Carolina Lambertini

Disentangling host, pathogen, and environmental determinants of a recently emerged wildlife disease: lessons from the first 15 years of amphibian chytridiomycosis research

Abstract The amphibian fungal disease chytridiomycosis, which affects species across all continents, recently emerged as one of the greatest threats to biodiversity. Yet, many aspects of the basic biology and epidemiology of the pathogen, Batrachochytrium dendrobatidis (Bd), are still unknown, such as when and from where did Bd emerge and what is its true ecological niche? Here, we review the ecology and evolution of Bd in the Americas and highlight controversies that make this disease so enigmatic. We explore factors associated with variance in severity of epizootics focusing on the disease triangle of host susceptibility, pathogen virulence, and environment. Reevaluating the causes of the panzootic is timely given the wealth of data on Bd prevalence across hosts and communities and the recent discoveries suggesting co‐evolutionary potential of hosts and Bd. We generate a new species distribution model for Bd in the Americas based on over 30,000 records and suggest a novel future research agenda. Instead of focusing on pathogen “hot spots,” we need to identify pathogen “cold spots” so that we can better understand what limits the pathogens distribution. Finally, we introduce the concept of “the Ghost of Epizootics Past” to discuss expected patterns in postepizootic host communities.

Partitioning the net effect of host diversity on an emerging amphibian pathogen

The ‘dilution effect’ (DE) hypothesis predicts that diverse host communities will show reduced disease. The underlying causes of pathogen dilution are complex, because they involve non-additive (driven by host interactions and differential habitat use) and additive (controlled by host species composition) mechanisms. Here, we used measures of complementarity and selection traditionally employed in the field of biodiversity–ecosystem function (BEF) to quantify the net effect of host diversity on disease dynamics of the amphibian-killing fungus Batrachochytrium dendrobatidis (Bd). Complementarity occurs when average infection load in diverse host assemblages departs from that of each component species in uniform populations. Selection measures the disproportionate impact of a particular species in diverse assemblages compared with its performance in uniform populations, and therefore has strong additive and non-additive properties. We experimentally infected tropical amphibian species of varying life histories, in single- and multi-host treatments, and measured individual Bd infection loads. Host diversity reduced Bd infection in amphibians through a mechanism analogous to complementarity (sensu BEF), potentially by reducing shared habitat use and transmission among hosts. Additionally, the selection component indicated that one particular terrestrial species showed reduced infection loads in diverse assemblages at the expense of neighbouring aquatic hosts becoming heavily infected. By partitioning components of diversity, our findings underscore the importance of additive and non-additive mechanisms underlying the DE.

Amphibian-killing chytrid in Brazil comprises both locally endemic and globally expanding populations.

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is the emerging infectious disease implicated in recent population declines and extinctions of amphibian species worldwide. Bd strains from regions of disease‐associated amphibian decline to date have all belonged to a single, hypervirulent clonal genotype (Bd‐GPL). However, earlier studies in the Atlantic Forest of southeastern Brazil detected a novel, putatively enzootic lineage (Bd‐Brazil), and indicated hybridization between Bd‐GPL and Bd‐Brazil. Here, we characterize the spatial distribution and population history of these sympatric lineages in the Brazilian Atlantic Forest. To investigate the genetic structure of Bd in this region, we collected and genotyped Bd strains along a 2400‐km transect of the Atlantic Forest. Bd‐Brazil genotypes were restricted to a narrow geographic range in the southern Atlantic Forest, while Bd‐GPL strains were widespread and largely geographically unstructured. Bd population genetics in this region support the hypothesis that the recently discovered Brazilian lineage is enzootic in the Atlantic Forest of Brazil and that Bd‐GPL is a more recently expanded invasive. We collected additional hybrid isolates that demonstrate the recurrence of hybridization between panzootic and enzootic lineages, thereby confirming the existence of a hybrid zone in the Serra da Graciosa mountain range of Paraná State. Our field observations suggest that Bd‐GPL may be more infective towards native Brazilian amphibians, and potentially more effective at dispersing across a fragmented landscape. We also provide further evidence of pathogen translocations mediated by the Brazilian ranaculture industry with implications for regulations and policies on global amphibian trade.

Chytrid fungus acts as a generalist pathogen infecting species-rich amphibian families in Brazilian rainforests.

The fungus Batrachochytrium dendrobatidis (Bd) is among the main causes of declines in amphibian populations. This fungus is considered a generalist pathogen because it infects several species and spreads rapidly in the wild. To date, Bd has been detected in more than 100 anuran species in Brazil, mostly in the southern portion of the Atlantic forest. Here, we report survey data from some poorly explored regions; these data considerably extend current information on the distribution of Bd in the northern Atlantic forest region. In addition, we tested the hypothesis that Bd is a generalist pathogen in this biome. We also report the first positive record for Bd in an anuran caught in the wild in Amazonia. In total, we screened 90 individuals (from 27 species), of which 39 individuals (from 22 species) were Bd-positive. All samples collected in Bahia (2 individuals), Pernambuco (3 individuals), Pará (1 individual), and Minas Gerais (1 individual) showed positive results for Bd. We found a positive correlation between anuran richness per family and the number of infected species in the Atlantic forest, supporting previous observations that Bd lacks strong host specificity; of 38% of the anuran species in the Atlantic forest that were tested for Bd infection, 25% showed positive results. The results of our study exemplify the pandemic and widespread nature of Bd infection in amphibians.

Seasonal Variation in Population Abundance and Chytrid Infection in Stream-Dwelling Frogs of the Brazilian Atlantic Forest

Enigmatic amphibian declines were first reported in southern and southeastern Brazil in the late 1980s and included several species of stream-dwelling anurans (families Hylodidae and Cycloramphidae). At that time, we were unaware of the amphibian-killing fungus Batrachochytrium dendrobatidis (Bd); therefore, pollution, habitat loss, fragmentation and unusual climatic events were hypothesized as primary causes of these declines. We now know that multiple lineages of Bd have infected amphibians of the Brazilian Atlantic forest for over a century, yet declines have not been associated specifically with Bd outbreaks. Because stream-dwelling anurans occupy an environmental hotspot ideal for disease transmission, we investigated temporal variation in population and infection dynamics of three stream-adapted species (Hylodes asper, H. phyllodes, and Cycloramphus boraceiensis) on the northern coast of São Paulo state, Brazil. We surveyed standardized transects along streams for four years, and show that fluctuations in the number of frogs correlate with specific climatic variables that also increase the likelihood of Bd infections. In addition, we found that Bd infection probability in C. boraceiensis, a nocturnal species, was significantly higher than in Hylodes spp., which are diurnal, suggesting that the nocturnal activity may either facilitate Bd zoospore transmission or increase susceptibility of hosts. Our findings indicate that, despite long-term persistence of Bd in Brazil, some hosts persist with seasonally variable infections, and thus future persistence in the face of climate change will depend on the relative effect of those changes on frog recruitment and pathogen proliferation.

Low resistance to chytridiomycosis in direct-developing amphibians

Host-generalist pathogens sporadically infect naive hosts, potentially triggering epizootics. The waterborne fungus Batrachochytrium dendrobatidis (Bd) is linked to declines of hundreds of amphibian species with aquatic larvae. Although several population declines and extinctions attributed to Bd have been reported among cryptic species undergoing direct development away from water, epidemiological studies focused on these terrestrial frogs are lacking. Our field data support that terrestrial direct-developing hosts are less exposed to Bd during their ontogeny than species with aquatic larvae, and thus they might lack adaptive responses against waterborne chytrids. Using controlled laboratory experiments, we exposed wild-caught amphibian species with terrestrial and aquatic life histories to Bd and found that direct developers showed more rapid increases in infection loads and experienced higher mortality rates than species with aquatic larvae. Our findings provide novel information about host responses to generalist pathogens and specifically show that our focal direct developing species have low resistance to Bd infections. Finally, our results underscore that we should not ignore Bd as a potential threat to direct developing species simply because they are less exposed to Bd in nature; instead future amphibian conservation plans should include efforts to safeguard hundreds of direct-developing amphibian species globally.

Crossing the threshold: an amphibian assemblage highly infected with Batrachochytrium dendrobatidis in the southern Brazilian Atlantic forest

ABSTRACT The fungal infection caused by Batrachochytrium dendrobatidis (Bd) in amphibians is known to be lethal when infection intensity values exceed loads of 10,000 zoospores per individual. We investigated Bd infection intensity in 100 anurans of southern Brazil. Almost half of the individuals were infected and the intensity ranged from four to about 156,000 zoospore genomic equivalents. We found no clinical signs of chytridiomycosis and no evidence of mortality. However, we observed a reduction in the number of infected individuals with loads above 10,000 zoospores. This fact could be considered indirect evidence that individuals with high loads are removed from the population.

Development and worldwide use of a non-lethal and minimal population-level impact protocols for the isolation of chytrids from amphibians

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into sterile culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to interested researchers worldwide as part of the BiodivERsA project RACE – here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been widely applied across at least 5 continents, 23 countries and in 62 amphibian species, and have been successfully used to isolate chytrids in remote field locations. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this important group of emerging fungal pathogens.

Development and worldwide use of non-lethal, and minimal population-level impact, protocols for the isolation of amphibian chytrid fungi.

Parasitic chytrid fungi have emerged as a significant threat to amphibian species worldwide, necessitating the development of techniques to isolate these pathogens into culture for research purposes. However, early methods of isolating chytrids from their hosts relied on killing amphibians. We modified a pre-existing protocol for isolating chytrids from infected animals to use toe clips and biopsies from toe webbing rather than euthanizing hosts, and distributed the protocol to researchers as part of the BiodivERsA project RACE; here called the RML protocol. In tandem, we developed a lethal procedure for isolating chytrids from tadpole mouthparts. Reviewing a database of use a decade after their inception, we find that these methods have been applied across 5 continents, 23 countries and in 62 amphibian species. Isolation of chytrids by the non-lethal RML protocol occured in 18% of attempts with 207 fungal isolates and three species of chytrid being recovered. Isolation of chytrids from tadpoles occured in 43% of attempts with 334 fungal isolates of one species (Batrachochytrium dendrobatidis) being recovered. Together, these methods have resulted in a significant reduction and refinement of our use of threatened amphibian species and have improved our ability to work with this group of emerging pathogens.

Hybrids of amphibian chytrid show high virulence in native hosts

Hybridization of parasites can generate new genotypes with high virulence. The fungal amphibian parasite Batrachochytrium dendrobatidis (Bd) hybridizes in Brazil’s Atlantic Forest, a biodiversity hotspot where amphibian declines have been linked to Bd, but the virulence of hybrid genotypes in native hosts has never been tested. We compared the virulence (measured as host mortality and infection burden) of hybrid Bd genotypes to the parental lineages, the putatively hypovirulent lineage Bd-Brazil and the hypervirulent Global Pandemic Lineage (Bd-GPL), in a panel of native Brazilian hosts. In Brachycephalus ephippium, the hybrid exceeded the virulence (host mortality) of both parents, suggesting that novelty arising from hybridization of Bd is a conservation concern. In Ischnocnema parva, host mortality in the hybrid treatment was intermediate between the parent treatments, suggesting that this species is more vulnerable to the aggressive phenotypes associated with Bd-GPL. Dendropsophus minutus showed low overall mortality, but infection burdens were higher in frogs treated with hybrid and Bd-GPL genotypes than with Bd-Brazil genotypes. Our experiment suggests that Bd hybrids have the potential to increase disease risk in native hosts. Continued surveillance is needed to track potential spread of hybrid genotypes and detect future genomic shifts in this dynamic disease system.