Aspects of amphibian chytrid infections in South Africa

Abstract

The waterborne pathogen Batrachochytrium dendrobatidis (Bd), amphibian chytrid, is implicated as being the causative agent for global amphibian declines. The fungus attacks the keratinized skin of adult and postmetamorphic animals and the keratinized mouthparts of tadpoles. Postmetamorphic animals seem to be more susceptible to Bd than tadpoles and adult frogs. Hypotheses exist that the origin of the fungus is in Africa. During the study different aspects of Bd infections in South African frogs were examined including the distribution of Bd, cultivation of Bd, preservation of cultures, the morphology of Bd as an infection as well as in culture and finally differences in host defense. Positive and negative localities for Bd were identified through surveys conducted in South Africa. These data will be contributed to the Bd Mapping Project and the African Bd Database in order to determine whether chytrid has any environmental preferences. Cultures obtained from the positive localities were maintained and cryopreserved for use in numerous experiments. In a future study, DNA extractions from the cultures will be analyzed using multilocus sequence typing in order to determine the sequence type of South African strains in comparison with global strains. This will provide important epidemiological information concerning the origin and control of Bd. The morphology of Bd was also examined using scanning electron microscopy and laser scanning confocal microscopy. Damage due to Bd infections was more severe on the larval mouthparts of Amietia vertebralis than that of Hadromophryne natalensis. The adverse effect of Bd is therefore not limited to postmetamorphic animals. Confocal microscopy uses fluorescent stains and lasers to examine specific structures within organisms. An especially effective stain used during confocal microscopy on Bd is Calcofluor White M2R. Due to its specificity this stain can be used as an effective screening tool for Bd in tissue. The role of antimicrobial skin peptides as a defense against Bd was also examined. A. vertebralis experiences die-offs due to chytrid, while H. natalensis does not experience the same effect in the presence of Bd. H. natalensis possess more antimicrobial skin peptides against Bd with a higher effectiveness than peptides extracted from A. vertebralis. This may explain the observed susceptibility of A. vertebralis to Bd. The relevance of this study is in order to identify areas in South Africa in which the probability of finding Bd is high. This will help in the surveillance of Bd and in the identification of susceptible species to be monitored and protected against the fungus. The effect of Bd on frog species can also be determined by means of exposure experiment using cultures isolated during this study. Through the identification of peptides effective against Bd, predictions can be made with regard to the susceptibility of different frogs to Bd, improving our ability to protect the amphibian biodiversity in South Africa. With the use of confocal microscopy in the examination of Bd, we became the first group to use the method. By the identification of a stain with a high potential as a screening tool, we also contributed to the more efficient identification of Bd in tissue. Keywords: Batrachochytrium dendrobatidis, Bd, amphibian chytrid, distribution, cultivation, antimicrobial skin peptides, laser scanning confocal microscopy, Amietia vertebralis, Hadromophryne natalensis, South Africa