No description available.

Chytridiomycosis is an emerging cutaneous fungal disease that contributes to recent global declines and extinction of amphibian species, caused by infection of the skin with a fungus known as Batrachochytrium dendrobatidis (Bd). Many species of frogs secrete antimicrobial peptides onto their skin that are capable of killing Bd. This thesis is an investigation of the effect of corticosteroids on cutaneous innate immunity in frogs, in the context of infection with Bd. The general hypothesis was that injections of glucocorticoids would impair the cutaneous synthesis of these antimicrobial peptides, thereby increasing susceptibility to Bd infection. The objective of the first experiment was to measure and compare gene expression levels of cutaneous AMP’s in frogs treated with glucocorticoids with sham-treated controls. Wild-caught Lithobates pipiens were acclimatized and administered either the corticosteroid methylprednisolone or saline every 48 hours. Norepinephrine-elicited cutaneous secretions were collected prior to the first injection of corticosteroid or saline, and then every 8 days for 40 days. Gene expression of the AMP’s brevinin and ranatuerin in the cutaneous secretions was quantified relative to the reference genes EF1-α and RPL8 using reverse transcription quantitative polymerase chain reaction (RT-qPCR). Corticosteroid treatment was associated with a significant (P<0.027) increase in brevinin gene expression, which was most notable at 24-40 days of corticosteroid administration. Ranatuerin expression followed a similar but nonsignificant trend. The second experiment was a pilot study intended to establish a Bd challenge protocol in L. pipiens. Frogs were immersed in water containing 0, 104, 105 or 106 zoospores of Bd strain JEL 423. Cutaneous swabs were collected prior to challenge and tested for Bd by qPCR; unexpectedly, some tested positive, indicating pre-challenge infection. The analysis was complicated by an identified cross-reactivity of the assay with other fungi. The findings of the first experiment refuted the hypothesis, and suggested that corticosteroids promote rather than impair AMP gene expression in the skin of L. pipiens, under these experimental conditions. Further, the second study demonstrated that none of the frogs showed clinical abnormalities or died, despite exposure to Bd zoospores and despite molecular and histologic evidence of cutaneous Bd infection in some frogs. / NSERC Discovery Grant

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 / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.

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 / Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2009.

Amphibienpopulationen sind weltweit bedroht. Für fundierte Entscheidungen im Naturschutz ist ein wissenschaftliches Hintergrundwissen notwendig. Eine wichtige Komponente ist die Verbreitung der Arten und die Gründe hierfür. Dies setzt auch Klarheit bezüglich des taxonomischen Status voraus. Vernachlässigte Regionen liegen meist in den Tropen. Um diese Lücke zu füllen, untersucht die vorliegende Arbeit die Makroökologie westafrikanischer Amphibien. Eine neue Art der Familie Phrynobatrachidae wird beschrieben. Anschließend werden die taxonomischen Unterschiede zweier Mitglieder der Familien der Hyperoliidae und der Arthroleptidae untersucht. Dies ebnet den Weg für die Makroökologie. Es wird analysiert, ob Westafrika eine einzigartige biogeographische Region ist. Die Untersuchung zeigen, dass Westafrika in der Tat einzigartig ist. Die Ähnlichkeiten innerhalb der Region sind größer als die innerhalb ähnlicher Habitate aus unterschiedlichen Regionen. Der Cross River ist die wichtigste Barriere. Mehrere geographische Zonierungen innerhalb Westafrikas werden entdeckt. Es wird untersucht, ob der Chytrid Pilz, Batrachochytrium dendrobatidis, (Bd), ein wichtiger Faktor beim Rückgang der Amphibien Populationen, in West Afrika vorkommt. Bisher gibt es keinen positiven Nachweis westlich Nigerias. Dennoch sagen Modelle geeignete Habitate für Bd vorher. Die wahrscheinlichste Erklärung ist, dass die Dahomey Gap als natürliche Barriere die Ausbreitung von Bd verhindert. Als letztes werden die Nischen westafrikanischer Amphibien eruiert. Für die meisten Arten werden Nischenmodelle berechnet. Dies bestätigt bereits bekannte Gebiete hoher Alpha Diversität und zeigt bisher unbekannte Gebiete auf. Des Weiteren werden Erklärungen für unterschiedliche Verbreitungsgebietsgrößen gesucht. Generell wird die Nischenbreite hierfür verantwortlich gemacht. Die vorliegenden Daten lassen allerdings auch den Schluss zu, dass das Ausbreitungsvermögen das beobachtete Muster ebenfalls erklären kann. / Amphibian populations are declining globally. For informed conservation decisions a sound scientific background is needed. One major component is species distribution and the underlying causes. This also requires clarity on the taxonomic status. Often neglected regions are located in the tropics. In order to fill this gap, the present thesis examines the macroecology of West African amphibians. A new species of the family Phrynobatrachidae is described. Afterwards, the taxonomic differences between morphologically similar members of the families Hyperoliidae and Arthroleptidae are discussed. Beside other studies, this sets the field for macroecology. It is tested whether West Africa is a unique biogeographic region. The similarity of amphibian assemblages from Sub-Saharan Africa is analysed and it is shown that West Africa contains unique assemblages. Similarities within the region are higher than similarities between habitats across different regions. The main barrier towards Central Africa is the Cross River. Several geographic divisions within West Africa are detected. It is examined whether the chytrid fungus Batrachochytrium dendrobatidis (Bd), one important factor for amphibian population declines in many other regions, occurs in West Africa. So far there is no positive record west of Nigeria. However, models predict that environmental suitability for Bd is high. The most plausible explanation for the absence is that the Dahomey Gap acted as a natural barrier against the spread of the Bd. Finally, the niches of West African amphibians are investigated. For most species environmental niche models are calculated. This confirms previously known areas of high alpha diversity and so far unknown species rich areas are detected. In a further study, explanations for differing range sizes are searched for. Niche breadth is commonly assumed to be the general cause. However, the analysed data also suggests that dispersal ability can also explain the observed pattern.

<p dir="ltr">In this dissertation, I explore how human actions (climate change, road salt, land use change, species invasions) interact with and influence morphology, disease, and population dynamics in freshwater organisms (amphibians and aquatic crustaceans). First, I examined how the incidence and timing of disease epidemics in native species (<i>Daphnia dentifera</i>) caused by a generalist parasite (<i>Metschnikowia bicuspidata</i>) influenced the success and impact of an invasive species (<i>Daphnia lumholtzi</i>) in freshwater zooplankton (Chapter 1). In the following chapter, I explored how host-parasite interactions are affected by the interactive effects of multiple environmental stressors, focusing on American bullfrog tadpoles (<i>Lithobates catesbeianus</i>), two of their common parasites (<i>Batrachochytrium dendrobatidis</i> (<i>Bd</i>) and trematode parasites in the family Echinostomatidae), and two common stressors (fluctuating temperatures and sublethal road salt pollution; Chapter 2). Finally, I investigated how the combination of climate (temperature and precipitation) and land use (developed and/or forested area) change have influenced the body size of a common toad (Fowler’s toad, <i>Anaxyrus fowleri</i>) from 1930 – 2020 utilizing museum specimens (Chapter 3). Together, this research establishes how emerging and persistent anthropogenic environmental stressors will interact to affect morphology, disease, and population dynamics in vulnerable freshwater organisms.</p>