protein in the skin waslower than in uninfected frogs, suggesting that either proteinsynthesis was being inhibited or that newly synthesised proteinswere being lost. Interestingly, sloughing stimulated a transientupregulation of Na+ uptake in both infected and uninfected animals,and the newly exposed skin of infected frogs had a similarabundance of ENaC protein compared with uninfected frogs. Thissuggests the new skin layer initially possesses a full complement offunctional Na+-transport machinery that facilitates the temporaryrestoration of normal physiological function. However, thisrestoration period is brief; it appears that ENaC proteins in theskin are destroyed by exposure to Bd during the intermoult period,contributing to the ongoing loss of ionic homeostasis.

Physiological disruption of the skinConsistent with previous findings by Voyles et al. (2009), infectedL. caerulea during the intermoult period had lower transcutaneouselectrophysiological parameters (TEP, amiloride-sensitive ISC, skinresistance) and lower levels of plasma Na+ and Cl− than uninfectedfrogs. Small changes in the electrical properties of an epithelia canhave large consequences for the maintenance of physiologicalhomeostasis (Wood and Grosell, 2008); for example, a −10 mVchange in the TEP is correlated with a reduction in plasma [Na+] by33.5% in killifish (Wood and Grosell, 2015). The combination ofreduced TEP, reduced active uptake and increased skin permeabilitycould explain the low plasma Na+ (hyponatremia) and Cl−

(hypochloremia) levels seen in infected L. caerulea. The reducedTEP is likely a result of the reduction in both skin resistance andactive transcutaneous Na+ uptake (amiloride-sensitive ISC) acrossthe skin of non-sloughing infected frogs. Several lines of evidencesuggest that the secretion of proteolytic enzymes and toxins by Bd(Rosenblum et al., 2008; Symonds et al., 2008; Brutyn et al., 2012)breaks down proteins and intercellular junctions, leading toincreased skin permeability (Voyles et al., 2009). The loss of skinresistance and reduced uptake capacity is consistent with the ideathat Bd may directly damage local proteins in the skin. Further

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Fig. 3. Relative abundance of epithelial iontransporter proteins in the ventral skin ofinfected and uninfected Litoria caeruleaduring the intermoult and sloughingperiods. Abundance of the (A) ENaC-αsubunit (SCNN1A) and (B) NKA-α subunit(ATPA1A) expressed relative to the uninfectedintermoult group (dashed line). Western blotanalysis detected major bands at ∼80 kDafor ENaC-α, and ∼112 kDa for NKA-α.Representative immunofluorescence blotsassociated with each transport protein (top)show the molecular mass for the respectivetreatment groups. Bars represent means±s.e.m., with individual values overlain forintermoult (uninfected n=6, infected n=7) andsloughing (uninfected n=8, infected n=6)animals. Different letters represent significantdifferences between treatments and groups(P<0.05). Summary statistics are provided inTable S5.

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evidence for this hypothesis is that Bd had no effect on the activityor abundance of NKA in the skin. In frog skin, the multiple layers ofthe epithelium function as an interconnected syncytium (Heatwoleet al., 1994); active ion uptake occurs in the more apical skin layers,while the deeper skin layers contain the basolateral membrane-associated proteins (i.e. NKA) responsible for the generation of theelectrochemical gradient that facilitates the apical transporteractivities (Nielsen, 1979). The impairment of active Na+ uptake isconsistent with the hypothesis that Bd infection in the apical layersof the skin (stratum corneum and upper granulosum) (Greenspanet al., 2012) directly affects the functional capacity of ENaC and the

paracellular junctions, whereas ion transporters in the deeper layersof the epidermis (i.e. NKA) are largely unaffected by infection.

Responses in ENaC subunits to Bd infectionBd infection appears to alter the functional capacity of Na+ transportpathway by directly destroying these channels in the skin. Bdincreased the sensitivity of the cutaneous active ion transport pathwayto amiloride in intermoult frogs, reducing the IC50 by 70% relative tothe uninfected group. A reduction in amiloride-sensitive Na+

transport indicates that there are fewer functional ENaC proteins inthe skin of infected animals and so saturation of pumps is achieved at

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Fig. 4. Relative mRNA expression of epithelial ion transporters in the ventral skin of infected and uninfected Litoria caerulea during the intermoult andsloughing period. (A) mRNA expression of epithelial sodium channel (ENaC) subunits (α, β and γ). Absolute gene expression (ΔCT) was normalised toexpression of the housekeeping gene β-actin, and is presented as fold change relative to uninfected intermoult groups (dashed line). Diagrammatic representationof the ENaC structure (based on Canessa et al., 1994) with the position of subunits on the cell surface of the epidermis. (B) mRNA expression of Na+/K+-ATPase(NKA) subunits (α1, β1 and β3). Diagrammatic representation of the NKA structure (Suhail, 2010) with the position of subunits on the cell surface of the epidermis.All data points are presented with mean±s.e.m. bar chart overlay for intermoult (uninfected n=7, infected n=7) and sloughing (uninfected n=7, infected n=5)animals. Different letters represent significant differences between treatments and groups (P<0.05). Summary statistics are provided in Table S6.

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lower drug concentrations. This is consistent with our data showingthat there was less ENaC-α protein in the skin of infected (intermoult)frogs compared with healthy frogs. ENaC exists as an obligateheterotrimer with α, β and γ subunits (or β, γ, δ subunits in someorganisms; Hanukoglu and Hanukoglu, 2016), and although we didnot quantify the protein-level abundance of all ENaC subunits, it islikely that the reduction in ENaC-α reflects similar changes in theabundance of the functional heterotrimer. The skin of infectedanimals appears to try to compensate for the loss of functionalproteins by increasing the expression of the ENaC-α gene. However,the gene expression of other ENaC subunits did not follow the samepattern: Bd infection reduced the expression of ENaC-β and had noeffect on the expression of ENaC-γ. Whether Bd differentially affectsthe abundance of ENaC subunit proteins is unknown. ENaC subunitexpression is under tight control by hormones and extracellularfactors (Butterworth et al., 2008), including stress hormones, sofluctuations in circulating hormone levels as a consequence of naturalcycles or disease-related processes may contribute to the differentialregulation of ENaC subunit expression. It is also possible that onlyENaC-α protein is affected by Bd and, because ENaC-α is arequirement for ENaC channel activity,ENaC-β gene expressionmaybe downregulated to prevent an oversupply of the β-subunit in theabsence of the corresponding α-subunit.

Physiological alteration during sloughingIn infected animals, Na+ transport into the skin increasedimmediately after sloughing. The stark difference in Na+ uptakerates between recently sloughed and intermoult animals,particularly in infected frogs, likely reflects the exposure of thenew skin layer. Sloughing removes the most degraded outermostlayer of skin, effectively ‘resetting’ the skin surface (Larsen, 1976).However, until the newly exposed stratum corneum layer becomesfully keratinised, the skin remains relatively permeable to ions (i.e. ithas low resistance to ion loss). Therefore, the increase in ISC mayserve to offset the transient increase in skin permeability thataccompanies sloughing. Indeed, we and others have observed asimilar change in ISC across the skin immediately after sloughing inother amphibian species (Larsen, 1970; Nielsen and Tomilson,1970; Larsen, 1971; Wu et al., 2017). The increase in ISC post-sloughing in cane toads occurred commensurate with an increase inthe abundance of ENaC-α and NKA proteins in the epithelium (Wuet al., 2017). This is consistent with the idea that the increase in ISCpost-sloughing is the result of more functional transporters presentin the new skin, although isotope binding studies (e.g. Cramp et al.,2009) would be needed to quantify this.Importantly, for frogs infected with Bd, the increase in transporter

numbers in the skin post-sloughing was sufficient to restore activeNa+ uptake (ISC), albeit transiently. This finding supports the ideathat Bd directly damages ion-transport proteins in the apical skinlayers (Campbell et al., 2012), and that sloughing restores skinfunction by both exposing relatively undamaged cells and reducingthe abundance of Bd on the skin, which may reduce fungal toxins orsecretions. However, the increased expression of ENaC-α, NKA-αand NKA-β mRNA in the tissues of recently sloughed frogsindicates that Bd infection does exert some influence over iontransporter systems, even in the more basal skin layers. Althoughtissues were collected as soon after sloughing as possible, there wasoften a gap of up to 1 h between sloughing and when tissues werecollected, during which time the newly exposed skin would havebeen in direct contact with Bd in the surrounding environment. So,although increased mRNA expression in newly exposed skin of Bd-infected frogs might be a pre-sloughing compensatory response to

elevated rates of protein degradation in the skin, contact between thenewly exposed skin and Bd zoospores or toxins in the environmentmay have also stimulated ion transporter mRNA synthesis.

In the present study, infected frogs sloughing showed an increasein skin permeability. Increased permeability following sloughing isprolonged compared with uninfected frogs, which could be due to thecombination of leaky skin from the act of sloughing (Wu et al., 2017),structural disruption from infection (Nichols et al., 2001; Berger et al.,2005) and reduction in the expression of skin integrity genes from Bdinfection (Rosenblum et al., 2012; Ellison et al., 2015). Futureelectrophysiological studies could determine epithelial tight junctionpermeability by replacing Cl− Ringer’s solution with sulphateRinger’s solution in Ussing chamber set-ups. To compensate forthe high leakage demonstrated in this study, we found that NKAactivity and abundance increased during sloughing. An increase inNKA activity is associated with maintaining osmotic balance whenanimals are subjected to osmotic stressors (Choi and An, 2008;Cramp et al., 2010). However, because NKA actively exchanges ionsacross cellular membranes against their concentration gradients viaATP hydrolysis (Lingrel and Kuntzweiler, 1994), there may be anincrease in cellular energy expenditure to uptake Na+ into the cells(Harvey and Kernan, 1984). Susceptible amphibian species infectedwith Bd have greater demand on cellular metabolic processescompared with non-susceptible species (Poorten and Rosenblum,2016). Thus, the combination of increased skin permeability, cellularenergy expenditure, ionic disruption and rate of sloughing may haveadditional cumulative consequences for maintaining electrolytehomeostasis in highly infected frogs.

ConclusionsOur results reveal a complex interaction between sloughing andchytridiomycosis. The cumulative effect of increased skinpermeability during non-sloughing periods and increasedsloughing frequency effectively counteracted any benefit thatmight have be gained from the temporary, post-sloughing increasein electrolyte uptake capacity. Given that the magnitude of effects ofBd on skin function and sloughing frequency are proportional to Bdloads, species that fail to regulate Bd loads effectively are likely tosuffer greater physiological disruption and more rapid progressionof the disease than those species that can regulate their fungal loads(Ohmer et al., 2017). This work demonstrates the mechanismsunderlying cutaneous disruption during Bd infection in susceptibleamphibian species and highlights the intrinsic role of sloughing onthis process. Importantly, this study explains why sloughing can bedetrimental for susceptible species that develop high infectionloads, and may accelerate disease progression.

AcknowledgementsWe would like to acknowledge all volunteers who have assisted with animal care,maintenance and video surveillance, S. Bloomberg (University of Queensland) foradvice on statistical analysis, and E. P. Symonds for isolating strain EPS4. Specialthanks to J. Gauberg (York University), E. Watson and A. T. Khalid for assisting withexperiments.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: N.C.W., R.L.C., M.E.O., C.E.F.; Methodology: N.C.W., R.L.C.,M.E.O., C.E.F.; Software: N.C.W.; Validation: N.C.W., R.L.C.; Formal analysis:N.C.W.; Investigation: N.C.W.; Resources: R.L.C., C.E.F.; Data curation: N.C.W.;Writing - original draft: N.C.W.; Writing - review & editing: N.C.W., R.L.C., M.E.O.,C.E.F.; Visualization: N.C.W.; Supervision: R.L.C., C.E.F.; Project administration:N.C.W., R.L.C., C.E.F.; Funding acquisition: C.E.F.

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FundingThis research was funded by the Australian Government Research TrainingProgram (RTP) scholarship and the Peter Rankin Trust Fund for Herpetologyawarded to N.C.W., and a University of Queensland research grant awardedto C.E.F.

Supplementary informationSupplementary information available online athttp://jeb.biologists.org/lookup/doi/10.1242/jeb.191817.supplemental

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