Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists

Abstract

The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a "marine" protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.

Keywords: alveolates; emerging disease; frog decline; molecular diversity; parasite.

Fig. 1.

(A) Phylogenetic tree of Perkinsea SSU rDNA sequences focusing on the NAG01 group that includes two separate phylogenetic groups recovered from tadpole liver tissue samples. The phylogeny is estimated from a masked alignment consisting of 292 taxa and 776 characters. Bayesian posterior probability (6,000 samples from 2,000,000 MCMCMC generations), LogDet distance bootstrap (1,000 pseudoreplicates), and maximum likelihood bootstrap (1,000 pseudoreplicates) values are added to each node using the following convention: support values are summarized by black circles when all are equal to or greater than 0.9/80%/80%, and white circles when the topology support is less but equal to or greater than 0.6/50%/50%. Five sequences of Amoebophrya sp. were used as outgroup. Each square represents one environmental operational taxonomic unit (OTU), and the provenance of the OTUs is indicated by colored boxes (see key for the detail of sample provenance) (SI Appendix, Table S2 provides more details on the environments sampled). A red diamond indicates the individual clone sequences from the L. sphenocephalus 2006 mass mortality event in Georgia (United States) (12). A subset of the published environmental sequences have been reduced to representative triangles (see SI Appendix, Table S10 for detail of each environmental clade). (B) Representation of the V4 hyper-variable region of the template SSU rDNA and the relative position of the different primers used in this study (not to scale). (C) Histogram representing the percentage of clones per clade A, B, and C within each host superfamily from infected tadpoles. n₁ represents the number of total clones sequenced per host superfamily/number of infected tadpoles per host superfamily. (D) Geographical distribution of clade A, B, and C. Pie charts represent the proportion of clones per clade A, B, and C in each of the five geographical locations where NAG01 was detected (United Kingdom, French Guiana, São Tomé, Cameroon, and Tanzania). n₂ represents the number of clones sequenced per geographical location/number of infected tadpoles per geographical location.

Fig. 2.

A maximum likelihood phylogenetic circle tree (inverted) showing clades A, B, and C and demonstrating the provenance of NAG01 phylotypes detected in tadpole liver tissue. The RAxML phylogeny is estimated from a masked alignment consisting of 177 NAG01 sequences and 806 characters from infected tadpoles. Branches proportionally shortened by 1/2 are labeled with a double-slashed line. Each clone sequence detected from the same liver sample is connected across the central circle. The colors of the connected lines were defined by the geographical location of tadpoles sampled (see the key). Black lines on the branches mark sequence variation confirmed across multiple samples and therefore cannot be the product of PCR error during clone library construction.

Fig. 3.

Bayesian 16S rDNA phylogenetic tree of tadpole diversity sampled in this study, with histograms showing prevalence of NAG01 detection. The phylogeny is inferred from a masked alignment consisting of 247 taxa and 440 characters. Bayesian posterior probability (6,000 samples from 2,000,000 MCMCMC generations), LogDet distance bootstrap (1,000 replicates), and maximum likelihood bootstrap (1,000 replicates). Support values are summarized by black circles when all are equal to or greater than 0.9/80%/80%, and a white circle when topology support is weaker but all values are equal to or greater than 0.6/50%/50%. Sequences of Ambystoma sp. and Pleurodeles sp. (salamanders) were used as outgroup. Some frog species with multiple nonidentical 16S rDNA sequences recorded in GenBank are retained. The color-coded histogram represents the number of NAG01-negative tadpole samples (uncolored bars) and the number of NAG01-positive samples (colored bars). Each color corresponds to the tadpole’s country of origin as detailed in the key. The superfamily and suborder of the tadpoles tested is indicated on the histogram. The circled star indicates the host species described by Davis et al. (12) during the 2006 mortality event.