Patricia A. Burrowes

POTENTIAL CAUSES FOR AMPHIBIAN DECLINES IN PUERTO RICO

We monitored 11 populations of eight species of Eleutherodactylus in Puerto Rico from 1989 through 2001. We determined relative abundance of active frogs along transects established in the Caribbean National Forest (El Yunque), Carite Forest, San Lorenzo, and in the vicinity of San Juan. Three species (Eleutherodactylus karlschmidti, E. jasperi, and E. eneidae) are presumed to be extinct and eight populations of six different species of endemic Eleutherodactylus are significantly declining at elevations above 400 m. Of the many suspected causes of amphibian declines around the world, we focused on climate change and disease. Temperature and precipitation data from 1970–2000 were analyzed to determine the general pattern of oscillations and deviations that could be correlated with amphibian declines. We examined a total of 106 tissues taken from museum specimens collected from 1961–1978 and from live frogs in 2000. We found chytrid fungi in two species collected at El Yunque as early as 1976, this is the first report of chytrid fungus in the Caribbean. Analysis of weather data indicates a significant warming trend and an association between years with extended periods of drought and the decline of amphibians in Puerto Rico. The 1970s and 1990s, which represent the periods of amphibian extirpations and declines, were significantly drier than average. We suggest a possible synergistic interaction between drought and the pathological effect of the chytrid fungus on amphibian populations.

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.

A fungal pathogen of amphibians, Batrachochytrium dendrobatidis, attenuates in pathogenicity with in vitro passages.

Laboratory investigations into the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), have accelerated recently, given the pathogen’s role in causing the global decline and extinction of amphibians. Studies in which host animals were exposed to Bd have largely assumed that lab-maintained pathogen cultures retained the infective and pathogenic properties of wild isolates. Attenuated pathogenicity is common in artificially maintained cultures of other pathogenic fungi, but to date, it is unknown whether, and to what degree, Bd might change in culture. We compared zoospore production over time in two samples of a single Bd isolate having different passage histories: one maintained in artificial media for more than six years (JEL427-P39), and one recently thawed from cryopreserved stock (JEL427-P9). In a common garden experiment, we then exposed two different amphibian species, Eleutherodactylus coqui and Atelopus zeteki, to both cultures to test whether Bd attenuates in pathogenicity with in vitro passages. The culture with the shorter passage history, JEL427-P9, had significantly greater zoospore densities over time compared to JEL427-P39. This difference in zoospore production was associated with a difference in pathogenicity for a susceptible amphibian species, indicating that fecundity may be an important virulence factor for Bd. In the 130-day experiment, Atelopus zeteki frogs exposed to the JEL427-P9 culture experienced higher average infection intensity and 100% mortality, compared with 60% mortality for frogs exposed to JEL427-P39. This effect was not observed with Eleutherodactylus coqui, which was able to clear infection. We hypothesize that the differences in phenotypic performance observed with Atelopus zeteki are rooted in changes of the Bd genome. Future investigations enabled by this study will focus on the underlying mechanisms of Bd pathogenicity.

Susceptibility to the amphibian chytrid fungus varies with ontogeny in the direct-developing frog, Eleutherodactylus coqui.

Abstract Age-related differences in susceptibility to infectious disease are known from a wide variety of plant and animal taxonomic groups. For example, the immature immune systems of young vertebrates, along with limited prior exposure to pathogens and behavioral factors, can place juveniles at greater risk of acquiring and succumbing to a pathogen. We studied the ontogenetic susceptibility of terrestrial direct-developing frogs (Eleutherodactylus coqui) to the fungal pathogen, Batrachochytrium dendrobatidis (Bd), which is responsible for the decline of amphibian species worldwide. By exposing juvenile and adult frogs to the same dose and strain of Bd, we uncovered ontogenetic differences in susceptibility. Froglets exposed to the pathogen had significantly lower survival rates compared with control froglets, while adult frogs largely cleared infection and had survival rates indistinguishable from control frogs, even when exposed to a much higher dose of Bd. The high disease-induced mortality rate of juveniles may explain ongoing population declines in eastern Puerto Rico, where Bd is endemic and juveniles experience higher prevalence and infection intensity compared to adults. Our results have important implications for understanding and modeling the decline, possibly to extinction, of amphibian populations and species.

Teaching Biology through Statistics: Application of Statistical Methods in Genetics and Zoology Courses

Incorporation of mathematics into biology curricula is critical to underscore for undergraduate students the relevance of mathematics to most fields of biology and the usefulness of developing quantitative process skills demanded in modern biology. At our institution, we have made significant changes to better integrate mathematics into the undergraduate biology curriculum. The curricular revision included changes in the suggested course sequence, addition of statistics and precalculus as prerequisites to core science courses, and incorporating interdisciplinary (math–biology) learning activities in genetics and zoology courses. In this article, we describe the activities developed for these two courses and the assessment tools used to measure the learning that took place with respect to biology and statistics. We distinguished the effectiveness of these learning opportunities in helping students improve their understanding of the math and statistical concepts addressed and, more importantly, their ability to apply them to solve a biological problem. We also identified areas that need emphasis in both biology and mathematics courses. In light of our observations, we recommend best practices that biology and mathematics academic departments can implement to train undergraduates for the demands of modern biology.

Genomic Correlates of Virulence Attenuation in the Deadly Amphibian Chytrid Fungus, Batrachochytrium dendrobatidis.

Emerging infectious diseasespose a significant threat to global health, but predicting disease outcomes for particular species can be complicated when pathogen virulence varies across space, time, or hosts. The pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused worldwide declines in frog populations. Not only do Bd isolates from wild populations vary in virulence, but virulence shifts can occur over short timescales when Bd is maintained in the laboratory. We leveraged changes in Bd virulence over multiple generations of passage to better understand mechanisms of pathogen virulence. We conducted whole-genome resequencing of two samples of the same Bd isolate, differing only in passage history, to identify genomic processes associated with virulence attenuation. The isolate with shorter passage history (and greater virulence) had greater chromosome copy numbers than the isolate maintained in culture for longer, suggesting that virulence attenuation may be associated with loss of chromosome copies. Our results suggest that genomic processes proposed as mechanisms for rapid evolution in Bd are correlated with virulence attenuation in laboratory culture within a single lineage of Bd. Moreover, these genomic processes can occur over extremely short timescales. On a practical level, our results underscore the importance of immediately cryo-archiving new Bd isolates and using fresh isolates, rather than samples cultured in the laboratory for long periods, for laboratory infection experiments. Finally, when attempting to predict disease outcomes for this ecologically important pathogen, it is critical to consider existing variation in virulence among isolates and the potential for shifts in virulence over short timescales.

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.

Conserving the Puerto Rican herpetofauna

With a total area of 8900 km2, Puerto Rico is the smallest of the Greater Antilles. It is divided in three physiographic regions or areas of relief: the mountainous interior, the karst region, and the coastal plains and valleys. The island comprises six ecological life zones: subtropical dry forest, subtropical moist forest, subtropical wet forest, subtropical rain forest, lower montane wet forest and lower montane rain forest. The herpetofauna of Puerto Rico consists of 25 species of amphibians (19 native, six introduced) and 56 species of reptiles (52 native, four introduced). The goal of this paper is to describe some of the present studies directed towards the conservation of Puerto Rican herpetofauna. Eleutherodactylus karlschmidti, E. jasperi and E. eneidae have not been seen or heard since 1976, 1981 and 1990, respectively, and are probably extinct. Since 2000, the potential causes of amphibian declines in Puerto Rico have been studied, and a synergistic interaction between climate change (increased dry periods) and disease (chytridiomycosis) have been proposed as an explanation for the patterns observed. Recovery efforts for Peltophryne lemur include a captive-breeding program, reintroductions island-wide educational outreach, protection and restoration of existing habitat, and the creation of new breeding ponds. Among reptiles, the first conservation efforts to protect Epicrates inornatus were limited to trying to halt collection and hunting. However, current strategies to preserve the boa include gathering basic biological information, habitat conservation, and educational outreach. Recent efforts for the conservation of Trachemys s. stejnegeri combine three research approaches to clarify the status of local populations: a mark-recapture-release study, field monitoring of reproductive activity (i. e., nocturnal patrolling to identify nesting activity), and field assessment of the potential impact of introduced species, particularly identification of predatory species and exotic turtles. Recovery initiatives for Cyclura stejnegeri include management of invasive mammals, a headstart program for hatchling iguanas, and the assessment of the etiology of a condition causing blindness in adult iguanas. A reforestation project aimed at recovering a local herpetofaunal assemblage after disturbances in a limestone valley in northern Puerto Rico is discussed. As population sizes of common colonizers such as Eleutherodactylus and Anolis increased, larger forest-interior and predatory species like Epicrates inornatus, Alsophis portoricensis and Anolis cuvieri followed. Finally, the Mona Island marine turtle monitoring program is discussed and compared to other similar programs in Puerto Rico. As these and other similar conservation efforts provide scientifically based management recommendations, we hope to succeed in conserving the diverse herpetofauna that characterizes Puerto Rico.