Ana V. Longo

Seasonality of Batrachochytrium dendrobatidis infection in direct-developing frogs suggests a mechanism for persistence.

Batrachochytrium dendrobatidis (Bd), a disease-causing amphibian-specific fungus, is widely distributed in Puerto Rico, but is restricted to elevations above 600 m. The effect of this pathogen in the wild was studied by monitoring Eleutherodactylus coqui and E. portoricensis in 2 upland forests at El Yunque, a site characterized by historic population declines in the presence of chytridiomycosis. We tested a potential synergistic interaction between climate and Bd by measuring prevalence of infection and level of infection per individual sampled (number of zoospores), across the dry and wet seasons for 2 yr (between 2005 and 2007). Infection levels in adult frogs were significantly higher during the dry season in both species studied, suggesting a cyclic pattern of dry/ cool-wet/warm climate-driven synergistic interaction. These results are consistent with ex situ experiments in which E. coqui infected with Bd were more susceptible to chytridiomycosis when subjected to limited water treatments resembling drought. Long-term data on the prevalence of Bd in the populations studied versus intensity of infection in individual frogs provided contradictory information. However, the conflicting nature of these data was essential to understand the status of Bd in the species and geographical area studied. We conclude that in Puerto Rico, Bd is enzootic, and vulnerability of eleutherodactylid frogs to this pathogen is related to seasonal climatic variables. Our data suggest a mechanism by which this disease can persist in tropical frog communities without decimation of its hosts, but that complex interactions during severe droughts may lead to population crashes.

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.

ITS1 Copy Number Varies among Batrachochytrium dendrobatidis Strains: Implications for qPCR Estimates of Infection Intensity from Field-Collected Amphibian Skin Swabs

Genomic studies of the amphibian-killing fungus (Batrachochytrium dendrobatidis, [Bd]) identified three highly divergent genetic lineages, only one of which has a global distribution. Bd strains within these linages show variable genomic content due to differential loss of heterozygosity and recombination. The current quantitative polymerase chain reaction (qPCR) protocol to detect the fungus from amphibian skin swabs targets the intergenic transcribed spacer 1 (ITS1) region using a TaqMan fluorescent probe specific to Bd. We investigated the consequences of genomic differences in the quantification of ITS1 from eight distinct Bd strains, including representatives from North America, South America, the Caribbean, and Australia. To test for potential differences in amplification, we compared qPCR standards made from Bd zoospore counts for each strain, and showed that they differ significantly in amplification rates. To test potential mechanisms leading to strain differences in qPCR reaction parameters (slope and y-intercept), we: a) compared standard curves from the same strains made from extracted Bd genomic DNA in equimolar solutions, b) quantified the number of ITS1 copies per zoospore using a standard curve made from PCR-amplicons of the ITS1 region, and c) cloned and sequenced PCR-amplified ITS1 regions from these same strains to verify the presence of the probe site in all haplotypes. We found high strain variability in ITS1 copy number, ranging from 10 to 144 copies per single zoospore. Our results indicate that genome size might explain strain differences in ITS1 copy number, but not ITS1 sequence variation because the probe-binding site and primers were conserved across all haplotypes. For standards constructed from uncharacterized Bd strains, we recommend the use of single ITS1 PCR-amplicons as the absolute standard in conjunction with current quantitative assays to inform on copy number variation and provide universal estimates of pathogen zoospore loads from field-caught amphibians.

Disease Risk in Temperate Amphibian Populations Is Higher at Closed-Canopy Sites

Habitat loss and chytridiomycosis (a disease caused by the chytrid fungus Batrachochytrium dendrobatidis - Bd) are major drivers of amphibian declines worldwide. Habitat loss regulates host-pathogen interactions by altering biotic and abiotic factors directly linked to both host and pathogen fitness. Therefore, studies investigating the links between natural vegetation and chytridiomycosis require integrative approaches to control for the multitude of possible interactions of biological and environmental variables in spatial epidemiology. In this study, we quantified Bd infection dynamics across a gradient of natural vegetation and microclimates, looking for causal associations between vegetation cover, multiple microclimatic variables, and pathogen prevalence and infection intensity. To minimize the effects of host diversity in our analyses, we sampled amphibian populations in the Adirondack Mountains of New York State, a region with relatively high single-host dominance. We sampled permanent ponds for anurans, focusing on populations of the habitat generalist frog Lithobates clamitans, and recorded various biotic and abiotic factors that potentially affect host-pathogen interactions: natural vegetation, canopy density, water temperature, and host population and community attributes. We screened for important explanatory variables of Bd infections and used path analyses to statistically test for the strength of cascading effects linking vegetation cover, microclimate, and Bd parameters. We found that canopy density, natural vegetation, and daily average water temperature were the best predictors of Bd. High canopy density resulted in lower water temperature, which in turn predicted higher Bd prevalence and infection intensity. Our results confirm that microclimatic shifts arising from changes in natural vegetation play an important role in Bd spatial epidemiology, with areas of closed canopy favoring Bd. Given increasing rates of anthropogenic habitat modification and the resulting declines in temperate and tropical frogs, understanding how vegetation cover and disease interact is critical for predicting Bd spread and developing appropriate management tools for wild populations.

Seasonal and ontogenetic variation of skin microbial communities and relationships to natural disease dynamics in declining amphibians

Recently, microbiologists have focused on characterizing the probiotic role of skin bacteria for amphibians threatened by the fungal disease chytridiomycosis. However, the specific characteristics of microbial diversity required to maintain health or trigger disease are still not well understood in natural populations. We hypothesized that seasonal and developmental transitions affecting susceptibility to chytridiomycosis could also alter the stability of microbial assemblages. To test our hypothesis, we examined patterns of skin bacterial diversity in two species of declining amphibians (Lithobates yavapaiensis and Eleutherodactylus coqui) affected by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). We focused on two important transitions that affect Bd susceptibility: ontogenetic (from juvenile to adult) shifts in E. coqui and seasonal (from summer to winter) shifts in L. yavapaiensis. We used a combination of community-fingerprinting analyses and 16S rRNA amplicon sequencing to quantify changes in bacterial diversity and assemblage composition between seasons and developmental stages, and to investigate the relationship between bacterial diversity and pathogen load. We found that winter-sampled frogs and juveniles, two states associated with increased Bd susceptibility, exhibited higher diversity compared with summer-sampled frogs and adult individuals. Our findings also revealed that hosts harbouring higher bacterial diversity carried lower Bd infections, providing support for the protective role of bacterial communities. Ongoing work to understand skin microbiome resilience after pathogen disturbance has the potential to identify key taxa involved in disease resistance.

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.

Kinship, inbreeding and fine‐scale spatial structure influence gut microbiota in a hindgut‐fermenting tortoise

Herbivorous vertebrates rely on complex communities of mutualistic gut bacteria to facilitate the digestion of celluloses and hemicelluloses. Gut microbes are often convergent based on diet and gut morphology across a phylogenetically diverse group of mammals. However, little is known about microbial communities of herbivorous hindgut‐fermenting reptiles. Here, we investigate how factors at the individual level might constrain the composition of gut microbes in an obligate herbivorous reptile. Using multiplexed 16S rRNA gene sequencing, we characterized the faecal microbial community of a population of gopher tortoises (Gopherus polyphemus) and examined how age, genetic diversity, spatial structure and kinship influence differences among individuals. We recovered phylotypes associated with known cellulolytic function, including candidate phylum Termite Group 3, suggesting their importance for gopher tortoise digestion. Although host genetic structure did not explain variation in microbial composition and community structure, we found that fine‐scale spatial structure, inbreeding, degree of relatedness and possibly ontogeny shaped patterns of diversity in faecal microbiomes of gopher tortoises. Our findings corroborate widespread convergence of faecal‐associated microbes based on gut morphology and diet and demonstrate the role of spatial and demographic structure in driving differentiation of gut microbiota in natural populations.

Lability in Host Defenses: Terrestrial Frogs Die from Chytridiomycosis under Enzootic Conditions

Chytridiomycosis-induced mortalities often occur upon the emergence of Batrachochytrium dendrobatidis (Bd) in naïve amphibian populations. We report chytridiomycosis-associated mortalities in the wild of the coqui (Eleutherodactylus coqui), a declining direct-developing frog with persistent Bd infections. These findings provide additional evidence of decreased host defenses during cool-dry seasons in Puerto Rico.

Environmental fluctuations and host skin bacteria shift survival advantage between frogs and their fungal pathogen.

Fluctuating environments can modulate host–pathogen interactions by providing a temporary advantage to one of the interacting organisms. However, we know very little about how environmental conditions facilitate beneficial interactions between hosts and their microbial communities, resulting in individual persistence with a particular pathogen. Here, we experimentally infected Eleutherodactylus coqui frogs with the fungal pathogen Batrachochytrium dendrobatidis (Bd) under environmental conditions known to confer the survival advantage to the host during the warm-wet season, or alternatively to the pathogen during the cool-dry season. We used 16S rRNA amplicon sequencing to quantify changes in bacterial richness and phylogenetic diversity, and identified operational taxonomic units (OTUs) that became overrepresented or suppressed as a consequence of Bd infection. During the warm-wet season, frogs limited Bd infections, recruited putatively beneficial bacteria and returned to pre-infection levels of richness and phylogenetic diversity. In contrast, during the cool-dry season, Bd infections kept increasing through time, and bacterial diversity remained constant. Our findings confirm that infection outcome not only depends on abiotic factors, but also on biotic interactions between hosts and their associated bacterial communities.

Green Iguanas (Iguana iguana) in Puerto Rico: is it time for management?

Iguana iguana is native to Central and South America, and was introduced into Puerto Rico in the 1970s as a result of pet trade. The invasive biology of this reptile has not been studied in Puerto Rico, where its negative effects may threaten local biodiversity. The purposes of this study were to: (1) estimate population densities of I. iguana; (2) describe some aspects of its reproductive biology; and (3) assess its potential impacts. Visual-encounter surveys were performed at Parque Lineal in San Juan and Canal Blasina in Carolina, while nesting activity data were collected at Las Cabezas de San Juan in Fajardo. Densities of I. iguana in Puerto Rico reached a maximum of 223 individuals ha−1, higher than in any known locality in its native range, and showed fluctuations related to seasonality. Our 2008–2009 observations at the nesting sites document that this population of I. iguana is a reproductively successful species, producing more than 100 egg clutches and 2,558 eggs with a 91.4% egg viability. The ability to proliferate in a low predation environment and the absence of good competitors are the major drivers of the population densities observed in Puerto Rico. We found evidence that I. iguana is threatening native biodiversity and impacting infrastructure, agriculture and human safety. Thus, a management program to control the species must soon be developed to prevent this invasive reptile from becoming more widespread and dominant in other localities around the island.