Vol. 117: 245–252, 2016 DISEASES OF AQUATIC ORGANISMS Published January 13 doi: 10.3354/dao02940 Dis Aquat Org Physiological responses of Brazilian amphibians to an enzootic infection of the chytrid fungus Batrachochytrium dendrobatidis Rafael P. Bovo1,*, Denis V. Andrade1, Luís Felipe Toledo2, Ana V. Longo3, David Rodriguez4, Célio F. B. Haddad1, Kelly R. Zamudio3, C. Guilherme Becker1 1Departamento de Zoologia, c. p. 199, Universidade Estadual Paulista, 13506-900, Rio Claro, SP, Brazil 2Laboratório de História Natural de Anfíbios Brasileiros (LaHNAB), Departamento de Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, 13083-862, Campinas, SP, Brazil 3Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY 14853, USA 4Department of Biology, Texas State University, San Marcos, TX 78666, USA ABSTRACT: Pathophysiological effects of clinical chytridiomycosis in amphibians include dis - orders of cutaneous osmoregulation and disruption of the ability to rehydrate, which can lead to decreased host fitness or mortality. Less attention has been given to physiological responses of hosts where enzootic infections of Batrachochytrium dendrobatidis (Bd) do not cause apparent population declines in the wild. Here, we experimentally tested whether an enzootic strain of Bd causes significant mortality and alters host water balance (evaporative water loss, EWL; skin resistance, Rs; and water uptake, WU) in individuals of 3 Brazilian amphibian species (Dendropso- phus minutus, n = 19; Ischnocnema parva, n = 17; Brachycephalus pitanga, n = 15). Infections with enzootic Bd caused no significant mortality, but we found an increase in Rs in 1 host species con- comitant with a reduction in EWL. These results suggest that enzootic Bd infections can indeed cause sub-lethal effects that could lead to reduction of host fitness in Brazilian frogs and that these effects vary among species. Thus, our findings underscore the need for further assessment of physiological responses to Bd infections in different host species, even in cases of sub-clinical chytridiomycosis and long-term enzootic infections in natural populations. KEY WORDS: Chytridiomycosis · Amphibian declines · Fitness · Evaporative water loss · Water uptake · Amphibian skin · Mortality Resale or republication not permitted without written consent of the publisher INTRODUCTION et al. 2015) since at least the late 1800s (Rodriguez et al. 2014). Studies of Bd epidemiology in Brazil are on The fungal pathogen Batrachochytrium dendroba- the rise (Becker & Zamudio 2011, Becker et al. 2014, tidis (Bd) causes chytridiomycosis and has led to 2015, Lambertini et al. 2016, Ruggeri et al. 2015, amphibian population declines and extinctions in Valencia-Aguilar et al. 2015), but to date no records several parts of the globe (Berger et al. 1998, Lips et of amphibian declines in Atlantic Forest frogs have al. 2008, Vredenburg et al. 2010). Sub-lethal chytrid- been attributed to Bd (Toledo et al. 2006, Haddad iomycosis, however, is common where Bd is enzootic, et al. 2013, James et al. 2015). Because the Atlantic such as in Brazil’s Atlantic Forest, where multiple Bd Forest harbors ~10% of all described amphi bian spe- strains have infected several hosts (Valencia-Aguilar cies (Haddad et al. 2013, Toledo et al. 2014, IUCN *Corresponding author: [email protected] © Inter-Research 2016 · www.int-res.com 246 Dis Aquat Org 117: 245–252, 2016 2015), experimentally testing the effect of locally iso- alters water balance in 3 amphibian species native lated Bd on both mortality and physiological re - to Brazil’s Atlantic Forest. Specifically, we tested sponses of local host species is critical for biodiversity whether experimental Bd infections affected 3 physi- conservation. ological parameters key to host water balance: (1) Clinical infections of Bd often impair amphibians EWL, (2) skin resistance (Rs), and (3) WU. If endemic from maintaining adequate homeostasis and directly Bd infection is detrimental for Brazilian amphibians, impact host physiological and functional responses we would expect to observe either some evidence (Voyles et al. 2009, Salla et al. 2015). Specifically, Bd of disease-associated mortality (clinical chytridio - infection causes hyperkeratosis (skin thickening) mycosis) or some evidence of sub-clinical pathophy - and impairs host ability to absorb and regulate body siology that may compromise host osmoregulatory fluids, leading to electrolyte imbalance and cardiac capabilities. arrest in infected hosts (Voyles et al. 2011). Skin permeability is critically important in regulating water transport and exchange of electrolytes and MATERIALS AND METHODS respiratory gases in amphibians (Wells 2007, Hill- man et al. 2009, Voyles et al. 2011). In cases of Study species aclinical chytridiomycosis (i.e. when hosts do not show gross pathological changes in the superficial We captured adult amphibians of 3 species in epidermis; Voyles et al. 2011), the potential impair- October 2012 at Parque Estadual da Serra do ments in physiological function remain, for the most Mar−Núcleo Santa Virginia, municipality of São part, unmeasured (but see also Salla et al. 2015). Luiz do Paraitinga, state of São Paulo, Brazil However, previous studies indicate that even under (23.35° S, 45.16° W). Dendropsophus minutus has aclinical conditions, Bd may interfere with host aquatic larvae, breeds in aquatic habitats, and occu- water balance (Carver et al. 2010, Wardziak et al. pies the emergent vegetation at the edge of water 2013). If that is the case, even minor shifts in host bodies. Ischnocnema parva and Brachycephalus osmoregulation — a physiological process intimately pitanga deposit eggs on land, undergo direct devel- associated with amphibian life history, habitat use, opment, and are active on the forest leaf-litter. By and evolutionary ecology — could lead to long-term using wild-caught amphibians in laboratory experi- decreases in host population fitness. mental infections, we are not able to control for age, A number of studies have investigated the impact previous exposure, and potential acquired resist- of chytridiomycosis on host water balance (Voyles et ance (McMahon et al. 2014); however, randomly al. 2009, Carver et al. 2010, Voyles 2011, Campbell et assigning individual hosts for each treatment can al. 2012, Wardziak et al. 2013). Water balance para - minimize these uncontrolled factors. meters are particularly relevant in amphibians due to their highly permeable skin and the associated risk of incurring excessive evaporative water loss (EWL; Experimental design Shoemaker et al. 1992, Spotila et al. 1992, Lillywhite 2006). While laboratory studies have shown impair- We treated field-collected animals with itracona- ments of normal skin functions in Bd-infected zole 0.01% for 7 d to ensure study organisms were amphibians, the magnitude of these effects across Bd-negative prior to the beginning of the experiment hosts depends on a complex array of factors, includ- (Pessier & Mendelson 2010, Moreno et al. 2015); ing differences in Bd strains (Gahl et al. 2012), previ- treated hosts all tested negative prior to experimental ous exposure of the host (Murphy et al. 2011), host infections. We cultured an enzootic Bd strain isolated life history (Gervasi et al. 2013), and host susceptibil- from Minas Gerais, Brazil (CLFT 023; Schloegel et al. ity (Searle et al. 2011). It is uncertain whether low 2012, Longo et al. 2013) on tryptone agar Petri plates infection intensities, such as those in localities where at ~19°C for 7 d. We harvested Bd by flooding plates Bd is an enzootic pathogen, can significantly inter- with distilled water and waiting ~3 h for zoospore fere with EWL rates and/or influence the rates of release. The strain used in experiments belongs to water uptake (WU) through the ventral skin, and the global panzootic lineage (GPL), a highly virulent whether such effects vary among host species. strain causing amphibian declines and extinctions in In this study, we experimentally tested whether several regions of the globe (Rosenblum et al. 2013). infection by an enzootic strain of Batrachochytrium In Brazil, it has been present for at least a century dendrobatidis causes (1) significant mortality and (2) (Rodriguez et al. 2014). We then pooled inoculum Bovo et al.: Amphibian responses to enzootic chytrid infection 247 from plates and quantified zoospores with a hemo - with 0.5 cm of water, in a climate-controlled chamber cytometer. (122FC model, Eletrolab) at 20°C for 1 h to ensure We added 4 conspecific amphibians to rectangular that they were fully hydrated at the beginning of the experimental units measuring 40 × 29 × 13.5 cm and experiment. Immediately after this period, each frog kept temperatures at 19.74°C (0.55 SD). These tem- was carefully blotted with paper tissue, its urinary perature records were measured by a Hobo® data bladder was emptied by gently pressing the ab - logger at 30 min intervals and on a 12 h light:12 h domen, and body mass was recorded (±0.0001 g). We dark cycle. Each experimental unit consisted of a placed each individual frog in a circular PVC cham- plastic terrarium with terrestrial habitat covering one ber (8 cm diameter) connected to an automated open end of the container (i.e. autoclaved moist sphag- flow system that measures EWL. A pump combined num) and aquatic habitat covering the other end of to a mass flow meter (SS-3 Subsampler, Sable Sys- the container (i.e. 250 ml of Bd inoculum, 106 zoo- tems) provided stable airflow current of 21.66 cm3 s−1, spores total, in the treatment group or 250 ml of dis- with relative humidity (RH) controlled at 30% by a tilled water in the control group). RH/Dewpoint Controller (DG-4, Sable Systems). Air- We monitored amphibians daily and fed them pin- flow was directed into the experimental chamber, head crickets ad libitum. We swabbed all individuals and relative humidity of excurrent air was continu- after 18 d of exposure, a period encompassing several ously monitored by a water vapor analyzer (RH-300 replication cycles of the pathogen (Longcore et al. RH/Dewpoint Analyzer, Sable Systems).