Mycologia
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DOI: 10.3852/mycologia.100.1.1
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Mycologia, 100(1), 2008, pp. 1-5.
© 2008 by The Mycological Society of America

Chemotaxis of the amphibian pathogen Batrachochytrium dendrobatidis and its response to a variety of attractants


Angela S. Moss
Nikla S. Reddy
Ida M. Dortaj
Michael J. San Francisco 1

     Department of Biological Sciences, Box 43131, Texas Tech University, Lubbock, Texas, 79409-3131

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

Batrachochytrium dendrobatidis is a fungal pathogen of amphibians that is increasingly implicated as a major cause of large-scale mortalities of amphibian species worldwide. Previous studies indicate that motile zoospores of B. dendrobatidis colonize the keratinized tissues of susceptible amphibians. Infections spread to adults and cause destruction of epidermal tissue. In an effort to understand how the chytrid cues into its host we developed an assay to study chemotaxis in the fungus. Here we show that zoospores exhibit positive movement toward a variety of attractants including sugars, proteins and amino acids. These observations suggest that the chytrid can respond to nutritional cues, including those of host origin. Implications of these observations to amphibian susceptibility to infection and chytrid virulence are discussed.

Key words: amphibians, chemotaxis, chytrids


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Since the late 1800s we have known that some microorganisms have the ability to exhibit positive or negative chemotaxis toward chemicals in their microenvironment (see Adler 1966Go). Within the past 30 y numerous studies have demonstrated positive chemotaxis in fungi to a variety of substrates including specific hosts and various nutrients (Held 1974Go, Muehlstein et al 1988Go, Deacon and Saxena 1977).

The pathogen Batrachochytrium dendrobatidis has been implicated as the main cause for the increasing decline of amphibian population worldwide (Berger et al 1998Go, Lips et al 2006Go, Daszak et al 1999Go). Widespread mortalities and declines have been reported in countries including Panama, Venezuela, Australia, Spain and parts of the western United States (Berger et al 1998Go, Lips 1999Go, Bonaccorso et al 2003Go, Bosh et al 2000Go, Bradley et al 2002Go, Fellers et al 2001Go, Rachowicz et al 2006Go). On infection motile zoospores of B. dendrobatidis colonize the keratinized layer of the stratum corneum in adults, eventually resulting in hyperkeratosis and sloughing of the epidermis (Berger et al 1998Go). Because B. dendrobatidis grows in keratinized tissues of susceptible animals we hypothesized that the fungus may exhibit positive chemotaxis toward attractants of nutritional relevance as well as those of host origin. We therefore developed a novel method to test for chemotaxis toward sugars, proteins and amino acids.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Cultures.— – Batrachochytrium dendrobatidis (isolate VM1) was kindly provided by Louise Rollins-Smith. This isolate was obtained from a diseased Western chorus frog (Pseudacris triseriata) by Verma Miera and Elizabeth Davidson of Arizona State University. B. dendrobatidis was grown and maintained on TGhL agar (1.6% tryptone, 0.2% gelatin hydrolysate, 0.4% lactose, 0.8% agar) and H-broth (1% tryptone, 0.32% glucose) according to methods outlined in Boyle et al 2003.

Chemotaxis assays.— – Chemotaxis of B. dendrobatidis was examined as follows: filter sterilized solutions of hydrolyzed casein (Fisher Scientific, FairLawn, New Jersey), keratin (MP Biomedicals, Aurora, Ohio), glucose (Amresco, Solon, OH), lactose (Fisher Scientific), gelatin hydrolysate (Sigma-Aldrich, St Louis, Missouri), glycine (Sigma-Aldrich), cysteine (Matheson Coleman, Norwood, Ohio) and glutamic acid (United States Biochemical Corp., Cleveland, Ohio) were tested at concentrations of 0.2% and 2% (wt/ vol) in water. Water also was tested as a vehicle control. Assays were carried out by manual counting of zoospores with a No. 4099-A (Scott) hemacytometer with improved Neubauer ruling, similar in design to the bright line hemacytometer model by American Optical (Buffalo, New York). The slide contains two counting chambers, each of which is divided into nine large 1 mm squares on an etched and silvered surface separated by a trough. Each chamber has a V-shaped filling notch at one end for sample loading. Raised edges on either side of each chamber provide support for a cover slip. The slide was viewed with a Fisher Scientific Micromaster® I microscope, using a 40x phase-contrast objective or an Olympus BH-2 microscope with 20x and 10x objectives. To facilitate zoospore monitoring on the slide, columns of the counting grid were designated A–D and rows were designated 1–5 (FIG. 1Go). Row 1 was closest to the attractant being studied and row 5 most distant from the attractant.


Figure 1
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  		FIG. 1. Layout of cell counting grid used for chemotaxis assays. Zoospores of B. dendrobatidis were placed close to rows 5A–E at the start of each assay. Migration of zoospores from starting point to rows 1A–E was observed for 90 min.

 
Whatman filter paper was used first to make attractant disks. These however proved to be too thick and interfered with microscope resolution. Therefore sheets of white, acid-free printing paper (Boise Cascade, Boise, Idaho) were cut into circular disks with a hole-punch. Each disk was 6 mm diam. Appropriate attractant solution (approximately 0.1 mL) was applied to the sterile paper disks and allowed to air dry 24 h. The disks were dipped briefly in 0.1 mL of sterile distilled water immediately before each assay in an effort to moisten the attractant disk and prevent it from creating a wicking effect during the 90 min period. During the initial experiments we experienced occasional evaporation of the zoospore suspension. This most likely was caused by a combination of the heat from the microscope light and the paper disk drawing water toward itself. To counteract this effect we found that it was necessary first to dip each disk in 0.1 mL of sterile distilled water immediately before each assay. The counting slide was cleaned with 95% ethanol and sterile distilled water before the disk was placed at one end of the counting grid, closest to row 1 (FIG. 1Go). We harvested zoospores from TGhL agar plates (5–7 d old) by routine methods (Boyle et al 2003Go). The resulting zoospore suspension of 0.01 mL (containing varying densities up to 2.2 x 107 zoospores/mL) was placed directly on the surface of the counting grid (not in the loading notch) opposite the paper disk containing the attractant (closest to row 5). A cover slip was placed on the surface of the slide before counting. Adding the cover slip at this time caused an even dispersal of the zoospores from their original position to the entire surface of the counting chamber so that equivalent numbers of zoospores were found throughout rows 1–5 and columns A–E (at time 0).

Zoospore abundance was determined by counting total zoospore numbers within the counting grid at 45 min intervals for 90 min. This was done by taking pictures of the counting grid every 45 min with an Olympus DP70 digital camera (Olympus Corp., Tokyo, Japan) attached to the microscope. At the end of each assay representative squares (four) were counted and the zoospore number extrapolated to account for all the squares (25).

To determine whether the chemotactic response was affected by cell density, zoospores at varying concentrations were tested with several of the attractants (TABLE IGo).


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  		TABLE I. Zoospore numbers for chemotaxis assays. Zoospores within the the counting grid were counted at three time points

 
Statistical analysis.— – One-way ANOVA with Matlab (version 6.0.0.88 release 12) were conducted to determine whether the attractants had a significant effect as compared to water. Each attractant was assayed with five replicates with the exception of keratin which was replicated four times. All data were square-root/arcsine transformed to ensure homogeneity of variance. Tukey’s honestly significant difference criterion was used to determine differences among pairs of treatments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
B. dendrobatidis response to lactose, glucose.— – At concentrations of 0.2%, significant migration (P ≤ 0.001) toward lactose and glucose was observed (FIG. 2Go), suggesting zoospore migration toward these attractants. Using lactose and glucose as attractants at an initial concentration of 2.0% however resulted in no significant change in zoospore migration toward the disks during the experiments. A marginal increase in the zoospore numbers was noted, but the difference was not significant (data not shown). The apparent lack of positive movement in this time frame to these attractants might result from the high concentration of the sugars in the disks and rapid loss of the chemical gradient. Studies with the solvent control, water, also indicated no notable change in zoospore migration toward the disk during the experiment.


Figure 2
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  		FIG. 2. Movement of B. dendrobatidis zoospores toward glucose and lactose at concentrations of 0.2% (wt/vol). Zoospores within the grid were counted at 0, 45 and 90 min during the course of the assay. Means of five replicates are presented. Water was used as the control attractant.

 
B. dendrobatidis response to amino acids and proteins.— – Because gelatin hydrolysate is a constituent of B. dendrobatidis culture media, we tested chemotaxis of zoospores toward this compound. At a concentration of 0.2%, positive migration was seen toward the disk during 90 min (FIG. 3Go). Movement of B. dendrobatidis zoospores toward 2.0% gelatin hydrolysate were inconclusive (data not shown). Glycine is a predominant amino acid of gelatin. Positive movement toward 0.2% glycine was observed over 90 min (P ≤ 0.001) (FIG. 3Go). Positive movement also was observed with hydrolyzed casein that has been shown to support B. dendrobatidis growth (in the form of 1% skim milk) (Piotrowski et al 2004Go). At a concentration of 0.2%, migration toward the disk was noticeable during the 90 min (P ≤ 0.001) (FIG. 3Go). When the concentration was increased to 2.0% a significant effect was not observed (data not shown). Keratin, the major constituent of the amphibian epidermis, is sparingly soluble in water. When a water mixture of 2% keratin was used in the assay we observed positive movement toward the attractant (P ≤ 0.001) (FIG. 4Go). We also observed positive movement toward cysteine, an abundant amino acid of keratin, when used at a concentration of 0.2% (P ≤ 0.001) (FIG. 4Go). Of interest, a positive response was not observed when 0.2% glutamic acid was used as an attractant (FIG. 4Go). Glutamic acid is also a primary amino acid constituent of keratin.


Figure 3
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  		FIG. 3. Movement of B. dendrobatidis zoospores toward proteins casein and gelatin hydrolysate and the amino acid glycine. Substrates were tested at concentrations of 0.2% (wt/vol). Zoospores were counted as before and means of five replicates are presented. Water was used as the control attractant.

 

Figure 4
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  		FIG. 4. Movement of B. dendrobatidis zoospores toward the protein keratin at a mixture concentration of 2.0% (wt/ vol). The amino acids cysteine and glutamic acid were tested at concentrations of 0.2% (wt/vol). Zoospores were counted as before and means of five replicates are presented. Water was used as the control attractant.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Chemotaxis is an important virulence factor for many pathogenic microorganisms (Ottemann et al 1997Go, Hawes and Smith 1989Go, Ormonde et al 2000Go). Zoosporic fungi rely on the presence of one or more flagella to aid in their ability to swim through liquid environments (Bimpong and Clerk 1970Go, Koch 1968Go). In addition, several studies indicate that some species of motile, pathogenic microorganisms display positive migration toward suitable host and nutrient substrates, indicating that their ability to infect a host involves not only motility but also chemotaxis (Lux et al 2001Go, O’Toole et al 1996Go).

Zoospores of B. dendrobatidis are motile by means of a single, posteriorly located flagellum (Longcore et al 1999Go, Berger et al 1999Go). Amphibians are infected when the motile zoospores are dispersed and encyst within their keratinized tissues. These tissues include the mouthparts of tadpoles, and the epidermis of adult animals (Berger et al 1998Go, Marantelli et al 2004Go). The aquatic environment of B. dendrobatidis and its potential hosts led us to hypothesize that the organism might use chemical cues in its environment to locate potential host amphibians. Previous studies have investigated this possibility using an agar-plate method adapted from Pommerville 1978 and the capillary tube method (Muehlstein and Amon 1987Go) but found no evidence of chemotaxis toward the attractants tested (Piotrowski et al 2004Go, Piotrowski 2002Go). In this study we developed a novel chemotaxis assay that would let us test for positive migration toward a variety of suitable attractants including sugars, proteins and amino acids. Gelatin hydrolysate, lactose and glucose were used because they are included in chytrid culture media such as TGhL and H-broth (Boyle et al 2003Go). Keratin was chosen for its role in amphibian chytrid infections. It is a structural protein found in the epidermis of amphibians and is thought to be targeted by zoospores of B. dendrobatidis (Berger et al 1998Go). Casein was used because of the ability of B. dendrobatidis to grow in skim milk (Piotrowski et al 2004Go). During the assay zoospore swimming was monitored for 90 min. Zoospores of B. dendrobatidis displayed positive movement toward the sugars glucose and lactose, as well as toward casein hydrolysate, gelatin hydrolysate and keratin. Of interest, lower concentrations (0.2%) of glucose, lactose, gelatin hydrolysate and casein hydrolysate were more efficient at eliciting a response than higher concentrations (2.0%). Keratin induced positive movement in B. dendrobatidis at concentrations as high as 2.0%. Because keratin is sparingly soluble in water we postulate that the actual concentration of the protein in solution is markedly lower and this might set up a chemical gradient for chemotaxis. These findings suggest that B. dendrobatidis does display a chemotactic response toward certain nutritional cues in its immediate environment. Gelatin predominantly is made up of the amino acid glycine, while glutamic acid and cysteine are major components in keratin. For this reason they also were used as attractants in the chemotaxis assays. The amino acids glycine and cysteine did induce positive movement in the fungus while glutamic acid produced no significant effect. The lack of positive movement toward glutamic acid might indicate that the zoospores are primarily attracted to the cysteine component of keratin. Proline, the predominant amino acid found in casein, was not used due to its insolubility in solvents suitable for the bioassays. B. dendrobatidis zoospores did not demonstrate positive or negative movement when exposed to sterile distilled water. This also suggests that the paper disks used in the assay did not serve as chemotactic attractants or repellents.

When no attractant was present (i.e. with the water control) zoospores displayed normal swimming behavior and did not appear to be trapped or hindered by the cover slip placed on the surface of the counting slide. This behavior indicated to us that the zoospore attraction was not affected by the possibility of electrostatic binding to the glass cover slip.

While Piotrowski et al (2004)Go showed that B. dendrobatidis zoospores grew well at tryptone concentrations of 1% (with no additional nutrients), we postulate that the zoospores are migrating to the attractants tested because they are the only nutrients present at the time of each assay.

The positive movement of B. dendrobatidis toward the attractants used in these assays suggests that the organism is able to sense certain chemicals in its immediate environment. Thus B. dendrobatidis might be capable of positive chemotaxis. The above observations imply that motility, in addition to chemotactic responses, might let the fungus identify and infect susceptible amphibians. More definitive studies must be conducted to determine the precise mechanism of attraction of zoospores to each of the chemicals tested. In addition, the effect of repellants on zoospores movement should provide useful information.


   ACKNOWLEDGMENTS
 
The authors thank Adam Lord and Jordan Owens for their assistance in counting the chemotaxis assays and Dr Reynaldo Patiño for the use of his microscope. We also thank Heath Grizzle and Jennifer Huddleston for their help with the statistical analyses. This work is supported by the National Science Foundation under grant No. 0201105.


   FOOTNOTES
 
Accepted for publication September 24, 2007.

1 Corresponding author. E-mail: michael.sanfrancisco{at}ttu.edu


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