Elsevier

Fungal Ecology

Volume 4, Issue 3, June 2011, Pages 196-200
Fungal Ecology

Phenotypic profiling of Batrachochytrium dendrobatidis, a lethal fungal pathogen of amphibians

https://doi.org/10.1016/j.funeco.2010.12.003Get rights and content

Abstract

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is responsible for many amphibian declines worldwide. The determinants of Bd’s high virulence across a wide range of amphibian species are not well understood but include environmental factors, host responses to infection, and pathogenicity of Bd. Because there are clear differences among Bd isolates, genetic profiling of multiple strains is currently in progress. However, genetic analyses alone may not provide a functional explanation for differential virulence among strains. I aimed to quantify phenotypic traits, such as zoospores densities, that are important for disease development. Among the three isolates tested, zoospore densities were highest in GibboRiver-L.lesueuri-00-LB-P24, an isolate originally collected from a Litoria lesueuri tadpole and highly virulent in infection experiments. Because characterizing differences among isolates is central to understanding Bd pathogenicity, integrating genetic work with the phenotypic profiling methods provided in this study will offer insights into chytridiomycosis and may be important for amphibian conservation.

Introduction

In the past few decades several infectious fungal pathogens have gained considerable attention for causing dramatic die-offs in wildlife populations (e.g. amphibians, Lips et al. 2006; bats, Frick et al. 2010). The factors contributing to high virulence in these fungal disease systems are not well understood but are currently the subject of intense investigations owing to their significant threat to wildlife health (Daszak 2010).

The fungus Batrachochytrium dendrobatidis (Bd) causes the disease chytridiomycosis, which is highly lethal to many species of amphibians. Outbreaks of chytridiomycosis have reduced the abundance of amphibian species with dramatic declines, and even species extinctions, reported (Berger et al., 1998, Lips et al., 2006, Schloegel et al., 2006). However, not all species of amphibians succumb to disease in exposure experiments (Daszak et al., 2004, Retallick and Miera, 2007, Woodhams et al., 2007) and some species and populations of amphibians that have survived initial declines persist in the wild with a wide range of infection levels (Retallick et al., 2004, Woodhams and Alford, 2005). The differences in the effects of Bd infection on populations and species are probably due to a combination of factors that drive disease dynamics, including environmental conditions (Woodhams & Alford 2005), host behavioral characteristics (Rowley and Alford, 2007, Richards-Zawacki, 2010), host immune defences (Woodhams et al., 2007, Rosenblum et al., 2008, Ramsey et al., 2010) and differential pathogen virulence among strains of Bd (Berger et al., 2005, Fisher et al., 2009), a factor which has received relatively less attention (Fisher et al., 2009, Voyles et al., 2009a, Voyles et al., in press).

Preliminary genetic studies on Bd found little variation among globally collected isolates (Morehouse et al., 2003, Morgan et al., 2007), but further investigation has demonstrated clear differences among Bd isolates (Berger et al., 2005, Retallick and Miera, 2007, Fisher et al., 2009, James et al., 2009, Goka et al., 2009). Assessing genotypic and phenotypic differences among Bd strains is central to understanding the global epidemiology of chytridiomycosis and for directing amphibian conservation efforts. Specifically, investigating the degree of relatedness among strains should help to determine Bd’s point of origin (Goka et al., 2009, James et al., 2009), explain patterns of Bd emergence (Rohr et al. 2008) and direct efforts to limit the spread of genetically distinct strains. However, molecular analysis and genotypic profiling may not fully capture pathogen phenotypic characteristics that are important to disease development. Methods for examining phenotypic characteristics (i.e. traits that are important virulence factors; Fisher et al. 2009) will complement genetic work and offer additional insights into differential pathogenicity among Bd isolates.

The rate of production of infectious zoospores appears to be important in disease development and mortality in infected amphibians; local re-infection of individuals leads to an exponential increase in Bd-load, and infections that progress above a species-specific threshold may lead to mortality (Briggs et al., 2010, Vredenburg et al., 2010). This idea is further supported by exposure experiments in which increases in Bd-load correspond with the onset of clinical signs of disease and pathophysiogical changes that lead to mortality (Voyles et al., 2007, Voyles et al., 2009b). Thus, establishing methods that quantify zoospore production (Bd fecundity or rate of development/reproduction) will provide a way to profile phenotypic characteristics that may be critical to understanding Bd virulence and development of lethal chytridiomycosis.

Section snippets

Materials and methods

I hypothesized that phenotypic characteristics, including time to zoospore release and zoospore densities, would differ among the three Bd. To test this hypothesis, time to zoospore release (number of days to first observed zoospores) was recorded and zoospore densities were compared on the first day of zoospore release and on the day of maximum zoospore densities for three Bd isolates. Three Bd isolates with similar passage histories were selected to quantify and compare zoospore production:

Results

Microscopic inspection of the three isolates suggested that their rates of maturation and morphological characteristics were not substantially different (Fig 1), but zoospore densities among the three isolates differed significantly (repeated measures ANOVA, P < 0.001; Fig 2). New zoospores were visible and active after 2 d for the Rockhampton and Tully River isolates and after 3 d for the Gibbo River isolate. Zoospore densities in Gibbo River isolate (mean density SEM: 155.3 ± 50.82) and the Tully

Discussion

Differential pathogenicity of B. dendrobatidis has been reported in several studies, but it is unclear why this variation exists (Berger et al., 2005, Retallick and Miera, 2007, Fisher et al., 2009). This study quantified phenotypic differences among Bd isolates that are potentially important for the development of lethal chytridiomycosis. Comparing zoospore densities among three isolates originally cultured from different amphibian species revealed that zoospore production differed

Acknowledgments

This work was funded by the Australian Research Council Discovery Project DP0452826, and the Australian Government, Department of Environment and Heritage Project RFT 43/2004. I thank Lee Berger, Lee Skerratt, Ross Alford, Rick Speare, Erica Bree Rosenblum and Scott Cashins for their suggestions and assistance.

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