Thesis (Ph.D.) - James Cook University, 2006. / Typescript (photocopy) Bibliography: leaves 90-102.

Thesis (M.Sc.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2009/06/25). Includes bibliographical references.

The worldwide decline of amphibians is due to several interacting factors that vary in their involvement and severity according to species, geography, environment, and individual response (Wake and Vredenburg 2008; Gahl et al. 2011). One of those threats has caused population declines globally (Stuart et al. 2004), Batrachochytrium dendrobatidis (Bd), which is the fungal pathogen that causes chytridiomycosis in amphibians (Berger et al. 1998; Longcore et al. 1999). Bd's effects are not completely known since some areas of the world have been studied (e.g., Panama, Lips et al. 2006; United States Sierra Nevada, Briggs et al. 2010; Australia, Phillott et al. 2013) while other areas have received little or no attention, often because these systems appear stable or because the effect of threats are not known. In the Midwestern United States, widespread anuran population declines occurred historically and are in some cases ongoing (e.g., Vogt 1981, Oldfield and Moriarty 1995, Brodman and Kilmurry 1998, Casper 1998, Hay 1998, Moriarty 1998, Mossman et al. 1998, Varhegyi et al. 1998, Steiner and Lehtinen 2008, Zippel and Tabaka 2008). Large-scale habitat alterations, chemical contaminations, and other threats have likely caused some Midwestern U.S. amphibian declines (Lannoo, 1998), but the role of Bd in historic and current population declines has been limited to small population surveys or incidental discovery of Bd (e.g., Pessier et al. 1999; Beasley et al. 2005; Steiner and Lehtinen 2008). I investigated the current and historic Bd infection levels among amphibians in Illinois and identified species risk factors associated with likelihood of chytridiomycosis-related death. My research questions focused on which biotic and abiotic factors explained Bd prevalence and intensities among current populations, which species risk factors would make them more likely to suffer severe Bd infection, and what the historic Bd status was in Illinois. Working with Illinois amphibians presented the opportunity to answer these research questions because Bd was already known to occur in Illinois (Pessier et al. 1999), there were a variety of anecdotal examples of historic population declines in Illinois (Beasley et al. 2005; Lannoo 1998), and extensive museum holdings were available to document the spatial and temporal pattern of Bd among Illinois populations. In the chytridiomycosis-amphibian disease system, mortality is driven by intensity of infection. Intensity is affected by many factors, including environmental temperatures, amphibian community composition, and fungal traits. However, the relative importance of biotic and abiotic factors on Bd prevalence and intensity in multispecies, natural communities is unknown for any wild populations. In 2008-2009, I conducted one of the first large-scale strategic surveys of both current and historic presence of Bd. I sampled 4,691 Illinois amphibians from current and historic populations to provide a framework of historic Bd infection and current status, and used those results to identify at-risk populations based on natural history and species risk factors. I tested 2,804 amphibians from 12 species for Bd, and determined that Bd was present in all sites, wetlands, and in all species in both years. Statewide prevalence was relatively high (2008 &mu = 34%; 2009 &mu = 55%), as was average individual infection intensity (2008 &mu = 1,773 Zswab; 2009 &mu = 2,159 Zswab). Wetland water temperature best explained Bd prevalence, but several biological factors explained intensity. Higher Bd intensities were correlated with species richness and indicated an amplification effect (Ostfeld and Keesing 2012). Hylid treefrogs may be amplifying species because they had the highest infection intensities and their presence was correlated with increased infection in other taxa. Bd can cause declines and extinctions in amphibian populations (Stuart et al. 2004), but other threats may also be involved (Collins and Storfer 2003). In Illinois, amphibian populations may be threatened by a variety of assaults including disease, habitat loss, chemical contaminants, and invasive species (Lannoo 1998). Management for biodiversity typically focuses on identifying and mitigating threats and prioritizing species susceptibility by identifying risk factors. I proposed to study whether species risk factors for Bd also signal general susceptibility to other threats (e.g., Lips et al. 2003; Bielby et al. 2008; Cooper et al. 2008) in Illinois amphibians. I identified nine potential risk factors for each of 21 Illinois species form the literature, and compared association of those traits with disease intensity. I used Bd intensity data from 1,445 Bd-positive amphibians collected 2008-2010. As in Chapter 2, I found that both biological and environmental factors explained disease intensity at the species levels: air temperature during the breeding season was the best predictor of infection intensity with three species biological traits also contributing (i.e., longevity, clutch size, and aquatic index). Conservation status did not explain Bd intensities, likely because conservation status is based upon rarity, population trends, and presence of threats, but which does not always include Bd susceptibility. Since most of the study species were common prior to my disease survey with relatively stable populations with no prior Bd threat, the conservation statuses used in this analysis did not predict Bd risk. Now that I have shown Bd to be widespread and at high intensities in the state, a reassessment of data included in the species status would be timely and might be warranted. I found that Bd was geographically and taxonomically widespread in Illinois, which suggested an established infection status, perhaps longer than the first report from the 1990s (Pessier et al. 1999). Also, this suggests that population declines from chytridiomycosis might have occurred historically. I used museum holdings to determine spatial and temporal distributions of Bd in Illinois amphibians. I tested 1,008 museum specimens from the vertebrate collections at Southern Illinois University, Illinois Natural History Survey, and the University of Illinois Museum of Natural History to determine the oldest date of anuran Bd infection in Illinois. I detected 110 Bd positive specimens (10.7%, CI: 9.0-12.8%) in four species collected during the 1890s-1980s. The earliest Bd record was from a Lithobates sphenocephalus collected in southern Illinois in 1900. I determined that Illinois amphibians have been living endemically with Bd for at least 113 years, extending the date of the oldest U.S. record of Bd infection by 61 years. The long-term presence of Bd, coupled with multiple anecdotal reports of population declines, suggest that Bd may have been involved in historic population declines in Illinois amphibians. I found widespread taxonomic and geographic distribution of Bd among current and historic populations of Illinois amphibians. I found a surprisingly long history of Bd in Illinois that transforms the way we consider impacts on historic species and potential co-evolution of disease in Midwestern U.S. amphibians. My finding is as old as the oldest records from Brazil, Africa, and Asia (Weldon et al. 2004; Goka et al. 2009; Schloegel et al. 2010, 2012), suggesting a more ancient history of Bd and amphibians.

Amphibian populations have undergone unprecedented declines in recent decades. Many of these declines are due to the spread of the cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis. The Panamanian golden frog (Atelopus zeteki) has not been seen in the wild since Bd spread through western Panama. In response to initial declines, golden frogs were collected from wild populations and placed in captive colonies with the goal of future reintroductions. An understanding of this species' natural defense mechanisms against Bd is needed for reintroduction to be successful. Previous studies indicate that cutaneous bacteria are an important defense mechanism for some amphibians and applying antifungal bacteria to the skin of Bd-susceptible amphibians (probiotic therapy) can prevent chytridiomycosis. Therefore, the goals of my dissertation were to characterize the bacterial community of A. zeteki and determine if probiotic therapy could be used to prevent chytridiomycosis in this species. I initially characterized the bacterial community of wild and captive golden frogs using samples collected prior to the initial declines and after approximately eight years in captivity. I found that the community structure of the microbiota was significantly different between wild and captive frogs; however, the offspring of the original captive frogs still shared 70% of their microbial community with wild frogs. Then, I characterized the Bd-inhibitory properties of 484 bacteria isolated from 11 species of free-living Panamanian amphibians. I found a large proportion of bacteria (75.2%) had the ability to inhibit Bd and this trait was widely distributed among bacterial taxa, although there was also significant variation within bacterial genera in their ability to inhibit Bd growth. I then experimentally tested the ability of four of these isolates to prevent chytridiomycosis in captive golden frogs. None of them successfully prevented infection; however, there were several frogs that cleared infection and this was correlated with composition of the bacteria initially present on their skin. Overall these results demonstrate that the structure of microbial communities of A. zeteki are important to host health and building on this might provide the best hope for reintroducing this iconic species back to its native habitat. / Ph. D.

The amphibian pathogen, Batrachochytrium dendrobatidis (Bd), has been the cause of mass declines of amphibian populations worldwide (Berger et al. 1998). This pathogen has been shown to infect approximately 387 different amphibian species and causes declines in approximately 200 species (Skerratt et al. 2009). The total impact on amphibian biodiversity as well as their ecosystems has yet to be determined but it has already been suspected in some species extinctions (Schloegel et al. 2006). The distribution of this amphibian pathogen has been described by two competing hypotheses, the novel and endemic pathogen hypotheses. The endemic pathogen hypothesis states that the pathogen has always been a part of the ecosystem and has only recently become pathogenic due to environmental factors. The novel pathogen hypothesis states that the pathogen has just recently been introduced and has encountered a naïve host which has resulted in population declines (Rachowicz et al. 2005). Research into these two hypotheses has been very active yet the results have still been conflicted (Pounds et al. 2006; James et al. 2009). In our study we assess two relatively under surveyed locations for the presence of Bd, both in Peninsular Malaysia and British Columbia (BC). The results of the amphibian survey showed that Bd was currently ubiquitous throughout the province of BC. This was coupled with a population genetic evaluation of two Bd strains in British Columbia which led us to conclude that they were a part of a novel pathogen which may have been introduced through the amphibian trade possibly from the east coast of Canada. During the first two years of surveying for the presence of Bd in Peninsular Malaysia we found no evidence of the pathogen. In the third and final year of the survey we did discover low prevalence of the pathogen, which was supported by a recently published report of initial Bd detection in Peninsular Malaysia (Savage et al. 2011). We were not able to definitively state which of the competing hypotheses (NPH vs EPH) was correct for either collection region. Our population genetic results for two isolates collected from Bullfrogs on Vancouver Island suggest that Bd may have been introduced via the animal trade however the endemicity for the rest of the province remains unresolved. In peninsular Malaysia Bd may represent a novel pathogen or it could exist as an endemic pathogen with a low prevalence. / Graduate

Master of Science / Department of Clinical Sciences / James W. Carpenter / In many species, temporary increases in glucocorticoids (GC) can be used to identify changes in adrenal activity in response to acute stressors. For this research, GC metabolites were identified in fecal extracts from various Atelopus species. The objectives were to identify possible correlates between GCs and health status, assess the impact of husbandry protocols on adrenal activity, and evaluate the sub-lethal effects of antifungal bacteria used for protection of frogs against the chytrid fungus (Batrachochytrium dendrobatidis; Bd). The first study examined whether fecal GC concentrations can be correlated with animal health and behavior changes in a captive setting. Atelopus zeteki with varying degrees of dermatitis were categorized based on the severity of their skin abnormalities and GC metabolite concentrations were analyzed to detect correlations between severity of disease and GC metabolite concentrations. Similarly, behaviors that may indicate elevated stress levels (e.g., time spent in hide) were analyzed to detect correlation between disease and behavior changes. There was no correlation between fecal GC metabolites and health status of the animal or between health status and amount of time spent in hide. The second study established ex situ colonies of two Panamanian frog species, Atelopus certus and Atelopus glyphus, to determine how male group size affects behavior and GC levels. When housed in groups of eight, animals initially had elevated GC concentrations and interacted aggressively, but these instances declined substantially in the first 2 weeks of being housed together. Thus, captive Atelopus populations can be housed in same-sex enclosures without causing sub-lethal stress on the individuals involved. The third study examined the ability of antifungal bacterium from Central America to propagate on Atelopus skin as a preventative treatment for Bd and the sub-lethal effects of each bacteria species on adrenal function based on GC analysis. Four species of bacteria (Pseudomonas sp., Pseudomonas putida, Chryseobacterium indolgenes, and Stenotrophomonas maltophili) were found to be successful Bd inhibitors in vitro. There were no detectable effects of bacterial exposure with GC metabolite concentrations over time for any of the treatments assessed.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / There have been many cases reported of amphibian populations declining. These are often due to anthropogenic factors such as habitat destruction and pollution. However, some declines have not had an obvious cause and many of these have been investigated and found to be due to pathogenic disease. Batrachochytrium dendrobatidis is a recently described pathogen of frogs. The population declines that have been associated with chytridiomycosis have occurred in relatively undisturbed areas such as national parks. The declines tend to occur at higher altitudes or in colder climates. This is thought to be because of the frog immune system being slower at lower temperatures. Chyt1id fungus has been found in frog populations throughout the world. Little research has been carried out in Africa although, chytridiomycosis has already been seen in Kenya and South Africa. In this project frogs were sampled from selected transects in the Western Cape and three sites in the Northern Cape. The effect of altitude on the occurrence of infection was tested in the Western Cape. It was found that 18 frogs were infected in the Western Cape and the effect of altitude was not significant. Large numbers of dead and dying frogs were found in two of the Northern Cape sites and the incidence of chytridiomycosis was high in these populations. Chytrid was found in two Bufogariepensis from the Eastern Cape and in Xenopus petersii from Kasanka National Park, Zambia. More research is needed on chytridiomycosis in these populations. The frogs in the Western Cape seem to survive with chytrid fungus infection whereas, the frogs in the Northern Cape are dying. This suggests another factor acting on the Northern Cape frog populations.

Environmental and anthropogenic stressors negatively affect amphibians in a variety of ways, often increasing their vulnerability to pathogen infection and mortality. Sampling for the pathogenic fungus Batrachochytrium dendrobatidis (Bd) was conducted in order to: 1) determine the presence of chytrid infection in stream-associated plethodontid salamanders of southeastern Kentucky, and 2) evaluate differences in infection intensity between salamanders residing in intact forest streams, timber-harvested streams, and surface-mined streams. During 14 sampling sessions occurring between March, April and May of 2013, DNA samples from 306 individual salamanders within 8 species from the family Plethodontidae were collected; additional amphibians (i.e. frogs, newts) were opportunistically sampled when encountered. Approximately 2.1% of the salamanders and 50% of the frogs sampled from intact streams, 2.3% of the salamanders and 80% of the frogs sampled from the harvested streams, and none of the salamanders and 100% of the frogs sampled from the mined streams tested positive for Bd. No significant differences in occurrence of Bd or infection intensity were detected between the treatment sites (x2 = 0.59; p-value = 0.75), or between individuals of a species between different treatments (see tables). These findings are the first to demonstrate that Batrachochytrium dendrobatidis is present in amphibians of eastern Kentucky.

The sudden appearance of chytridiomycosis, as the cause of amphibian deaths and population declines in several continents suggests that its etiological agent, the amphibian chytrid fungus Batrachochytrium dendrobatidis, was introduced into the affected regions. However, the origin of this virulent pathogen is unknown. Efforts were directed to determine the occurrence of chytridiomycosis in Africa, whether the disease had been introduced into South Africa in recent years and how wild frog populations were affected by infection. A chytridiomycosis survey of 2,300 archived and live specimens involving members of the Pipidae family in sub-Saharan Africa, as well as a number of unrelated frog species in South Africa was conducted by histological diagnosis of skin samples. The epidemiological evidence indicated that chytridiomycosis has been a stable endemic infection in southern Africa for 23 years before any positive specimens were found outside Africa. The occurrence of chytridiomycosis in South Africa can be described as widespread both in terms of geographical distribution and host species and generally infection is not associated with adverse effects at the individual or population level. It was proposed that the amphibian chytrid originated in Africa and that the international trade in the African clawed toad Xenopus laevis that commenced in the mid 1930s was the means of dissemination. A risk assessment of the X. laevis trade demonstrated that chytridiomycosis could spread through this pathway and culminated in the development of a management protocol to reduce the risks of spreading disease through this animate commodity. Initial comparative genetic analysis of B. dendrobatidis strains isolated from South African frogs with a global set of 35 strains, suggests that analysis of a more geographically diverse set of southern African strains is needed before this line of argument can support or reject the "out of Africa" hypothesis. / Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005.

The sudden appearance of chytridiomycosis, as the cause of amphibian deaths and population declines in several continents suggests that its etiological agent, the amphibian chytrid fungus Batrachochytrium dendrobatidis, was introduced into the affected regions. However, the origin of this virulent pathogen is unknown. Efforts were directed to determine the occurrence of chytridiomycosis in Africa, whether the disease had been introduced into South Africa in recent years and how wild frog populations were affected by infection. A chytridiomycosis survey of 2,300 archived and live specimens involving members of the Pipidae family in sub-Saharan Africa, as well as a number of unrelated frog species in South Africa was conducted by histological diagnosis of skin samples. The epidemiological evidence indicated that chytridiomycosis has been a stable endemic infection in southern Africa for 23 years before any positive specimens were found outside Africa. The occurrence of chytridiomycosis in South Africa can be described as widespread both in terms of geographical distribution and host species and generally infection is not associated with adverse effects at the individual or population level. It was proposed that the amphibian chytrid originated in Africa and that the international trade in the African clawed toad Xenopus laevis that commenced in the mid 1930s was the means of dissemination. A risk assessment of the X. laevis trade demonstrated that chytridiomycosis could spread through this pathway and culminated in the development of a management protocol to reduce the risks of spreading disease through this animate commodity. Initial comparative genetic analysis of B. dendrobatidis strains isolated from South African frogs with a global set of 35 strains, suggests that analysis of a more geographically diverse set of southern African strains is needed before this line of argument can support or reject the "out of Africa" hypothesis. / Thesis (Ph.D. (Zoology))--North-West University, Potchefstroom Campus, 2005.