Introduction

Global loss of biodiversity is one of the most serious problems of our time, with Amphibia being the most affected vertebrate class [1, 2]. Habitat loss, introduction of exotic species, environmental contamination, increased UV radiation, climate change and infectious diseases have been identified as major causes of amphibian population declines [3–17]. Chytridiomycosis, an infectious fungal disease caused by Batrachochytrium dendrobatidis (Bd) [15], has been linked to many incidents of amphibian mass mortality worldwide [15,18,19], and is considered one of the greatest causes of global amphibian declines [20]. In amphibians, Bd infection occurs only in keratinized tissues, which are restricted to the oral region (jaw sheaths and teeth) of tadpoles, and the epidermis of metamorphs and adults [15, 21–24].

Several studies have established a relationship between Bd infection and the occurrence of anomalies in the oral region of tadpoles of a number of different amphibian species. Fellers et al. [25] found that 67% of Rana muscosa tadpoles with abnormally keratinized mouthparts were infected by Bd. In turn, Knapp and Morgan [26] found for the same species that 89% of tadpoles with less than 90% jaw sheath pigmentation were infected. This relationship was later reinforced by Drake et al. [27], who found clear nonrandom relationships between oral deformities and Bd presence in Lithobates sphenocephalus (= Rana sphenocephala) tadpoles. A similar result was reported for Hylodes japi tadpoles, for which 94.5% of infected individuals possessed depigmented mouthparts [28]. These results suggest the possibility of using tadpole oral deformities as a general proxy for Bd detection in tadpoles, as has been previously suggested by Fellers et al. [25] and recently applied by Carvalho et al. [29].

Despite this promising proxy for which only a quick visual inspection is required, there are many studies that have indicated that oral deformities are not always related to Bd infection. For example, no relationship was found between depigmentation of mouthparts and Bd infection of Lithobates catesbeianus (= Rana catesbeiana) and Pseudacris regilla [30]. Furthermore, a laboratory study found no differences in the proportions of tadpoles with mouthpart abnormalities between Bd infected and uninfected individuals of Anaxyrus boreas (= Bufo boreas) and Pseudacris regilla (= Hyla regilla) [31]. In addition, many other factors have been associated with oral deformities in tadpoles, including low temperatures [32], water contamination [33], nutrition [21], competition [34], and predation risk [35]. Herein we quantitatively evaluate, in detail, the use of oral deformities as a reliable proxy for determination of Bd infection in six anuran species from southeastern Brazil.

Material and methods

Identification of oral deformities

It is important to emphasize that studies regarding the relationship between Bd infection and oral deformities have used different, and sometimes ambiguous terms for describing the deformities. For example, while some of studies describe oral deformities as depigmentation of mouthparts (e.g. [28, 30]), others refer to them as dekeratinization (e.g. [27, 31]). This ambiguity was recognized by Altig [37], who pointed out that the “depigmentation” as described in some studies was, in fact, dekeratinization of mouthparts. Therefore, we have chosen to use the term dekeratinization of mouthparts.

All individuals studied were between Gosner stages 25 and 40, since the oral disc of tadpoles within these stages normally possesses completely keratinized tissues [24, 38]. Oral discs of tadpoles were examined in detail using a stereoscopic microscope (Leica 54 MZ75), and tadpoles were classified in accordance with dekeratinization (from partial to complete absence of keratinized structures) that presented in mouthparts: JS (tadpoles with lack of keratinization only in upper and/or lower jaw sheath), TR (tadpoles with lack of keratinization, with non-disrupted supporting tissue, only in upper and/or lower tooth rows), and JT (tadpoles with lack of keratinization in jaw sheaths and tooth rows) (Fig 1). All tadpole specimens were deposited in the Amphibia—Tadpoles collection of the Department of Zoology and Botany of UNESP–São José do Rio Preto (DZSJRP-Amphibia-Tadpoles).

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Fig 1. Example of oral deformities found among the studied tadpoles.

(A) and (B) = normal tadpoles; (C) = tadpole with dekeratinized jaw sheaths (black arrows) and generalized dekeratinization in tooth rows; (D) = tadpole with dekeratinized upper jaw sheath (black arrow). Upper images Boana albopunctata, and lower images Scinax hayii.

https://doi.org/10.1371/journal.pone.0190955.g001

Results

The tadpoles studied belonged to six different species: Aplastodiscus albosignatus (N = 721), Boana albopunctata (N = 793), Boana faber (N = 467), Scinax hayii (N = 80), Crossodactylus caramaschii (N = 34) and Physalaemus cuvieri (N = 61). At least one type of oral deformity was found in 9% (195) of the 2156 tadpoles inspected. Of the tadpoles that exhibited oral deformities, 51.3% had JT, while 29.2% had only TR, and 19.5% only JS.

There was no relationship between Bd infection and tooth rows dekeratinization (TR) in any of studied of species (Table 1). However, dekeratinization of the jaw sheath, accompanied or not with dekeratinization of teeth (JS or JT), was related to Bd infection in three of the species studied: A. albosignatus showed a nonrandom relationship with JS (Fisher’s exact test; N = 197, two-tailed p = 0.0023) and JT (N = 197, two-tailed p < 0.001); B. albopunctata showed nonrandom relationship with JT (N = 63, two-tailed p < 0.001); and S. hayii showed nonrandom relationships with JS (N = 32, two-tailed p = 0.0068). By contrast, B. faber, C. caramaschii and P. cuvieri did not exhibit relationship between any of the oral deformities studied and Bd infection (Table 1).

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Table 1. Oral deformities and related Batrachochytrium dendrobatidis (Bd) infections of tadpoles of six species of Atlantic Forest anurans in southeastern Brazil.

https://doi.org/10.1371/journal.pone.0190955.t001

In the light of these results, we decided to sum JS and JT into a single proxy, JSD-proxy (JSD = jaw sheaths dekeratinization with or without dekeratinization in tooth rows), in order to simplify the determination of oral dekeratinization of tadpoles in future studies. Same statistical analyses performed for TR, JS and JT were then done for this JSD-proxy. The results showed that JSD-proxy considerably improved the accuracy of identifying Bd infection. Our original proxies (JS, TR and JT) included 33.8% false positives and 20.1% false negatives for Bd infection; the JSD-proxy, on the other hand, included only 12.3% false positives and 18.7% false negatives (see details in Table 2). False positives corresponded to tadpoles that possessed oral deformities but were negative for Bd infection via qPCR analysis. False negatives, on the other hand, corresponded to tadpoles that possessed normal oral mouthparts but were positive for Bd infection via qPCR.

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Table 2. Cases of false positives and false negatives for the detection of Batrachochytrium dendrobatidis (Bd) infection produced by the use of the three oral deformities studied as proxies (JS, TR and JT) and for the JSD-proxy.

https://doi.org/10.1371/journal.pone.0190955.t002

Although the use of JSD-Proxy was more accurate at identifying Bd infection in tadpoles than our original proxies, the analyses of the relationships between Bd infection and JSD-proxy found a nonrandom relationship only for three species of the family Hylidae: A. albosignatus (Fisher’s exact test; N = 197, two-tailed p < 0.001), B. albopunctata (N = 63, two-tailed p<0.001), and S. hayii (N = 32, two-tailed p < 0.001) (see details in Table 1).

Discussion

This is the first study to report the presence of Bd in Núcleo Curucutu, one of the largest and most preserved remnants of Atlantic Forest in São Paulo State, which is also a refuge to one of the most biodiverse amphibian faunas of a single locality [41]. Therefore, this virtually pristine area is added to the many others around the world that possess Bd (e.g. [18, 42]).

Not all of the tadpoles that possessed oral deformities were infected by Bd, and so some of the factors that caused the deformities in our studied tadpoles remain unknown and deserve further investigation. Since our study area is a very well preserved remnant of Atlantic Forest, we considered that one might rule out chemical contamination as the cause of deformities. More importantly, our results showed that TR was the worst proxy for Bd infection; in fact, of the 57 tadpoles exhibited only TR deformities, only 8 (14%) were infected with Bd. On the other hand, of the 138 tadpoles possessing dekeratinization of the jaw sheaths, accompanied or not with dekeratinization of the teeth, 121 (87.7%) were infected. These results are similar to those obtained by Knapp and Morgan [26] and Vieira et al. [28], who also found a strong correlation between jaw sheaths dekeratinization and Bd infection. This finding is also consistent with Marantelli et al. [24], who found that the dekeratinization of jaw sheaths indicates a heavier infection than dekeratinization of tooth rows.

Despite the fact that the analyses of all studied tadpoles together found a strong relationship between tadpoles possessing dekeratinization of jaw sheaths and Bd infection, the results by species were less clear. Only three species of the family Hylidae exhibited a significant relationship between JSD-Proxy and Bd infection, which was not observed for B. faber (Hylidae), C. caramaschii (Hylodidae) and P. cuvieri (Leptodactylidae). These results agree with Blaustein et al. [31], who also detected varying relationships between Bd infection and oral deformities among species. Therefore, and in discordance with Padget-Flohr and Goble [30], who concluded that Bd infection and anuran larval mouthpart deformities are two separate processes, we found that a relationship between dekeratinization in jaw sheaths and Bd infection does indeed exist in three species of family Hylidae.

Although the relationship between jaw sheaths and Bd infection was found for some of our studied species, the use of JSD-proxy remains unreliable for purposes of assessing Bd prevalence in tadpoles populations. Based on this proxy, Bd detection in species that exhibited a positive relationship with jaw sheaths dekeratinization resulted in up to 30.8% false positives in B. albopunctata and up to 29.3% false negatives in A. albosignatus. Furthermore, the use of the JSD-proxy in species for which no relationship was found, reached 100% of false positives. Further illustrating the unreliability of JSD-proxy, in an open area pond where we sampled 23 tadpoles of B. albopunctata (two of them with dekeratinized jaw sheaths), the use of JSD-proxy would have classified the pond as a Bd positive site although qPCR analyses did not confirm this assumption.

Thus, we conclude that the use of oral dekeratinization as a generalized proxy for Bd detection in tadpoles should not be used as a single diagnosis technique. We then recommend the use of more accurate techniques such as histology, histochemistry, or PCR analyses in order to obtain accurate diagnosis. However, it is worth noting the usefulness of using JSD-proxy as a screening tool in those species for which the relationship between Bd-infection and oral dekeratinization has been proven, since its use minimize the number of individuals evaluated by more costly or time-intensive methods.

Acknowledgments

We are in debt to FS Annibale, LR Malagoli, CE Sousa and D Garcia for their help in fieldwork, K Picheli for her help with tadpole identification and C Monsalve for her help with qPCR analyses.