First Survey of the Pathogenic Fungus Batrachochytrium dendrobatidis in Wild Populations of the Yunnan Caecilian (Ichthyophis bannanicus) in Guangxi, China

Batrachochytrium dendrobatidis (hereafter Bd) is a cause of chytridiomycosis, a disease that has led to the decline of at least 501 amphibian species, including 90 presumed extinctions (Scheele et al. 2019). With the aid of human activities and global trade, Bd has spread around the world rapidly and has been found on all continents except Antarctica (Liu et al. 2013; Olson et al. 2013). Initially Bd was thought to infect only anurans and caudates, but it has since been reported infecting caecilians (Gymnophiona) (Doherty-Bone et al. 2013; Gower et al. 2013). However, Bd field surveys of caecilians are rare, with infection known in only a dozen species (Doherty-Bone et al. 2013; Gower et al. 2013). The Yunnan caecilian (Ichthyophis bannanicus) is the only species of Gymnophiona in China (Pan et al. 1985). The population of I. bannanicus has declined noticeably (David et al. 2004), and it is classified as near threatened in the China Species Red List (Wang and Xie 2004). To date, only three (museum) specimens of I. bannanicus have been tested; these were negative (Zhu et al. 2014). Here we provide a survey and first report of Bd in free-living I. bannanicus.

We conducted our sampling in May–August 2019 at three locations in Guangxi Zhuang Autonomous Region, China: Bobai (22°19′49.26″N, 110°3′20.79″E), Cenxi (22°5′33.2″N, 110°52′53.59″E), and Beiliu (22°48′9.9″N, 110°17′43.93″E). A total of 71 adult I. bannanicus was collected. Each caecilian was placed in separate sterile sample bag with washed and bare hands, and kept for less than 1 h before swabbing. Each specimen was handled with fresh pairs of disposable gloves, rinsed with sterile water, and swabbed along the length of the body, contacting ventral, lateral, and dorsal surfaces with sterile cotton swabs 20 times. The swabs were placed in individual 1.5-mL microcentrifuge tubes with 70% ethyl alcohol and stored at –20 C until DNA extraction. We extracted DNA from swabs with the Rapid Fungi Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. We conducted nested PCR to amplify the Bdspecific internal transcribed spacer sequence (ITS)1–5.8S–ITS2 for each sample; PCR primers, reaction conditions, controls were as described previously (Wang et al. 2017), but reactions were made with minor modifications: total reaction volumes were 20 µL, consisting of 2 µL of DNA template, 1×PCR buffer, 0.4 mM of each dNTP, 0.5 mM of each primer, and 1.0 U of Taq DNA polymerase (Tiandz, Beijing, China).

A total of 6 of 71 samples tested positive for Bd: five from Bobai and one from Cenxi (Fig. 1). For further verification, all positive products were sent to a commercial company (Sangon Biotech) for sequencing by the Sanger sequencing method with primers Bd1a and Bd2a as previously described (Wang et al. 2017). Two haplotypes (GXBN-1 and GXBN-2) were identified from the six positive samples and were registered in GenBank (accession nos. MW093222 and MW093224). The GXBN-1 haplotype is completely identical to the CN18 haplotype previously reported in China (Bai et al. 2012), indicating that the Bd strain we found is not specific to caecilians. The GXBN-2 haplotype differs from GXBN-1 only at the 48th nucleotide (A or G). Blast analysis showed that the sequences of two haplotypes were 99% identical to the corresponding internal transcribed spacer sequences of Bd in GenBank (nos. MH745069, MG252075, MG601126), and the phylogenetic analysis further confirmed the samples are Bd (Fig. 2).

From nested PCR results and phylogenetic analysis, we concluded that Bd infection does exist in I. bannanicus in Guangxi, and the Bd strains are similar to the strains previously found in China (Bai et al. 2012).

We estimated infection prevalence at 8.4%, with a 95% confidence interval of 3–17%, which represents a low Bd infection prevalence and is similar to the overall prevalence (7.6%) in a previous study (Bai et al. 2012). None of the Bd-positive individuals showed signs of chytridiomycosis, which is consistent with other studies (Bai et al. 2012; Zhu et al. 2014, 2016). We propose some hypotheses that explain these results: 1) Seasonality. The optimal temperature for Bd growth in vitro is 17–25 C (Piotrowski et al. 2004). We sampled I. bannanicus in May, July, and August, when temperatures ranged from 25 C to 34 C, which is outside the range that supports the growth of Bd. The high temperatures inhibit Bd replication and may help protect I. bannanicus from chytridiomycosis. To exclude the influence of high temperature, we could attempt to sample in early spring in the future, when the temperature is more suitable for Bd, but it is difficult to find individuals during early spring because of hibernation and subterranean habits. 2) Co-evolution or low virulence of Bd strains. Batrachochytrium dendrobatidis potentially infected frogs (museum samples) as early as 1933 (Zhu et al. 2014), suggesting that Bd has been present in China for many years. Despite this, no clinical disease has been reported in any native amphibians in China. Thus, we hypothesize that Bd strains have established a commensal relationship with the native amphibians during a long period of co-evolution. It is also possible that Bd strains in China are of low virulence. 3) Host immunity. Previously, antimicrobial peptides (AMPs), symbiotic skin microbes, or skin sloughing have been reported to inhibit Bd infection (Cramp et al. 2014; Patocka et al. 2019; Kruger 2020). Several such antagonistic bacteria or AMPs may exist in I. bannanicus and inhibit Bd. Skin sloughing may be beneficial for I. bannanicus in the face of chytridiomycosis.

Bd has been hypothesized to have originated in East Asia (O'Hanlon et al. 2018).

Ichthyophis bannanicus is a very ancient Chinese species, and it has been infected by Bd. Further studies on Bd infection of I. bannanicus are important to understand the Bd Asian origin hypothesis.

Although Bd is widely distributed in China, there is no evidence that Bd is a novel pathogen presenting an immediate threat to Chinese amphibian populations. Nonetheless, more data are needed from other parts of the I. bannanicus range to assess fully the threat of Bd and whether this chytrid fungus needs to be considered in future conservation assessments. Future research should also explore whether skin secretions, skin sloughing, or microbial communities influence the presence or abundance of Bd on I. bannanicus.

We thank Yiming Li at the Chinese Academy of Sciences Institute of Zoology for providing Bd DNA and thank Caihong Bai for proofreading. This research was funded by the National Natural Science Foundation of China (32060239), the Natural Science Foundation of Guangxi (2018GXNSFBA281098), and the Basic Ability Improvement Project for young and middle-aged teachers in Guangxi universities (2019KY0589). We express our gratitude to EditSprings (https://www.editsprings.com/) for the expert linguistic services provided.