Body size influences energetic and osmoregulatory costs in frogs infected with Batrachochytrium dendrobatidis

Sloughing maintains the skins integrity and critical functionality in amphibians. Given the behavioural, morphological and osmoregulatory changes that accompany sloughing, this process is likely to be physiologically costly. Chytridiomycosis, a cutaneous disease of amphibians caused by the fungus Batrachochytrium dendrobatidis (Bd), disrupts skin function and increases sloughing rates. Moreover, mortality rates from chytridiomycosis are significantly higher in juveniles and so we hypothesised that smaller individuals maybe more susceptible to chytridiomycosis because of allometric scaling effects on the energetic and osmoregulatory costs of sloughing. We measured in-vivo cutaneous ion loss rates and whole animal metabolic rate (MR) of Green tree frogs, Litoria caerulea, over a range of body sizes both infected and uninfected frogs during sloughing. Infected animals had a greater rate of ion loss and mass-specific MR during non-sloughing periods but there were no additional effects of sloughing on either of these parameters. There were also significant interactions with body size and Bd load indicating that smaller animals with higher Bd loads have greater rates of ion loss and higher energetic demands. Our results shed light on why smaller Bd-infected anurans often exhibit greater physiological disruption than larger individuals.

ml aged water. Uninfected frogs were treated similarly, but with aged water only. At 2 weeks post-exposure and fortnightly thereafter, each frog was swabbed with a sterile fine-tipped cotton swab (MW100-100; Medical Wire & Equipment, Wiltshire, England) three times over the frog's ventral surface, thighs, armpit, forelimb feet, and hindlimb feet 1,3 to assess infection status. Samples were processed following Boyle et al. 2 . Swabs were extracted in 50µl PrepMan Ultra (Applied Biosystems, Foster City, CA, USA), and analysed in duplicate with Taqman qPCR in a thermal cycler (MiniOpticon TM Real-Time PCR Detection System, Bio-Rad Laboratories, Inc.) in a modified 15 µl reaction following Ohmer et al. 1 . If after 1 month, exposed frogs had no detectable Bd infection, they were re-exposed as above. Infection load or number of zoospore equivalent (ZE) on the skin surface were log + 1 transformed [Log(ZE+1)]. Prevalence of infection with Bd was high: in the first set of infection 65% of exposed frogs developed infection, and one month re-exposure 87% of exposed frogs developed an infection. The three frogs that did not develop infection after re-exposure were excluded from the analysis.

Ion loss measurements
Changes in ion loss during sloughing were measured by placing animals into a ventilated clear chamber (50-1000 ml, depending on the frogs' mass) containing between 30 and 200 ml of distilled water with a magnetic stirrer to circulate the solution. The rate of ion efflux from the animal was measured as change in the conductivity of the bathing solution (microsiemens per hour; Δ µS h -1 ). Conductivity was measured between two electrodes placed into the solution which were connected to a conductivity pod (ML307, ADInstruments, NSW, Australia). The output was digitised with PowerLab 4/35 interface (ADInstruments) and recorded onto Labchart software (ADInstruments). The baseline of the solution was measured for roughly an hour before the animal was placed into the chamber. Animals were at one of 5 points in their sloughing cycles when measurements were made: 1. intermoult (half way through slough cycle); 2. day of slough (3 h prior to slough, or day of predicted slough in infected frogs but did not slough); 3. pre-slough (10 -20 min prior to slough); 4. mid-slough (during a slough); and 5. post slough (30 -60 min post sloughing). Sloughing behaviour was monitored continuously via webcam (Microsoft VX-3000) and measurements for each group was defined: 1.
intermoult and 2. day of slough, where the animals remain still; 3. pre-slough, when animals started to extend their limbs to lift the abdomen off the ground; 4. mid-slough which begins with mouth gaping, followed by abdominal contractions, upper body wiping and removal of the old skin; and 5. post-slough, up to 1 h after sloughing when normal behaviour resumes (Fig. S1). All experiments were conducted at room temperature (20.5 ± 0.5°C), and animals were swabbed for Bd load prior to the introduction to the chamber.
Three dimensional (3D) agar models of 4 different sizes (20, 40, 60, and 80% of adult size) were made to examine the effect of surface area on the rate of cutaneous ion loss across a free-flowing permeable surface.
To make the agar replicas, a life size adult green tree frog plastic model was scanned using a HDI surface scanner on FlexScan3D software (LMI technologies Inc. BC, Canada), then cleaned, refined and filled using 3 % agar solution in Ringers solution (in mmol l -1 : NaCl (112), KCl (2.5), Na2HPO4 (2), CaCl2 (1), MgCl2 (1), HEPES salt (5), HEPES (5), pH 7.4, osmolarity 270 ± 20 mOsmol l -1 ) was poured into the molds and allowed to solidify. The agar replicas were placed into the ion loss water baths and subjected to the same conditions as the living frogs. Ion loss into the water bath was recorded for 1 h.
To estimate the overall effect of sloughing on the animals sodium budget, conductivity measures were converted to a rate of sodium loss (mmol h -1 ) based on the [Na + ] of bath water samples collected after sloughing (measured via flame photometry (BWB-XP flame photometer; BWB Technologies Ltd, UK)), and assuming that (1) sodium was the primary ion contributing to solution conductivity, and (2) that the proportion of sodium lost relative to other ions was equal 4 . The net amount of Na + lost during sloughing was approximated assuming an extracellular fluid (ECF) volume of approximately ~25 % of the body mass of the animal 5 . The actual Na + concentration of the ECF was measured from plasma samples collected from green tree frogs in a separate experiment following Wu et al. 4 . The proportion of the total extracellular fluid Na + (as % of ECF Na + h -1 ) lost during sloughing was calculated by dividing the rate of ion loss (mmol l h -1 ) by the total amount of ECF Na + (mmol) and multiplied by 100.
The effective ventral surface area (Av; cm 2 ) across which ion exchanges would occur was calculated by photographing the ventral side of the animal in contact with a glass surface and calculating the surface area (excluding surface area of limbs and head; Fig. S2). Images were analysed using Image J (http://imagej.nih.gov/ij/). For surface area-specific ion loss, data was presented as S cm 2 h -1 .

Respirometry set-up
Positive pressure flow-through respirometry was used to measure whole animal rates of oxygen consumption (̇2, ml O2 h -1 ) and carbon dioxide production (̇2, ml CO2 h -1 ), as a proxy for whole animal metabolism (the sum of both respiratory and cutaneous respiration). Atmospheric air scrubbed of CO2 (using soda lime, Chem-Supply, Adelaide, Australia) and water vapour (using Drierite, W. A. Hammond Drierite Co. Ltd, USA) was drawn in to the respirometry system via a sub-sampler pump (SS-3, Sable Systems International, Las Vegas, NV, USA), at a controlled flow rate of either 30 ml min -1 (for small frogs < 10 g), 50 ml min -1 (for medium frogs 10 -25 g) or 80 ml min -1 (for large frogs > 25 g) by a mass flow controller (GFC17, Aalborg Instruments & Controls Inc., Orangeburg, NY, USA). The dry, CO2-free air passed through the metabolic chamber (50 ml, 300 ml or 500 ml glass air-tight container (ClipFresh, Hong Kong)), before passing through a relative humidity (RH) analyser (RH-100; Sable Systems, Las Vegas, NE, USA). The air was then rescrubbed of water vapour before passing through an infrared CO2 gas analyser (LI-820, LI-COR ® Biosciences Inc., Lincoln, NE, USA) and an O2 analyser (Oxzilla II; Sable Systems, Las Vegas, NE, USA). The fractional concentrations of the CO2 and O2 in the excurrent air ( 2 and 2 ) were recorded in a PowerLab 4/35 interface and imported into Labchart software (ADinstruments).

Resting and sloughing metabolic measurements
Each frog was fasted for at least 4 days prior to measurement to ensure a post-absorptive state 6 . The background fractional CO2 and O2 concentration of the ex-current air from the respirometry chamber was recorded overnight prior to the introduction of the animal. Body mass (Mb, g) was recorded before and after the experiment, and animals were swabbed prior to the introduction of the chamber. The resting metabolic rate (RMR) was taken over the period when the animal was in a water conserving posture and behaviourally inactive which corresponded to the lowest O2 and CO2 readings observed. Metabolic rate during the day of slough, pre-slough, mid-slough, and post-slough stages were also recorded. Active metabolic rate, defined as the rate of CO2 production during a period of continuous movement in the chamber was also measured to compare with the relative energetic cost of sloughing. All activities and behaviours were monitored remotely with a webcam (Microsoft VX-3000) and recorded in Labchart. The temperature of the experimental room was maintained at 20.75 ± 0.4 °C. The mean ̇2 and ̇2 for all activities were calculated following Lighton 7 : Due to inconsistent drifts in the O2 analyser for some experiments, overall sample size for ̇2 was low, thus