<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> and the Decline and Survival of the Relict Leopard Frog

Epizootic disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd) is a major driver of amphibian declines, yet many amphibians declined before the pathogen was described. The Relict Leopard Frog, Rana onca (=Lithobates onca), was nearly extinct, with the exception of populations within a few geothermal springs. Growth of Bd, however, is limited by high water temperature, and geothermal springs may have provided refuge during outbreaks of chytridiomycosis. We conducted field surveys and laboratory experiments to assess the susceptibility of R. onca to Bd. In the field, we found Bd at one of the two areas where remnant populations of R. onca still occur, but not in the other. In the laboratory, we infected juvenile frogs from these two areas with two hypervirulent Bd isolates associated with declines in other ranid species. In our experiments, these Bd isolates did not affect survivorship of R. onca and most infections (64%) were cleared by the end of the experiments. We propose that R. onca either has inherent resistance to Bd or has recently evolved such resistance. These results may be important for conservation efforts aimed at establishing new populations of R. onca across a landscape where Bd exists. Resistance, however, varies among life stages, and we also did not assess Bd from the local environment. We caution that the resistance we observed for young frogs under laboratory conditions may not translate to the situation for R. onca in the wild.     

Adaptive tolerance to a pathogenic fungus drives major histocompatibility complex evolution in natural amphibian populations

Amphibians have been affected globally by the disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), and we are just now beginning to understand how immunogenetic variability contributes to disease susceptibility. Lineages of an expressed major histocompatibility complex (MHC) class II locus involved in acquired immunity are associated with chytridiomycosis susceptibility in controlled laboratory challenge assays. Here, we extend these findings to natural populations that vary both in exposure and response to Bd. We find that MHC alleles and supertypes associated with Bd survival in the field show a molecular signal of positive selection, while those associated with susceptibility do not, supporting the hypothesis that heritable Bd tolerance is rapidly evolving. We compare MHC supertypes to neutral loci to demonstrate where selection versus demography is shaping MHC variability. One population with Bd tolerance in nature shows a significant signal of directional selection for the same allele (allele Q) that was significantly associated with survival in an earlier laboratory study. Our findings indicate that selective pressure for Bd survival drives rapid immunogenetic adaptation in some natural populations, despite differences in environment and demography. Our field-based analysis of immunogenetic variation confirms that natural amphibian populations have the evolutionary potential to adapt to chytridiomycosis.     

Variation in the Presence of Anti-<Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> Bacteria of Amphibians Across Life Stages and Elevations in Ecuador

Amphibian populations are decreasing worldwide due to a variety of factors. In South America, the chytrid fungus Batrachochytrium dendrobatidis (Bd) is linked to many population declines. The pathogenic effect of Bd on amphibians can be inhibited by specific bacteria present on host skin. This symbiotic association allows some amphibians to resist the development of the disease chytridiomycosis. Here, we aimed (1) to determine for the first time if specific anti-Bd bacteria are present on amphibians in the Andes of Ecuador, (2) to monitor anti-Bd bacteria across developmental stages in a focal amphibian, the Andean marsupial tree frog, Gastrotheca riobambae, that deposits larvae in aquatic habitats, and (3) to compare the Bd presence associated with host assemblages including 10 species at sites ranging in biogeography from Amazonian rainforest (450 masl) to Andes montane rainforest (3200 masl). We sampled and identified skin-associated bacteria of frogs in the field using swabs and a novel methodology of aerobic counting plates, and a combination of morphological, biochemical, and molecular identification techniques. The following anti-Bd bacteria were identified and found to be shared among several hosts at high-elevation sites where Bd was present at a prevalence of 32.5%: Janthinobacterium lividum, Pseudomonas fluorescens, and Serratia sp. Bd were detected in Gastrotheca spp. and not detected in the lowlands (sites below 1000 masl). In G. riobambae, recognized Bd-resistant bacteria start to be present at the metamorphic stage. Overall bacterial abundance was significantly higher post-metamorphosis and on species sampled at lower elevations. Further metagenomic studies are needed to evaluate the roles of host identity, life-history stage, and biogeography of the microbiota and their function in disease resistance.     

Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian

Ontogenetic changes in disease susceptibility have been demonstrated in many vertebrate taxa, as immature immune systems and limited prior exposure to pathogens can place less developed juveniles at a greater disease risk. By causing the disease chytridiomycosis, Batrachochytrium dendrobatidis (Bd) infection has led to the decline of many amphibian species. Despite increasing knowledge on how Bd varies in its effects among species, little is known on the interaction between susceptibility and development within host species. We compared the ontogenetic susceptibility of post-metamorphic green and golden bell frogs Litoria aurea to chytridiomycosis by simultaneously measuring three host-pathogen responses as indicators of the development of the fungus—infection load, survival rate, and host immunocompetence—following Bd exposure in three life stages (recently metamorphosed juveniles, subadults, adults) over 95 days. Frogs exposed to Bd as recently metamorphosed juveniles acquired higher infection loads and experienced lower immune function and lower survivorship than subadults and adults, indicating an ontogenetic decline in chytridiomycosis susceptibility. By corresponding with an intrinsic developmental maturation in immunocompetence seen in uninfected frogs, we suggest these developmental changes in host susceptibility in L. aurea may be immune mediated. Consequently, the physiological relationship between ontogeny and immunity may affect host population structure and demography through variation in life stage survival, and understanding this can shape management targets for effective amphibian conservation.     

Assessing the ability of swab data to determine the true burden of infection for the amphibian pathogen Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd) is a pathogenic fungus which causes the disease chytridiomycosis in amphibians by infecting the animals’ epidermis. The most commonly applied method for the detection of Bd is the use of a sterile swab, rubbed over the keratinized areas of an amphibian and then processed to yield DNA for detection by qPCR. This method has been used to infer a threshold of lethal infection in some species; however, how reliable and reproducible the swabbing method is at detecting the true burden of infection suffered by individuals is not known. European midwife toads, Alytes obstetricans, are susceptible to chytridiomycosis and are highly parasitised by Bd across Europe. By quantifying Bd-load throughout the entire skin and comparing this to swab results taken from the same individual, we determined whether epidermal swabs provide a quantifiable and accurate indication of the true fungal burden suffered. Further, we examined whether we could infer a threshold for lethal infection based on comparison of swab data taken from infected A. obstetricans exhibiting different clinical states. From swab data, we detected significantly higher fungal burdens from moribund metamorphs compared to visually healthy individuals; however, the ability of these swab data to provide an accurate indication of the true fungal burden was not reliable. These data suggest that fungal load dynamics play an important role in disease-induced mortality in A. obstetricans at these sites, but that using swab data to infer an exact threshold for Bd-associated mortality might be inappropriate and misleading.     

Diversity in growth patterns among strains of the lethal fungal pathogen <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> across extended thermal optima

The thermal sensitivities of organisms regulate a wide range of ecological interactions, including host–parasite dynamics. The effect of temperature on disease ecology can be remarkably complex in disease systems where the hosts are ectothermic and where thermal conditions constrain pathogen reproductive rates. Amphibian chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis (Bd), is a lethal fungal disease that is influenced by temperature. However, recent temperature studies have produced contradictory findings, suggesting that our current understanding of thermal effects on Bd may be incomplete. We investigated how temperature affects three different Bd strains to evaluate diversity in thermal responses. We quantified growth across the entire thermal range of Bd, and beyond the known thermal limits (T _max and T _min). Our results show that all Bd strains remained viable and grew following 24 h freeze (?12 °C) and heat shock (28 °C) treatments. Additionally, we found that two Bd strains had higher logistic growth rates (r) and carrying capacities (K) at the upper and lower extremities of the temperature range, and especially in low temperature conditions (2–3 °C). In contrast, a third strain exhibited relatively lower growth rates and carrying capacities at these same thermal extremes. Overall, our results suggest that there is considerable variation among Bd strains in thermal tolerance, and they establish a new thermal sensitivity profile for Bd. More generally, our findings point toward important questions concerning the mechanisms that dictate fungal thermal tolerances and temperature-dependent pathogenesis in other fungal disease systems.     

Perceived Risk of Avian Influenza and Urbanization in Northern Vietnam

Decision-analytic models provide forecasts of how systems of interest will respond to management. These models can be parameterized using empirical data, but sometimes require information elicited from experts. When evaluating the effects of disease in species translocation programs, expert judgment is likely to play a role because complete empirical information will rarely be available. We illustrate development of a decision-analytic model built to inform decision-making regarding translocations and other management actions for the boreal toad (Anaxyrus boreas boreas), a species with declines linked to chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd). Using the model, we explored the management implications of major uncertainties in this system, including whether there is a genetic basis for resistance to pathogenic infection by Bd, how translocation can best be implemented, and the effectiveness of efforts to reduce the spread of Bd. Our modeling exercise suggested that while selection for resistance to pathogenic infection by Bd could increase numbers of sites occupied by toads, and translocations could increase the rate of toad recovery, efforts to reduce the spread of Bd may have little effect. We emphasize the need to continue developing and parameterizing models necessary to assess management actions for combating chytridiomycosis-associated declines.     

Population genetic diversity and geographical differentiation of MHC class II DAB genes in the vulnerable Chinese egret (<Emphasis Type="Italic">Egretta eulophotes</Emphasis>)

Major histocompatibility complex (MHC) genes are excellent markers for the study of adaptive genetic variation occurring over different geographical scales. The Chinese egret (Egretta eulophotes) is a vulnerable ardeid species with an estimated global population of 2600–3400 individuals. In this study, we sampled 172 individuals of this egret (approximately 6 % of the global population) from five natural populations that span the entire distribution range of this species in China. We examined their population genetic diversity and geographical differentiation at three MHC class II DAB genes by identifying eight exon 2 alleles at Egeu-DAB1, eight at Egeu-DAB2 and four at Egeu-DAB3. Allelic distributions at each of these three Egeu-DAB loci varied substantially within the five populations, while levels of genetic diversity varied slightly among the populations. Analysis of molecular variance showed low but significant genetic differentiation among five populations at all three Egeu-DAB loci (haplotype-based ?_ST: 0.029, 0.020 and 0.042; and distance-based ?_ST: 0.036, 0.027 and 0.043, respectively; all P < 0.01). The Mantel test suggested that this significant population genetic differentiation was likely due to an isolation-by-distance pattern of MHC evolution. However, the phylogenetic analyses and the Bayesian clustering analysis based on the three Egeu-DAB loci indicated that there was little geographical structuring of the genetic differentiation among five populations. These results provide fundamental population information for the conservation genetics of the vulnerable Chinese egret.     

<Emphasis Type="Italic">Batrachochytrium salamandrivorans</Emphasis> and the Risk of a Second Amphibian Pandemic

Amphibians are experiencing devastating population declines globally. A major driver is chytridiomycosis, an emerging infectious disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Bd was described in 1999 and has been linked with declines since the 1970s, while Bsal is a more recently discovered pathogen that was described in 2013. It is hypothesized that Bsal originated in Asia and spread via international trade to Europe, where it has been linked to salamander die-offs. Trade in live amphibians thus represents a significant threat to global biodiversity in amphibians. We review the current state of knowledge regarding Bsal and describe the risk of Bsal spread. We discuss regional responses to Bsal and barriers that impede a rapid, coordinated global effort. The discovery of a second deadly emerging chytrid fungal pathogen in amphibians poses an opportunity for scientists, conservationists, and governments to improve global biosecurity and further protect humans and wildlife from a growing number of emerging infectious diseases.     

Genetic markers of adaptive processes in the Far Eastern pink salmon <Emphasis Type="Italic">Oncorhynchus gorbuscha</Emphasis>: Allelic diversity at the locus of major histocompatibility complex MHC I-<Emphasis Type="Italic">A1</Emphasis>

To clarify allelic diversity at the locus of major histocompatibility complex MHC class I-A1 in the Far Eastern pink salmon Oncorhynchus gorbuscha, sequencing of the electrophoretic alleles isolated from the gel (DGGE alleles) was performed. In 47 individuals, the genotypes of which consisted of ten DGGE alleles, 18 MHC I-A1 nucleotide sequences were revealed, and thus, eight cryptic alleles not detected by electrophoresis were identified. Eleven of these alleles were identified earlier in pink salmon from Hokkaido, Alaska, and British Columbia, and seven, possibly, were unique to the populations from some Far Eastern regions. Six of the previously determined DGGE alleles corresponded to more than one nucleotide sequence. However, the sequences attributed to the same DGGE allele differed on average by less than 1 nucleotide. These findings point to sufficient sensitivity of the DGGE method, although the genetic diversity and differentiation estimates obtained with it will obviously be somewhat underestimated. Considerable predominance of nonsynonymous substitutions over the synonymous ones in the codons of the MHC I-A1 antigen-binding site confirms the presence of positive selection aimed at providing the population resistance to local spectrum of pathogens. Refinement of the allelic composition of the adaptively important MHC genetic marker will contribute to more complete understanding of the adaptive genetic structure of pink salmon as an important element of the overall population structure of the species.     

External Reinfection of a Fungal Pathogen Does not Contribute to Pathogen Growth

Chytridiomycosis is an emerging infectious disease of amphibians caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has led to devastating declines in amphibian populations worldwide. Current theory predicts that Bd infections are maintained through both reproduction on the host’s skin and reinfection from sources outside of the host. To investigate the importance of external reinfection on pathogen burden, we infected captive-bred individuals of the highly susceptible Panamanian Golden Frog, Atelopus glyphus, and wild-caught glass frogs, Espadarana prosoblepon, with Bd. We housed the animals in one of three treatments: individually, in heterospecific pairs, and in conspecific pairs. For 8 weeks, we measured the Bd load and shedding rate of all frogs. We found that Atelopus had high rates of increase in both Bd load and shedding rate, but pathogen growth rates did not differ among treatments. The infection intensity of Espadarana co-housed with Atelopus was indistinguishable from those housed singly and those in conspecific pairs, despite being exposed to a large external source of Bd zoospores. Our results indicate that Bd load in both species is driven by pathogen replication within an individual, with reinfection from outside the host contributing little to the amplification of host fungal load.     

Spatiotemporal adaptive evolution of an MHC immune gene in a frog-fungus disease system

Genetic diversity of major histocompatibility complex (MHC) genes is linked to reduced pathogen susceptibility in amphibians, but few studies also examine broad spatial and temporal patterns of MHC and neutral genetic diversity. Here, we characterized range-wide MHC diversity in the Northern leopard frog, Rana pipiens, a species found throughout North America that is experiencing disease-related declines. We used previously sequenced neutral markers (mitochondrial DNA and microsatellites), sequenced an expressed MHC class IIß gene fragment, and measured infection prevalence and intensity of the global fungal pathogen Batrachochytrium dendrobatidis (Bd) across 14 populations. Four populations were sampled across two decades, enabling temporal comparisons of selection and demography. We recovered 37 unique MHC alleles, including 17 that were shared across populations. Phylogenetic and population genetic patterns between MHC and neutral markers were incongruent, and five MHC codon positions associated with peptide binding were under positive selection. MHC heterozygosity, but not neutral marker heterozygosity, was a significant factor explaining spatial patterns of Bd prevalence, whereas only environmental variables predicted Bd intensity. MHC allelic richness (AR) decreased significantly over time but microsatellite-based AR did not, highlighting a loss of functional immunogenetic diversity that may be associated with Bd selective pressures. MHC supertype 4 was significantly associated with an elevated risk of Bd infection, whereas one supertype 2 allele was associated with a nearly significant reduced risk of Bd. Taken together, these results provide evidence that positive selection contributes to MHC class IIß evolution in R. pipiens and suggest that functional MHC differences across populations may contribute to disease adaptation.Subject terms: Genetic variation, Immunogenetics     

Detection of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> in Amphibians Imported into the UK for the Pet Trade

There is increasing evidence that the global spread of the fungal pathogen Batrachochytrium dendrobatidis (Bd) has been facilitated by the international trade in amphibians. Bd was first detected in the UK in 2004, and has since been detected in multiple wild amphibian populations. Most amphibians imported into the UK for the pet trade from outside the European Union enter the country via Heathrow Animal Reception Centre (HARC), where Bd-positive animals have been previously detected. Data on the volume, diversity and origin of imported amphibians were collected for 59 consignments arriving at HARC between November 2009 and June 2012, along with a surveillance study to investigate the prevalence of Bd in these animals. Forty-three amphibian genera were recorded, originating from 12 countries. It was estimated that 5000–7000 amphibians are imported through HARC into the UK annually for the pet trade. Bd was detected in consignments from the USA and Tanzania, in six genera, resulting in an overall prevalence of 3.6%. This suggests that imported amphibians are a source of Bd within the international pet trade.     

Recent Emergence of a Chytrid Fungal Pathogen in California Cascades Frogs (<Emphasis Type="Italic">Rana cascadae</Emphasis>)

The pathogenic fungus Batrachochytrium dendrobatidis (Bd) has been associated with global amphibian declines, but it is often difficult to discern the relative importance of Bd as a causal agent in declines that have already occurred. Retrospective analyses of museum specimens have allowed researchers to associate the timing of Bd arrival with the timing of past amphibian declines. Cascades frogs (Rana cascadae) have experienced dramatic declines in northern California, but it is not clear whether the onset of these declines corresponds to the arrival of Bd. We used quantitative real-time PCR assays of samples collected from museum specimens to determine historical Bd prevalence in the northern California range of Cascades frogs. We detected Bd in 13 of 364 (3.5%) Cascades frog specimens collected between 1907 and 2003, with the first positive result from 1978. A Bayesian analysis suggested that Bd arrived in the region between 1973 and 1978, which corresponds well with the first observations of declines in the 1980s.     

Unraveling the historical prevalence of the invasive chytrid fungus in the Bolivian Andes: implications in recent amphibian declines

We studied the historical prevalence of the invasive and pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) among amphibians from the Bolivian Andes. Our aim was also to determine its geographic pattern of dispersion, and a potential host taxonomic signature. We collected frog tissue samples from nine museum collections covering a period from 1863 to 2005 and from the field during 2009–2016. Bd was diagnosed via quantitative PCR in 599 individuals of 17 genera and 54 species. We found an overall Bd prevalence of 41% among 44 species tested. The first incidence of Bd was from a Telmatobius culeus in 1863; this is the earliest report of detection for this pathogen in the world. Results reveal a non-random historical and geographical pattern of Bd occurrence and amphibian declines that suggests the presence of two different invasive strains, an ancient endemic and a more recent introduction. Prevalence of Bd increased significantly by the mid-1990s, particularly in the cloud-forests, and this is coincident with the timing of drastic amphibian declines. In contrast, amphibians occurring in drier altiplano habitats have persisted in spite of Bd presence. We hypothesize that the early 1990s, and the cloud-forests in central Bolivia were the center of an epidemic surge of Bd that took its toll on many species, especially in the genus Telmatobius. Further sampling of cloud-forest species, and ongoing genetic studies of Bd isolates from Bolivia should help resolve the history of this invasive pathogen and test hypotheses on the differential response of endangered hosts.     

Genetic associations between ADHD and dopaminergic genes (<Emphasis Type="Italic">DAT1</Emphasis> and <Emphasis Type="Italic">DRD4</Emphasis>) VNTRs in Korean children

It is well known that dopaminergic genes affect the development of attention deficit hyperactivity disorder (ADHD) in various populations. Many studies have shown that variable number tandem repeats (VNTRs) located within the 3′-untranslated region of DAT1 and in exon 3 of DRD4 are associated with ADHD development; however, these results were inconsistent. Therefore, we investigated the genetic association between two VNTRs and ADHD in Korean children. We determined the VNTRs using PCR. We examined genotype and allele frequency differences between the experimental and control groups, along with the odds ratios, using Chi square and exact tests. We observed a significant association between the children with ADHD and the control group in the 10R/10R genotype of DAT1 VNTRs (p?=?0.025). In addition, the 11R allele of DAT1 VNTRs showed a higher frequency in the control group than in the ADHD group (p?=?0.023). Also, the short repeat (without 11R) and long repeat alleles (including 11R) were associated with ADHD (p?<?0.05). The analysis of DRD4 VNTRs revealed that the 2R allele is associated with ADHD (p?=?0.025). A significant result was also observed in long and short repeats (p?<?0.05). Additionally, ADHD subtypes showed that the DRD4 VNTRs are associated with combined and hyperactive-impulsive subtype groups (p?<?0.05). Therefore, our results suggest that DAT1 VNTRs and DRD4 VNTRs play a role in the genetic etiology of ADHD in Korean children.     

Local and Temporal Variation at the MHC I-<Emphasis Type="Italic">A1</Emphasis> Locus in Pink Salmon <Emphasis Type="Italic">Oncorhynchus gorbuscha</Emphasis> of Southern Sakhalin

In order to test the MHC I-A1 locus of the main histocompatiblity complex as an effective molecular marker for identification of populations of Far Eastern pink salmon, Oncorhynchus gorbuscha, the comparison of the allelic and genotypic frequencies between two neighboring populations of the Lyutoga and Taranay rivers (Aniva Bay, Sakhalin Island), as well as within the populations: within generation, between adjacent and distant generations, have been performed. It was revealed that temporal variation at MHC I-A1 is statistically insignificant in comparison between the samples of the juveniles sampled at different times during the downstream migration. It is also insignificant between the samples of juvenile and adult fish of the same generation, and between samples divided by a single and seven generations. At the same time, the samples of neighboring populations differ significantly. The possibility of differentiation of the populations on the local scale and temporal stability of the allele frequencies indicate the MHC I-A1 as valuable marker for the development of the population data bases. Inclusion of the MHC I-A1 into the panel of other markers (microsatellites, in particular) allows for considerable increase in the resolution power for identification as to which population an individual in the mixed sea catch belongs.     

Invasion of the Fungal Pathogen <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> on California Islands

Batrachochytrium dendrobatidis (Bd), an amphibian fungal pathogen, has infected >500 species and caused extinctions or declines in >200 species worldwide. Despite over a decade of research, little is known about its invasion biology. To better understand this, we conducted a museum specimen survey (1910–1997) of Bd in amphibians on 11 California islands and found a pattern consistent with the emergence of Bd epizootics on the mainland, suggesting that geographic isolation did not prevent Bd invasion. We propose that suitable habitat, host diversity, and human visitation overcome isolation from the mainland and play a role in Bd invasion.     

Genetic diversity of MHC class II <Emphasis Type="Italic">DRB</Emphasis> alleles in the continental and Japanese populations of the sable <Emphasis Type="Italic">Martes zibellina</Emphasis> (Mustelidae,Carnivora, Mammalia)

The sable (Martes zibellina) is a medium-sized mustelid inhabiting forest environments in Siberia, northern China, the Korean Peninsula, and Hokkaido Island, Japan. To further understand the molecular evolution of the major histocompatibility complex (MHC), we sequenced part of exon 2 in MHC class II DRB genes, including codons encoding the antigen binding site, from 33 individuals from continental Eurasia and Japan. We identified 16 MHC class II DRB alleles (Mazi-DRBs), some of which were geographically restricted and others broadly distributed, and eight putative pseudogenes. A single-breakpoint recombination analysis detected a recombination site in the middle of exon 2. A mixed effects model of evolution analysis identified five amino acid sites presumably under positive selection. These sites were all located in the region 3′ to the recombination site, suggesting that positive selection and recombination could be committed to the diversity of the M. zibellina DRB gene. In a Bayesian phylogenetic tree, all Mazi-DRBs and the presumed pseudogenes grouped within a Mustelidae clade. The Mazi-DRBs showed trans-species polymorphism, with some alleles most closely related to alleles from other mustelid species. This result suggests that the sable DRBs have evolved under long-lasting balancing selection.