Mycologia, 96(1), 2004, pp. 9–15. q 2004 by The Mycological Society of America, Lawrence, KS 66044-8897 Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians Jeffrey S. Piotrowski Seanna L. Annis1 Joyce E. Longcore drobatidis led to the death of poison dart frogs in a test of Koch’s postulates (Nichols et al 2001); however, the direct cause of death from chytridiomycosis is uncertain (Berger et al 1998). Not all amphibians develop chytridiomycosis or die when experimentally or naturally infected with B. dendrobatidis (P. Daszak, pers comm). Environmental factors also may affect the rate of mortality because many deaths from chytridiomycosis occur during the cooler time of the year at a given location, or in populations in cool, high-altitude regions (Berger et al 1998, Lips 1998, 1999, Bosch et al 2000, Bradley et al 2002). The pH of the aquatic environment also has been suggested as a possible cofactor in the development of chytridiomycosis (Berger et al 1998, Bosch et al 2000). Chytridiomycosis is considered an emerging infectious disease (Daszak et al 2000). The effects of B. dendrobatidis on some populations of amphibians have been devastating, and herpetologists, ecologists and epidemiologists are investigating its role in amphibian declines. Longcore et al (1999) reported on the morphology and development in pure culture and the zoospore ultrastructure of B. dendrobatidis, but additional physiological information about the fungus is needed. Herein we present data on the effects of temperature, pH, nutrient preferences, zoospore longevity, swimming ability and enzyme production for B. dendrobatidis. Department of Biological Sciences, University of Maine, Orono, Maine 04469 Abstract: Batrachochytrium dendrobatidis is a pathogen of amphibians that has been implicated in severe population declines on several continents. We investigated the zoospore activity, physiology and protease production of B. dendrobatidis to help understand the epidemiology of this pathogen. More than 95% of zoospores stopped moving within 24 h and swam less than 2 cm before encysting. Isolates of B. dendrobatidis grew and reproduced at temperatures of 4– 25 C and at pH 4–8. Growth was maximal at 17–25 C and at pH 6–7. Exposure of cultures to 30 C for 8 d killed 50% of the replicates. B. dendrobatidis cultures grew on autoclaved snakeskin and 1% keratin agar, but they grew best in tryptone or peptonized milk and did not require additional sugars when grown in tryptone. B. dendrobatidis produced extracellular proteases that degraded casein and gelatin but had no measurable activity against keratin azure. The proteases were active against azocasein at temperatures of 6–37 C and in a pH range of 6–8, with the highest activity at temperatures of 23–30 C and at pH 8. The implications of these observations on disease transmission and development are discussed. Key words: chytridiomycosis, Chytridiomycota, disease, fungal proteases MATERIALS AND METHODS Isolates. Isolates of Batrachochytrium dendrobatidis (TABLE I) were from the chytrid culture collection at the University of Maine, Orono. Isolates for temperature and pH experiments were selected to represent different regions of North America and were isolated from different amphibian species. Isolates are morphologically indistinguishable. Stock cultures were transferred at 5 mo intervals; they were grown in TG liquid medium (1% tryptone, 0.3% glucose) or 1% tryptone (Difco) liquid medium in screw-capped, glass culture tubes at 23 C until growth was evident and then stored at 4–5 C. TGhL (1.6% tryptone, 4% gelatin hydrolysate, 0.5% lactose, 1% agar) or 1% tryptone in 1% agar media were used as solid media unless otherwise indicated. INTRODUCTION Batrachochytrium dendrobatidis Longcore et al (1999) has been detected in dead or dying anurans in North America, Australia, Central America and Europe and causes chytridiomycosis, which is implicated as the cause of amphibian deaths and some population declines (Berger et al 1998, Lips 1999, Bosch et al 2000, Bradley et al 2002). The spherical thalli of B. dendrobatidis live within keratinized epidermal cells of amphibians (Pessier et al 1999). Inoculation with B. den- Zoospore motility. Zoospores of isolate 197 were examined for the time and distance they swam before encysting. Grids with 1 3 1 mm sectors were etched onto the bottom of 5 cm diam plastic culture dishes. Plates were soaked overnight in 70% ethanol, rinsed in sterile water and dried in a lam- Accepted for publication March 31, 2003. 1 Corresponding author. E-mail: sannis@maine.edu 9 10 MYCOLOGIA TABLE I. Isolates of Batrachochytrium dendrobatidis, amphibian host and geographic origin of host Isolate # Amphibian isolated from Location 197 type isolate Blue Poison Dart Frog (Dendrobates azureus) 215 230 231 274 275 277 Mountain Yellow-Legged Frog (Rana muscosa) Lowland Leopard Frog (R. yavapiensis) Lowland Leopard Frog (R. yavapiensis) Boreal Toad (Bufo boreas) Boreal Toad (B. boreas) Tiger Salamander (Ambystoma tigrinum) inar flow hood under UV light. Three mL of a zoospore suspension in sterile distilled water with antibiotics (400 mg/L streptomycin and 200 mg/L penicillin) containing approximately 200 000 zoospores were added to each of three plates. In two sectors per grid, motionless zoospores on the bottom of the plates were counted immediately. Settled zoospores were counted every 6 h for 24 h, and counts from two sectors per plate were averaged. Four (10 cm diam) 0.5% tryptone-agar plates were flooded with 3 mL of sterilized pond water. One drop of a zoospore suspension (;65 000 zoospores) was added to one side of each plate. The control plate was dried immediately and incubated. The other plates were covered and left in the hood 24 h. After 24 h the plates were dried and incubated at 23 C for 1 wk. After 1 wk, plates were photographed, and the distances of the growing colonies from the site of the initial drop were measured. Inoculation of cultures and measurement of growth. All cultures were grown in 30 mL of the appropriate liquid medium in 50 mL screw-top, Corning polypropylene centrifuge tubes (Corning, New York). Inoculum was 1 mL of a 2-wk-old liquid culture standardized with distilled water to an optical density of 0.100 or 0.050 at 495 nm. Growth was measured by absorbance at 495 nm of 1 mL of a gently shaken culture. Growth was measured at the end of the incubation periods unless otherwise noted. Cultures for all experiments were screened microscopically to check for live zoospores and contamination. In all growth experiments, each treatment consisted of four replicates and each experiment was repeated at least once. Temperature experiments. Batrachochytrium dendrobatidis cultures in TG medium were incubated at 10, 17, 23, 25 and 28 C. Beginning on day 0 and every 3 d thereafter for 3 wk, four cultures per isolate were removed and growth was measured as above. Cultures also were grown at 4 C for 6 mo. Four cultures per isolate were removed and measured monthly for their growth. Experiments were designed to examine the effect of exposure of B. dendrobatidis to 30 C. After inoculation, all cultures were incubated at 23 C for 4 d to establish actively growing colonies. Growth then was measured, and half of the culture tubes were transferred to 30 C; the rest were kept at 23 C as controls. Four replicates were measured for each temperature treatment at 2, 4, 6 and 8 d after transfer. The condition of the cultures was evaluated microscopically National Zoological Park, Washington, D.C. Sierra Nevada, California Santa Catalina Mountains, Arizona Maricopa County, Arizona Rocky Mountains, Colorado Rocky Mountains, Colorado Campina Mesa, Arizona and by inoculating TGhL plates with 1 mL of culture from each replicate and incubating the plates at 23 C for 6–10 d to observe colony growth. Effect of pH. Preliminary experiments indicated that several buffers affected the growth of B. dendrobatidis. Growth of the chytrid in unbuffered TG liquid medium changed the pH of the medium less than 0.5 pH units after 2 wk of incubation. We used unbuffered media in pH experiments adjusted with 1 N HCl or 1 M NaOH to the required pH and unadjusted TG medium (pH 6.8–7.0) served as the control. We tested growth at pH 4.0, 5.0, 6.0, 7.0 and 8.0. Inoculum was 1 mL of culture standardized to an optical density of 0.050 absorbance units at 495 nm. Cultures were incubated 2 wk at 23 C and shaken once. Nitrogen and carbon sources. Isolate 274 of B. dendrobatidis (from Colorado) was used in experiments to test the effect of nitrogen source on growth. This isolate had not been growing on artificial media as long as isolates 197 or 215 and therefore was expected to be the least adapted to tryptone of the three isolates. Each treatment contained 0.3% glucose and 1% of one of these nitrogen sources: asparagine, gelatin hydrolysate (Sigma), yeast extract (Difco), peptonized milk (BBl or Oxoid), malt extract (Difco), peptone (Difco) or tryptone. The control was 0.3% glucose with no added nitrogen source. Isolate 274 also was used to test the effect of the addition of carbohydrates to tryptone medium on growth. Each treatment consisted of 1% tryptone plus 0.3% of one of the following: sucrose, maltose, sorbitol, mannose, glucose, glycerol or lactose. The control was 1% tryptone with no added sugars. To determine the effect of different concentrations of glucose on growth, we incubated isolate 274 with 1% tryptone and 0, 0.15, 0.3, 0.9, 1.8 or 3.6% glucose. The effect of tryptone concentration on growth also was tested on 0.3% glucose medium supplemented with 0, 0.25, 0.5, 0.75, 1.0, 1.5, 2.0 or 3.0% tryptone. Growth of the cultures was measured after incubation for 2 wk at 23 C. Isolate 197 of Batrachochytrium was inoculated onto 1% keratin agar (from scleroproteins, ICN, Costa Mesa, California) and into snakeskin medium (1 g macerated snakeskin in 75 mL ddH2O) to test its ability to grow on complex protein sources. To examine the salt tolerances of B. dendrobatidis, isolate 197 was grown in 1% tryptone liquid medium with 0.5 or 1% NaCl for 2 wk at 23 C. PIOTROWSKI ET AL: PHYSIOLOGY Protease production. Evidence for protease activity was determined initially by inoculating protein-substrate agar plates. We inoculated TGhL plates with 1 mL of liquid culture of isolate 197, dried them in the laminar flow hood 1 h and then incubated them at 23 C for 4–6 d, until dense growth and live zoospores were visible. Ten mm diam plugs containing colonies from these plates were placed cultureside down on protease assay plates, which contained 1% agarose and either 1% skim milk (Carnation) or 1% gelatin (Sigma). Each assay plate received four plugs: three with B. dendrobatidis colonies and one uninoculated control. Assay plates were incubated at 23 C for 2–4 d. Clear zones in the medium surrounding the plugs from the breakdown of proteins indicated protease activity (Karaup et al 1994). Further tests for protease activity used culture supernatants of isolate 197 grown in 75 mL of 1% skim milk powder in water inoculated with 1 mL of a 2-wk-old liquid culture. Cultures were incubated 2–4 d at 23 C, until the skim milk became clear. Cultures were centrifuged at 6000 rpm for 20 min to remove cells, and the supernatant was frozen at 280 C. Samples were freeze-dried, resuspended to one-tenth of their original volume in 50 mM Tris-HCl (pH 7.0) and dialyzed against membranes (5 KDa pores) at 6 C in three changes of buffer (buffer volume greater than 20 times the volume of the samples). After dialysis, the 103 concentrated supernatants were stored in 1.5 mL aliquots at 220 C until used in the following tests to measure the temperature and pH response of the extracellular proteases. Temperature and pH response of extracellular proteases. The pH and temperature ranges of extracellular proteases were measured with an azo dye bonded to casein (Sigma). The reaction mixture for measuring the temperature range consisted of 500 mL of 0.2 M CaCl2 and 500 mL of 5% azocasein in 50 mM Tris HCl (pH 7.5) in 1.7 mL microfuge tubes. To the reactions, 200 mL of water (as control) or 103 concentrated supernatant were added, and reactions were incubated 36 h at 6, 15, 23, 30 or 37 C. Reactions were stopped with 5% trichloro acetic acid (TCA) and centrifuged at 11 000 rpm for 2 min to remove precipitated proteins. Absorbance of the supernatant was measured at 440 nm. Reactions with distilled water served as the blank, and boiled culture supernatant served as the control. Each temperature treatment consisted of three replicates with unaltered supernatant, three with boiled supernatant and three with water. The pH range of extracellular proteases was measured in reaction mixtures of 1 mL 5% azocasein adjusted to pH 6, 6.5, 7, 7.5 or 8 with Tris-HCl buffer and 200 mL of 103 concentrated supernatant. Reactions were incubated 36 h at 23 C then stopped with 200 mL of 5% TCA and measured as above. Statistical analysis of data. The Kruskal-Wallis rank test was used to detect significant differences. Differences between isolates or between treatments for an isolate in temperature experiments were tested at two points—during logarithmic growth and during stationary growth phase. Differences were considered significant if P , 0.050. OF BATRACHOCHYTRIUM DENDROBATIDIS 11 FIG. 1. Growth of Batrachochytrium dendrobatidis isolates 197, 215 and 274 at 4 C over 6 mo as measured by the optical density (absorbance at 495 nm). The mean of four replicates from each month and the standard error of the mean are presented. RESULTS Zoospore activity. In preliminary experiments, the rate of encystment was the same over 24 h for zoospores in distilled water, pond water or 1% tryptone liquid medium. Tryptone medium was used for the remainder of the experiments examining zoospore motility. The majority of zoospores swam less than 2.0 cm before encysting. Colonies on plates that were flooded for 24 h did not appear to be more dispersed than on plates that were dried immediately after inoculation. In tests to determine the length of time zoospores remain motile, approximately 50% of zoospores remained motile after 18 h. By 24 h, approximately 5% of the zoospores still were swimming. Temperature effects on growth. Preliminary observations indicated that colonies of B. dendrobatidis on TGhL agar could grow for up to 5 mo at approximately 5 C. In experiments to test the growth of B. dendrobatidis at low temperature, isolates 197, 215 and 274 were alive and producing zoospores after 6 mo of incubation at 4 C (FIG. 1). In both repetitions of the experiment, isolate 215 grew significantly less than the other isolates after 6 mo at 4 C. Temperatures of 10–25 C were suitable for growth of the three isolates (197, 215 and 274) tested. The pattern of growth of isolate 197 at 10–28 C (FIG. 2) is representative of that found with the other isolates. Growth was slow at 10 C with cultures still increasing in density at the end of the experiment at 23 d. Growth was faster at 17–25 C. Cultures reached stationary phase by day 12 when grown at 17 and 23 C and by day 9 when grown at 25 C. Growth rates varied among isolates, depending upon the temperature and the repetition of the experiment. All cultures at 10–25 C contained live zoospores during the experiments, but at 28 C none of the isolates grew or contained live zoospores after 2 d of incubation. The growth of isolates 230, 231, 275 and 277 were compared to that of isolate 197 at 23 C. All isolates pro- 12 MYCOLOGIA FIG. 2. Growth of Batrachochytrium dendrobatidis isolate 197 over 21 or 23 d at different temperatures as measured by optical density (absorbance at 495 nm). The mean of four replicates from each day and the standard error of the mean are presented. duced similar growth curves at 23 C, with some variability in the level of growth for each isolate among the repetitions of the experiment. Exposure to 30 C. After the initial 4 d incubation at 23 C, growth among the isolates did not differ significantly. After 2 d, growth of replicates kept at 23 C was significantly more dense than of replicates transferred to 30 C. After 8 d at 30 C, 50% of the replicates from each isolate were dead. Replicates kept at 23 C continued to grow and had live zoospores and thalli at the end of the experiment. Effect of pH on growth. In all experiments, isolates 197, 215 and 274 grew most at pH 6–7, with less growth at pH 8 and minimal growth at pH 4 and 5 (FIG. 3). Isolate 215 grew less at pH 6–7 than the other two isolates. At the end of the experiment, swimming zoospores were present in all cultures grown at all pH. Nutritional requirements. Of the synthetic media tested, asparagine-glucose agar (Stevens 1974), dilute salts solution (Fuller and Jaworski 1987) and yeast nitrogen base plus 1% glucose and thiamine (Sigma), did not support growth of B. dendrobatidis. Therefore, more complex media were used to determine the ability of isolate 274 to grow on different nitrogen and carbon sources. Nitrogen sources strongly influenced the growth of B. dendrobatidis (FIG. 4). The chytrid grew most in 1% tryptone in distilled water and second best in 1% peptonized milk; however, the growth was significantly less than that on tryptone. All other media supported less growth than the control, which contained 0.3% glucose, plus any nutrients that were transferred with the inoculum. Malt extract, yeast extract and asparagine supported trace amounts of growth. After 2 wk of incubation, live thalli were found in all tested media except asparagine and all except aspar- FIG. 3. Growth of Batrachochytrium dendrobatidis isolates at different pH at 23 C as measured by optical density (absorbance at 495 nm). Growth at pH 7 is the unadjusted control. The mean of four replicates from each pH and the standard error of the mean are presented except * 5 mean of two replicates. agine and gelatin hydrolysate media contained motile zoospores. Different carbon sources added to liquid medium with 1% tryptone did not increase the growth of B. dendrobatidis as compared to the control, which contained 1% tryptone. B. dendrobatidis grew less on glycerol than on other added carbon sources. All cultures contained live zoospores and thalli at the end of the experiments. Growth was affected when glucose concentrations were increased above 2% or at tryptone concentrations above 1.5%. In 0.3% glucose medium, B. dendrobatidis grew equally well in tryptone concentrations of 0.25–1.0%. At the highest concentration of tryptone (3%), the chytrid grew less than in the glucose control medium, which contained a small amount of nutrients from the inoculum. B. dendrobatidis grew equally well in a medium with 1% tryp- FIG. 4. Growth of Batrachochytrium dendrobatidis isolate 274 on different nitrogen sources as measured by optical density. Cultures were grown in 30 mL of 0.3% glucose and 1% of the nitrogen source. Optical density was measured at 495 nm. Means of four replicates and the standard error of the mean are presented. All treatments were significantly different from each other (P , 0.050). Key to nitrogen sources: gh 5 gelatin hydrolysate, ye 5 yeast extract, pm 5 peptonized milk, me 5 malt extract, asp 5 asparagine, p 5 peptone, t 5 tryptone, x 5 basal media of glucose with no nitrogen source added. PIOTROWSKI ET AL: PHYSIOLOGY FIG. 5. Clearings in the 1% skim milk agar plate incubated 1 wk at 23 C with agar plugs of Batrachochytrium dendrobatidis (isolate 197) indicate proteolytic activity. Control was an uninoculated plug of agar. tone and 0–1.8% glucose. Growth was significantly less in 3.6% glucose than in all the other glucose media. All cultures contained live zoospores and thalli after 2 wk of incubation. Isolates of B. dendrobatidis grew and produced zoospores on 1% keratin agar and in snakeskin liquid medium after 1 wk. Growth in snakeskin medium was sparse compared to growth in TG medium. Colonies were flatter and larger in diameter when grown on keratin agar than on TGhL medium. The fungus grew and formed motile zoospores in media supplemented with 0.5% NaCl and grew slowly in media containing 1% NaCl. Growth in media containing 0.5% NaCl was less robust than in 1% tryptone alone. Production of extracellular enzymes. After 2–4 d of incubation, distinct clear zones were visible around each plug containing B. dendrobatidis colonies on skim milk and gelatin assay plates, indicating the production of extracellular proteases (FIG. 5). Control plugs were devoid of activity. Skim milk assay plates supported the growth of numerous thalli and the production of zoospores. Ten3 concentrated supernatant from isolate 197 grown in 1% skim milk degraded azocasein after 24 h at 30 C and was used in subsequent experiments. The culture supernatant was most active in degrading azocasein at 23–30 C (FIG. 6) and at pH 6–8. The culture supernatant had no measurable activity against keratin azure after 48 h of incubation at 23 C or 30 C. DISCUSSION Zoospores are the primary method of dispersal for B. dendrobatidis, but their ability to infect a host is OF BATRACHOCHYTRIUM DENDROBATIDIS 13 FIG. 6. Degradation of azocasein at different temperatures by a 103 concentrated culture supernatant from Batrachochytrium dendrobatidis (isolate 197) as measured by absorbance (440 nm) of released dye. Means of three replicates and the standard error of the mean are presented. The difference in absorbance of the reactions containing the boiled supernatant and reactions containing just water was zero at 30 and 37 C. constrained by the limited time before they encyst and the short distance they swim. Some zoospores (,5%) swam for more than 24 h, which is longer than the ‘‘average’’ chytrid or oomycete zoospore (see Fuller 1986); however, zoospore longevity in pure culture may differ from that in the presence of bacteria, ingestive protists and other microbiota found in the environment. In still water, zoospores of B. dendrobatidis swam less than 2 cm before they encysted, suggesting that zoospores are unable to swim long distances to find a host. On the skin of moderately infected amphibians, thalli are in clusters of infected skin cells (Longcore, unpubl obs) rather than being spread more evenly over the surface of the skin. This distribution probably develops because many zoospores encyst and infect cells in the immediate area from which they were released. Batrachochytrium dendrobatidis may spread from amphibian to amphibian by close or direct contact during mating, schooling of larvae or other aggregative behaviors. Zoospores could be spread longer distances if carried in water currents, but this also would decrease the chances of a zoospore contacting a host, because the spores would be diluted to low concentrations. The chance of zoospores finding an amphibian may be increased if the zoospores are attracted to their host, as with some other parasitic chytrids (Sparrow 1960, Held 1974, Muehlstein et al 1988, Deacon and Saxena 1997). We tested tryptone, gelatin hydrolysate, glucose and lactose as potential attractants because they were in media used to culture chytrid; keratin and gelatin were tested because they are similar to components of amphibian skin. Our experiments did not reveal evidence of chemotaxis to the tested compounds (Piotrowski 2002). However, we did not test amphibian 14 MYCOLOGIA skin. If B. dendrobatidis is attracted to amphibian skin or compounds released from it, zoospores may swim farther than our results suggest. Even though we used inoculum of a standard age and optical density, significant variation was observed in the growth of isolates between repeats of an experiment. We believe this variation was due to an ‘‘inoculum effect’’ caused by the ratio of zoospores to thalli in liquid cultures changing from day to day. Because we measured inoculum by optical density, batches of inoculum were not identical. When liquid medium is inoculated with a portion of a stock culture, it could contain mostly zoospores, mostly thalli or varying proportions of each. This may affect the length of time before cultures achieve logarithmic and stationary phases of growth. Several of the experiments differed among repetitions, which we believe resulted from an inoculum effect, but overall growth trends were similar. Batrachochytrium dendrobatidis grows within a wide range of temperatures (4–25 C) and grows optimally at 17–25 C. This wide range of permissive and optimum temperatures should let this pathogen persist in many environments. The ability to persist and even grow slowly at 4 C would let B. dendrobatidis overwinter in its hosts, even in mid-latitude, temperate climates where temperatures of the aquatic environments are low. As temperatures rise in the environment, the chytrid then may reproduce rapidly, as it did when cultures were transferred to 23 C after incubation at 4 C. Batrachochytrium dendrobatidis does not grow well above 25 C, and higher temperatures do not favor epidemics (Berger et al 1998, Bosch et al 2000). Unless some isolates have different temperature constraints, outbreaks of chytridiomycosis in the tropics probably will be limited to cooler areas, as has been observed in Australia and Panama (Berger et al 1998, Lips 1998). In temperate zones, outbreaks could occur in montane areas in warmer months (Bosch et al 2000) or lowlands during the winter (Bradley et al 2002). At temperatures of 28 C or above, or below 10 C, B. dendrobatidis does not grow or grows slowly; infections at these temperatures may not be fatal because growth of the fungus is not favored. Pure cultures that did not grow at 28 C revived when returned to optimal temperatures (Longcore et al 1999), and the same may happen when B. dendrobatidis is within skin cells. Although exposure to 30 C killed cultures of B. dendrobatidis, half the replicates still were alive even after 8 d at 30 C. If a species of amphibians can survive elevated temperatures, exposure to temperatures above 30 C for more than 8 d may be an effective treatment for chytridiomycosis if the fungus is not protected from this temperature extreme by being within skin cells. Outbreaks of chytridiomycosis may be affected by pH, but the pH optimum (pH 6–7) for B. dendrobatidis is not outside common pHs of freshwater systems. Although the fungus grows poorly below pH 6, its zoospores can live at that pH, and once inside the host, the fungus may be buffered from external conditions. It is not surprising that all the isolates have similar physiological requirements; different genera of chytrids, even in different orders, have similar temperature and pH tolerances as B. dendrobatidis (Barr 1969, 1970a, b). Nitrogen source has a strong effect on the growth of B. dendrobatidis. This may be a result of the micronutrient, carbon and nitrogen levels of the nitrogen sources tested. B. dendrobatidis grew more on tryptone than on peptone, both of which are digests of casein protein with similar amino nitrogen content, total nitrogen content and carbohydrate content (product information from BD, Franklin Lakes, New Jersey). However, they differ in that peptone has only 0.1 mg/g thiamine compared to 0.4 mg/g for tryptone. It is not unusual for chytrids to require exogenous thiamine (Barr 1969, 1970a, b), and this difference between the two media may account for differences in growth. The chytrid grew almost as much on peptonized milk as on tryptone. Even though both are digests of casein, peptonized milk has less than half the total nitrogen of tryptone (product information from Oxoid Ltd., Hampshire, England). The pH and amount of carbohydrates in the different nitrogen sources may have had an effect on growth. Gelatin hydrolysate (pH 5.7) and malt-extract (pH 5.6) media have pH slightly below the growth optimum for B. dendrobatidis. The high sugar content of malt extract (60 to 63% reducing sugars) and yeast extract (17.5% carbohydrate) (product information from BD, Franklin Lakes, New Jersey) compared to tryptone (7.7%) may explain the lower growth in these liquid media. The high level of nitrogen and low pH (4.5) or the limited nutrient complexity of asparagine medium could explain the poor growth in this medium. Although growth was sparse in media other than tryptone and peptonized milk, live zoospores were present in all media except asparagine and gelatin hydrolysate, suggesting that the chytrid can grow, but not thrive, on many different nitrogen sources. We suggest 0.5 or 1% tryptone liquid or solid (1% agar) medium for culturing B. dendrobatidis. The results from the carbon/nitrogen ratio experiments suggest that B. dendrobatidis does not require sugars other than those in tryptone and that high percentages of sugar or tryptone (greater than 2%) PIOTROWSKI ET AL: PHYSIOLOGY hinder growth. Although B. dendrobatidis grew on snakeskin and keratin media, we cannot conclude that it was using the keratin or producing a keratinase, because some of the keratin might have been degraded by autoclaving. Batrachochytrium dendrobatidis produces extracellular proteases that degraded casein and gelatin but did not degrade keratin azure. However, many types of keratin exist and the form of keratin in the keratin azure might be more resistant to the chytrid’s protease attack than the keratin in amphibian skin. Although the chytrid is found only in the keratinized cells of amphibians, it is uncertain if it actually degrades the keratin. It is possible that B. dendrobatidis is found in keratinized epidermal cells because these cells are dead and easier to invade. Isolate 197 was the only isolate studied for enzyme production. Preliminar y experiments, however, showed that isolates 215 and 274 also produced casein-degrading proteases. Different isolates may produce different levels or types of proteases, and the differences may make some isolates more virulent. The temperature and pH ranges of the enzymes are similar to the temperature and pH optima for growth of B. dendrobatidis on defined media. The proteases produced by B. dendrobatidis may be nonspecific because they can degrade skim milk proteins, gelatin and snakeskin. This might let the chytrid survive saprobically on protein substrates in the environment. ACKNOWLEDGMENTS NSF Division of Biological Sciences supported this research as part of Integrated Research Challenges in Environmental Biology Grant IBN-9977063. LITERATURE CITED Barr DJS. 1969. 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