We present gross and histologic evidence of coinfection in amphibians by fungal-like parasites of the order Dermocystida (Amphibiocystidium sp.) and the fungus Batrachochytrium dendrobatidis. The condition was observed in frogs Hypsiboas pulchellus (Hylidae) from Uruguay in 2009 to 2012. This report is the first of dermocystids in Neotropical amphibians since 1940.
Amphibian fungal-like pathogens of the order Dermocystida have been known since the early 20th century (Pérez 1907). These parasites, grouped in the genus Amphibiocystidium, are present in Europe and North and South America and cause a disease that is recognized by the presence of characteristic skin nodules (González-Hernández et al. 2010). Their life cycle and the mechanisms of infection are virtually unknown (González-Hernández et al. 2010). In contrast, much research has focused on the chytrid fungus Batrachochytrium dendrobatidis (Chytridiomycota), which is associated with amphibian mortality and declines worldwide (Vredenburg et al. 2010). This fungus may cause skin erosions and hyperkeratosis (Voyles et al. 2009).
We present evidence of natural coinfection of both pathogens in frogs from Uruguay. While doing fieldwork in southern and eastern Uruguay to inventory amphibians, we observed three adult males of Hypsiboas pulchellus presenting a nodular skin disease. They were collected and transported individually to the laboratory, euthanized by cutaneous application of lidocaine 20%, fixed in formalin, and deposited at the herpetologic collection of Museo Nacional de Historia Natural de Montevideo (MNHN). Voucher specimens and collection data are the following: MNHN 9477, Valentines (33°15′S, 55°06′W) September 2009; MNHN 9478, La Paloma (34°38′S, 54°12′W) August 2011; MNHN 9479, Río Tacuarí (32°35′S, 54°04′W) August 2012 (Fig. 1). Skin samples were embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. Some nodules were fixed in 3% glutaraldehyde, embedded in epoxy resin, and ultrathin sections stained with lead citrate and uranyl acetate for electron microscope examination.
Hypsiboas pulchellus is a common species at the study sites, and we did not observe dead or moribund frogs. By the time studied specimens were collected, we could only capture a few additional individuals. The numbers of specimens with skin nodules/specimens captured at each site were 1/2, 1/1, and 1/15 for Valentines, La Paloma, and Río Tacuarí, respectively. Studied specimens (Fig. 2A) were collected while calling at breeding sites in temporary ponds and did not present abnormal postures or behavior; they were apparently in good condition according to their general aspect and muscle mass. Spherical nodules, some covered by ulcerated skin, were present mostly on the head and dorsum (MNHN 9477, 9479) or the belly (MNHN 9478). One specimen presented clusters of nodules in the skin over the urostyle (MNHN 9477). Nodules contained spherical cysts of approximately 520–540 µm (Fig. 2B) full of eosinophilic fungal-like microorganisms of 5–9 µm (Fig. 2C, D), and limited by a capsule (Fig. 2E). Vegetative reproductive structures were observed (Fig. 2C). The spores usually presented a large inclusion that pushed the cytoplasm and nucleus to the periphery (Fig. 2D). There were no evident inflammatory reactions in the surrounding tissue. Active B. dendrobatidis infection was observed in the three frog specimens, as determined by the presence of mature sporangia in histologic preparations (Fig. 2E).
The nodular disease observed is similar to previous reports of amphibian dermocystid skin infection (Pascolini et al. 2003; González-Hernández et al. 2010). Until new evidence is obtained, we tentatively refer to the parasites found in H. pulchellus as Amphibiocystidium sp. The only previous report of dermocystids in Neotropical amphibians is the description of Amphibiocystidium hylarum in frogs from southeastern Brazil (Carini 1940). Our specimens were collected approximately 1,500 km further south, suggesting a wide distribution of dermocystid infection in Neotropical amphibians. Infection by B. dendrobatidis in native amphibians is known from a few localities in northern and coastal southern Uruguay (Borteiro et al. 2009). Our findings of this pathogen at Valentines and Río Tacuarí are the first for central and northeastern Uruguay, respectively.
Coinfection of B. dendrobatidis with other eukaryotic skin pathogens has not been previously documented in free-living amphibians. Green and Kagarise Sherman (2001) identified B. dendrobatidis and Amphibiocystidium penneri in toads (Anaxyrus canorus) inhabiting the same site but did not observe a true coinfection. Groner and Relyea (2010) reported dermal cysts presumably due to Amphibiocystidium viridescens in populations of newts Notophthalmus viridescens infected by B. dendrobatidis but without further detail.
We are unaware on how dermocystid parasites and the coinfection with B. dendrobatidis can affect native amphibians in Uruguay. Further research is needed to study the possible interaction between the pathogens in amphibian skin and whether skin damage by B. dendrobatidis would facilitate dermocystid infection or vice versa.
We thank M.J. Sabalsagaray and J.C. Rosillo for assistance at the laboratory; T. Raffel and R. Tadeu kindly shared literature. We acknowledge financial support from Agencia Nacional de Investigación e Innovación and Japan International Cooperation Agency.