Tiffany A. Kosch

Evidence for acquisition of virulence effectors in pathogenic chytrids

BackgroundThe decline in amphibian populations across the world is frequently linked to the infection of the chytrid fungus Batrachochytrium dendrobatidis (Bd). This is particularly perplexing because Bd was only recently discovered in 1999 and no chytrid fungus had previously been identified as a vertebrate pathogen.ResultsIn this study, we show that two large families of known virulence effector genes, crinkler (CRN) proteins and serine peptidases, were acquired by Bd from oomycete pathogens and bacteria, respectively. These two families have been duplicated after their acquisition by Bd. Additional selection analyses indicate that both families evolved under strong positive selection, suggesting that they are involved in the adaptation of Bd to its hosts.ConclusionsWe propose that the acquisition of virulence effectors, in combination with habitat disruption and climate change, may have driven the Bd epidemics and the decline in amphibian populations. This finding provides a starting point for biochemical investigations of chytridiomycosis.

Sex-Chromosome Homomorphy in Palearctic Tree Frogs Results from Both Turnovers and X–Y Recombination

Contrasting with birds and mammals, poikilothermic vertebrates often have homomorphic sex chromosomes, possibly resulting from high rates of sex-chromosome turnovers and/or occasional X-Y recombination. Strong support for the latter mechanism was provided by four species of European tree frogs, which inherited from a common ancestor (∼ 5 Ma) the same pair of homomorphic sex chromosomes (linkage group 1, LG1), harboring the candidate sex-determining gene Dmrt1. Here, we test sex linkage of LG1 across six additional species of the Eurasian Hyla radiation with divergence times ranging from 6 to 40 Ma. LG1 turns out to be sex linked in six of nine resolved cases. Mapping the patterns of sex linkage to the Hyla phylogeny reveals several transitions in sex-determination systems within the last 10 My, including one switch in heterogamety. Phylogenetic trees of DNA sequences along LG1 are consistent with occasional X-Y recombination in all species where LG1 is sex linked. These patterns argue against one of the main potential causes for turnovers, namely the accumulation of deleterious mutations on nonrecombining chromosomes. Sibship analyses show that LG1 recombination is strongly reduced in males from most species investigated, including some in which it is autosomal. Intrinsically low male recombination might facilitate the evolution of male heterogamety, and the presence of important genes from the sex-determination cascade might predispose LG1 to become a sex chromosome.

Swabbing Often Fails to Detect Amphibian Chytridiomycosis under Conditions of Low Infection Load

The pathogenic chytrid fungus, Batrachochytrium dendrobatidis (denoted Bd), causes large-scale epizootics in naïve amphibian populations. Intervention strategies to rapidly respond to Bd incursions require sensitive and accurate diagnostic methods. Chytridiomycosis usually is assessed by quantitative polymerase chain reaction (qPCR) amplification of amphibian skin swabs. Results based on this method, however, sometimes yield inconsistent results on infection status and inaccurate scores of infection intensity. In Asia and other regions where amphibians typically bear low Bd loads, swab results are least reliable. We developed a Bd-sampling method that collects zoospores released by infected subjects into an aquatic medium. Bd DNA is extracted by filters and amplified by nested PCR. Using laboratory colonies and field populations of Bombina orientalis, we compare results with those obtained on the same subjects by qPCR of DNA extracted from swabs. Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected. Infection loads determined from filtered water were at least 1000 times higher than those estimated from swabs. Subjects significantly varied in infection load, as they intermittently released zoospores, over a 5-day period. Thus, the method might be used to compare the infectivity of individuals and study the periodicity of zoospore release. Sampling methods based on water filtration can dramatically increase the capacity to accurately diagnose chytridiomycosis and contribute to a better understanding of the interactions between Bd and its hosts.

Techniques for minimizing the effects of PCR inhibitors in the chytridiomycosis assay

Chytridiomycosis is an amphibian disease of global conservation concern that is caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Since the discovery of Bd in 1998, several methods have been used for detection of Bd; among these polymerase chain reaction (PCR) from skin swabs is accepted as the best method due to its noninvasiveness, high sensitivity and ease of use. However, PCR is not without problems – to be successful, this technique is dependent upon the presence of nondegraded DNA template and reaction contents that are free from inhibitors. Here, we report on an investigation of several techniques aimed at improving the reliability of the Bd PCR assay by minimizing the effects of humic acid (HA), a potent PCR inhibitor. We compared the effectiveness of four DNA extraction kits (DNeasy, QIAamp DNA Stool, PowerLyzer Power Soil and PrepMan Ultra) and four PCR methods (Amplitaq Gold, bovine serum albumin, PowerClean DNA Clean‐up and inhibitor resistant Taq Polymerase). The results of this and previous studies indicate that chytridiomycosis studies that use PCR methods for disease detection may be significantly underestimating the occurrence of Bd. Our results suggest that to minimize the inhibitory effects of HA, DNeasy should be used for sample DNA extraction and Amplitaq Gold with bovine serum albumin should be used for the Bd PCR assay. We also outline protocols tested, show the results of our methods comparisons and discuss the pros and cons of each method.

Characterization of MHC class IA in the endangered southern corroboree frog

Southern corroboree frogs (Pseudophryne corroboree) have declined to near extinction in the wild after the emergence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in southeastern Australia in the 1980s. A major captive breeding and reintroduction program is underway to preserve this iconic species, but improving resistance to B. dendrobatidis would help the wild population to be self-sustaining. Using 3′ and 5′ rapid amplification of complementary DNA ends (RACE), we characterized the major histocompatibility complex (MHC) class IA locus in this species. We then used sequences generated from RACE to design primers to amplify the peptide-binding region (PBR) of this functional genetic marker. Finally, we analysed the diversity, phylogeny, and selection patterns of PBR sequences from four P. corroboree populations and compared this with other amphibian species. We found moderately high MHC class IA genetic diversity in this species and evidence of strong positive and purifying selection at sites that are associated with putative PBR pockets in other species, indicating that this gene region may be under selection for resistance to Bd. Future studies should focus on identifying alleles associated with Bd resistance in P. corroboree by performing a Bd laboratory challenge study to confirm the functional importance of our genetic findings and explore their use in artificial selection or genetic engineering to increase resistance to chytridiomycosis.

Major histocompatibility complex selection dynamics in pathogen-infected túngara frog (Physalaemus pustulosus) populations

Pathogen-driven selection can favour major histocompatibility complex (MHC) alleles that confer immunological resistance to specific diseases. However, strong directional selection should deplete genetic variation necessary for robust immune function in the absence of balancing selection or challenges presented by other pathogens. We examined selection dynamics at one MHC class II (MHC-II) locus across Panamanian populations of the túngara frog, Physalaemus pustulosus, infected by the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). We compared MHC-II diversity in highland túngara frog populations, where amphibian communities have experienced declines owing to Bd, with those in the lowland region that have shown no evidence of decline. Highland region frogs had MHC variants that confer resistance to Bd. Variant fixation appeared to occur by directional selection rather than inbreeding, as overall genetic variation persisted in populations. In Bd-infected lowland sites, however, selective advantage may accrue to individuals with only one Bd-resistance allele, which were more frequent. Environmental conditions in lowlands should be less favourable for Bd infection, which may reduce selection for specific Bd resistance in hosts. Our results suggest that MHC selection dynamics fluctuate in túngara frog populations as a function of the favourability of habitat to pathogen spread and the vulnerability of hosts to infection.

Introduced bullfrogs are associated with increased Batrachochytrium dendrobatidis prevalence and reduced occurrence of Korean treefrogs

Bullfrogs, Lithobates catesbeianus, have been described as major vectors of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). Bd is widespread throughout the range of amphibians yet varies considerably within and among populations in prevalence and host impact. In our study, the presence of L. catesbeianus is correlated with a 2.5 increase in Bd prevalence in treefrogs, and the endangered Dryophytes suweonensis displays a significantly higher Bd prevalence than the more abundant D. japonicus for the 37 sites surveyed. In addition, the occurrence of L. catesbeianus was significantly correlated with a decrease in presence of D. suweonensis at sites. We could not determine if it is the presence of bullfrogs as competitors or predators that is limiting the distribution of D. suweonensis or whether this is caused by bullfrogs acting as a reservoir for Bd. However, L. catesbeianus can now be added to the list of factors responsible for the decline of D. suweonensis populations.

Selective constraint acting on TLR2 and TLR4 genes of Japanese Rana frogs

Toll-like receptors (TLRs) are an important component of innate immunity, the first line of pathogen defence. One of the major roles of TLRs includes recognition of pathogen-associated molecular patterns. Amphibians are currently facing population declines and even extinction due to chytridiomycosis caused by the Batrachochytrium dendrobatidis (Bd) fungus. Evidence from other vertebrates shows that TLR2 and TLR4 are involved in innate immunity against various fungi. Such genes therefore may play a functional role in amphibian-chytridiomycosis dynamics. Frogs from East Asia appear to be tolerant to Bd, so we examined the genetic diversity that underlies TLR2 and TLR4 from three Japanese Ranidae frog species, Rana japonica, R. ornativentris and R. tagoi tagoi (n = 5 per species). We isolated 27 TLR2 and 20 TLR4 alleles and found that these genes are evolutionarily conserved, with overall evidence supporting purifying selection. In contrast, site-by-site analysis of selection identified several specific codon sites under positive selection, some of which were located in the variable leucine rich repeat domains. In addition, preliminary expression levels of TLR2 and TLR4 from transcriptome data showed overall low expression. Although it remains unclear whether infectious pathogens are a selective force acting on TLRs of Japanese frogs, our results support that certain sites in TLRs of these species may have experienced pathogen-mediated selection.

The efficacy and pharmacokinetics of terbinafine against the frog-killing fungus (Batrachochytrium dendrobatidis)

Captive and wild amphibians are under threat of extinction from the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd). The antifungal drug terbinafine (TBF) is used by pet owners to treat Bd-infected frogs; however, it is not widely used in academic or zoological institutions due to limited veterinary clinical trials. To assess TBFs efficacy, we undertook treatment trials and pharmacokinetic studies to investigate drug absorption and persistence in frog skin; and then we correlated these data to the minimal lethal concentrations (MLC) against Bd. Despite an initial reduction in zoospore load, the recommended treatment (five daily 5 min 0.01% TBF baths) was unable to cure experimentally infected alpine tree frogs and naturally infected common eastern froglets. In vitro and in vivo pharmacokinetics showed that absorbed TBF accumulates in frog skin with increased exposure, indicating its suitability for treating cutaneous pathogens via direct application. The MLC of TBF for zoosporangia was 100 μg/ml for 2 h, while the minimal inhibitory concentration was 2 μg/ml, suggesting that the drug concentration absorbed during 5 min treatments is not sufficient to cure high Bd burdens. With longer treatments of five daily 30 min baths, Bd clearance improved from 12.5% to 50%. A higher dose of 0.02% TBF resulted in 78% of animals cured; however, clearance was not achieved in all individuals due to low TBF skin persistence, as the half-life was less than 2 h. Therefore, the current TBF regime is not recommended as a universal treatment against Bd until protocols are optimized, such as with increased exposure frequency.

Genetic potential for disease resistance in a critically endangered frog decimated by chytridiomycosis.

Southern corroboree frogs (Pseudophryne corroboree) have been driven to functional extinction in the wild after the emergence of the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd) in southeastern Australia in the 1980s. This species is currently maintained in a captive assurance colony and is managed to preserve the genetic diversity of the founding populations. However, it is unlikely that self-sustaining wild populations can be re-established unless Bd resistance increases. We performed a Bd-challenge study to investigate the association between genetic variants of the major histocompatibility complex class IA (MHC) and genome-wide single nucleotide polymorphisms (SNPs). We also investigated differences in Bd susceptibility among individuals and populations, and the genetic diversity and population genetic structure of four natural P. corroboree populations. We found several MHC alleles and SNPs associated with Bd infection load and survival, provide evidence of significant structure among populations, and identified population-level differences in the frequency of influential variants. We also detected evidence of positive selection acting on the MHC and a subset of SNPs as well as evidence of high genetic diversity in P. corroboree populations. We suggest that low interbreeding rates may have contributed to the demise of this species by limiting the spread of Bd resistance genes. However, our findings demonstrate that despite dramatic declines there is potential to restore high levels of genetic diversity in P. corroboree. Additionally, we show that there are immunogenetic differences among captive southern corroboree frogs, which could be manipulated to increase disease resistance and mitigate the key threatening process, chytridiomycosis.