Introduction

Figure 1: This image shows growing clusters of Batrachochytrium dendrobatidis grown at 24°C on 1% tryptone agar for seven days.  On solid media, the mature organism appears as small vanilla-colored colonies ranging in size from 1mm to 5mm in diameter.  Each colony visible on the plate arose when one or more zoospores encysted in the media and grew into multinucleated, fertile sporangia (1,5).  A sporangium, by definition, is a structure that produces and contains asexual spores.  The ideal temperature range in which B. dendrobatidis grows is between 17°C and 25°C (6).

Figure 2: This image shows the fungal morphology of Batrachochytrium dendrobatidis at 1000X magnification using bright field imaging.  The specimen was stained using Lactophenol-Cotton Blue Stain (VWR) and reveals a mature, asexual sporangium with its associated zoospores and discharge papillae.

As a given sporangium on the plate matures, the plug at the end of the discharge papillae dissolves allowing retained zoospores to be ejected (1,5).  Note that a given sporangium may have one or more discharge papillae to eject the zoospores.  Next, the zoospores are released at the base of the colony towards the nutrient source and encyst to form mature sporangium (1,6).  As new sporangia form at the base of the colony immediately adjacent to the nutrient source, older, empty sporangia (those that do not contain any zoospores) are pushed upwards forming the visible, macroscopic clusters as seen in Figure 1(1).

Methods

Figure 1: A culture of Batrachochytrium dendrobatidis was obtained from Dr. Joyce Longcore from University of Maine and inoculated onto plain agar supplemented with 1% tryptone.  The new culture was then grown in the dark at 20ºC for seven days.  The image was taken with a 100mm f2.8 macro lens @f11 ISO 800 using a Pentax K-7, 14 megapixel DSLR.  The image was cropped and labeled using Adobe Photoshop CS5.

Figure 2: Batrachochytrium dendrobatidis was stained using Lactophenol-Cotton Blue and imaged through an Olympus CX41 microscope.  The specimen was viewed via bright field imaging using a 100X Oil Objective (1.25, Plan CN).  From a live-view feed of the sample using a Spot Idea™ digital camera (1.4 megapixels), an image of the specimen was captured using Spot computer software.  The image was cropped and labeled using Adobe Photoshop CS5.

Discussion

Batrachochytrium dendrobatidis is a pathogenic fungus that causes the potentially fatal amphibian disease chytridiomycosis (1,2,5).  Being highly infectious (1,2) and a having low host-specificity (4), this fungus infects the keratinized skin tissues of amphibians and has been a large concern among the scientific community in recent years (2,3,4).  Note that keratinized skin tissue is the outer layer of skin that becomes thickened when cells die and are filled with the protein keratin.  Since the fungus' first documented appearance in 1998 among declining Anuras (frogs) in the rain forests of Australia and Panama (2), B. dendrobatidis has been identified globally on six continents and has resulted in the decimation and extinction of several species of frogs and toads (3,4,6).  Estimates project that upwards of a third of all amphibian species are at risk from extinction largely as a result of lethal infections caused by the pandemic chytrid fungus (10).  Because of the catastrophic consequences of loosing such a vital component of the world’s biota, international efforts have been undertaken to understand the extent of the epidemic and save as many at-risk species of amphibians as possible.

References

1. Berger, L., Hyatt, A.D., Speare, R., and Longcore, J.E. 2005.  Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis Diseases of Aquatic Organisms.  68: 51–63.

2. Berger, L., Speare, R., Daszak, P., Green, D. E., Cunningham, A. A., Goggin, C. L., Slocombe, R., Ragan, M. A., Hyatt, A. D., McDonald, K. R., Hines, H. B., Lips, K. R., Marantelli, G., Parkes, H.  1998.  Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America.  Proceedings of the National Academy of Science, USA.  95: 9031-9036.

3. Carey, C., Cohen, N., Rollins-Smith, L.  1999. Amphibian declines: an immunological perspective.  Developmental and Comparative Immunology 23:459-472.

4. Daszak, P., Berger, L., Cunningham, A. A., Hyatt, A. D., Green, D. E., Speare, R.  1999.  Emerging Infectious Diseases and Amphibian Population Declines.  Emerging Infectious Diseases.  5: 735-748.

5. Longcore, Joyce E.; Pessier, A.P.; and Nicholas, D.K. 1999.  Batrachochytrium dendrobatidis gen. et.  sp. nov., a Chytrid Pathogen to Amphibians.  Mycologia 91: 219-227.

6. Piotrowski, Jeffrey S., Annis,  Seanna L., and Longcore, J. E. 2004.  Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians.  Mycologia, 96: 9-15.

7. Skerratt, Lee Francis; Berger, Lee; Speare, Richard; Cashins, Scott; McDonald, Keith Raymond; Phillott, Andrea Dawn; Hines, Harry Bryan; Kenyon, Nicole.  2007. Spread of chytridiomycosis has caused the rapid global decline and extinction of frogs.  EcoHealth 4:125-134. 

8. Stuart, S. N.; Chanson, J. S.; Cox, N. A.; Young, B. E.; Rodrigues, A. S. L.; Fischman, D. L.; Waller, R. W.  2004.  Status and trends of amphibian declines and extinctions worldwide.  Science 306:1783-1786.

9. Voyles, J., S. Young, L. Berger, C. Campbell, W.F. Voyles, A. Dinudom, D. Cook, R. Webb, R.A. Alford, L.F. Skerratt, and R. Speare.  2009.  Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines.  Science 326:582-585.

10.  Zippel, K. C., Mendelson III, J. R.  2008.  The Amphibian Extinction Crisis: A Call To Action.  Herpetological Review 39: 23-29.