Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 139-163). Also available on the World Wide Web.

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.

Os anfíbios apresentaram um severo declínio de suas populações nas últimas décadas. Dentre as diversas causas dos declínios estão as doenças infecciosas. O presente trabalho tem o objetivo de estudar a ocorrência de enfermidades selecionadas que podem acometer estes animais na Mata Atlântica brasileira, e, desta forma, contribuir para a melhor compreensão das conseqüências destas enfermidades para a conservação dos anfíbios em nosso meio. Foram coletados 120 animais da Estação Biológica da Boracéia e realizaram-se exames necroscópicos, histopatológicos, microbiológicos, parasitológicos e PCR convencional para Batrachochytrium dendrobatidis em grande parte dos animais. Coletou-se 33 espécies diferentes pertencentes a sete famílias diferentes. Encontrou-se, no exame necroscópico, endoparasitas em 45,8% (55/120) dos espécimes examinados e ectoparasita (sanguessuga) em um espécime. Do total de animais analisados para Batrachochytrium dendrobatidis, 19,1% (22/115) foram positivos para Bd. O exame necroscópico assim como o histopatologicos da grande maioria dos animais estudados não exibiu alterações relevantes, sendo que os sistemas mais acometidos foram o digestório e tegumentar seguidos pelos urinário e linfóide. A somatória dos resultados aqui exibidos sugere que, nas condições específicas deste trabalho, os agentes identificados não induziram processos patológicos relevantes, sinalizando uma relação hospedeiro x parasita simétrica. Porém, face ao frágil equilíbrio existente no ambiente da Mata Atlântica e a suscetibilidade dos anfíbios às mudanças dos ecossistemas, cremos que monitoramentos de longo prazo sejam necessários para que se possa acompanhar, com alguma margem de segurança, a evolução desta delicada relação. / Amphibians suffered a severe decline of their populations in recent decades. Among the various causes of the declines are infectious diseases. The present work aims to study the occurrence of selected diseases that can affect these animals in the Brazilian Atlantic Forest, and thus contribute to a better understanding of the consequences of these diseases for the conservation of these animals. We collected 120 animals from the Biological Station of Boracéia and performed necropsy, histopathology, microbiology, parasitology and conventional PCR for Batrachochytrium dendrobatidis exams in most animals. We collected 33 different species belonging to seven different families. It was found at necropsy, endoparasites in 45.8% (55/120) of specimens examined and an ectoparasite in only one specimen. Of the total number of animals analyzed for Batrachochytrium dendrobatidis, 19.1% (22/115) were positive for this fungus. The necropsy and histopathological exams from the vast majority of animals studied did not show significant changes. The most affected systems were the digestive and integumentary followed by the lymphoid and urinary. The results shown in this work suggests that the infections agents did not induce relevant pathological processes in the specific conditions of this research. However, given the fragile balance of the Atlantic Forest and the susceptibility of amphibians to ecosystem change, we believe that long-term monitoring is needed to follow with some safety margin, the evolution of this delicate relationship.

Os anfíbios apresentaram um severo declínio de suas populações nas últimas décadas. Dentre as diversas causas dos declínios estão as doenças infecciosas. O presente trabalho tem o objetivo de estudar a ocorrência de enfermidades selecionadas que podem acometer estes animais na Mata Atlântica brasileira, e, desta forma, contribuir para a melhor compreensão das conseqüências destas enfermidades para a conservação dos anfíbios em nosso meio. Foram coletados 120 animais da Estação Biológica da Boracéia e realizaram-se exames necroscópicos, histopatológicos, microbiológicos, parasitológicos e PCR convencional para Batrachochytrium dendrobatidis em grande parte dos animais. Coletou-se 33 espécies diferentes pertencentes a sete famílias diferentes. Encontrou-se, no exame necroscópico, endoparasitas em 45,8% (55/120) dos espécimes examinados e ectoparasita (sanguessuga) em um espécime. Do total de animais analisados para Batrachochytrium dendrobatidis, 19,1% (22/115) foram positivos para Bd. O exame necroscópico assim como o histopatologicos da grande maioria dos animais estudados não exibiu alterações relevantes, sendo que os sistemas mais acometidos foram o digestório e tegumentar seguidos pelos urinário e linfóide. A somatória dos resultados aqui exibidos sugere que, nas condições específicas deste trabalho, os agentes identificados não induziram processos patológicos relevantes, sinalizando uma relação hospedeiro x parasita simétrica. Porém, face ao frágil equilíbrio existente no ambiente da Mata Atlântica e a suscetibilidade dos anfíbios às mudanças dos ecossistemas, cremos que monitoramentos de longo prazo sejam necessários para que se possa acompanhar, com alguma margem de segurança, a evolução desta delicada relação. / Amphibians suffered a severe decline of their populations in recent decades. Among the various causes of the declines are infectious diseases. The present work aims to study the occurrence of selected diseases that can affect these animals in the Brazilian Atlantic Forest, and thus contribute to a better understanding of the consequences of these diseases for the conservation of these animals. We collected 120 animals from the Biological Station of Boracéia and performed necropsy, histopathology, microbiology, parasitology and conventional PCR for Batrachochytrium dendrobatidis exams in most animals. We collected 33 different species belonging to seven different families. It was found at necropsy, endoparasites in 45.8% (55/120) of specimens examined and an ectoparasite in only one specimen. Of the total number of animals analyzed for Batrachochytrium dendrobatidis, 19.1% (22/115) were positive for this fungus. The necropsy and histopathological exams from the vast majority of animals studied did not show significant changes. The most affected systems were the digestive and integumentary followed by the lymphoid and urinary. The results shown in this work suggests that the infections agents did not induce relevant pathological processes in the specific conditions of this research. However, given the fragile balance of the Atlantic Forest and the susceptibility of amphibians to ecosystem change, we believe that long-term monitoring is needed to follow with some safety margin, the evolution of this delicate relationship.

Amphibian diversity has declined, in part, due to the infectious disease chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). Andean water frogs in the genus Telmatobius are particularly vulnerable to the disease and the genus has been extirpated from Ecuador and in Andean cloud forests, yet populations of species persist in the high Andes of Peru and Bolivia. The Alpaca Water Frog (Telmatobius intermedius), endemic to the Peruvian Andes, can be found infected with Bd. Alpaca Water Frogs inhabit high elevation open canopy freshwater systems. My overall goal was to study the effect of chytrid infection on the trophic ecology of Telmatobius tadpoles. I used stable isotope ratios of carbon (δ13C) and nitrogen (δ15N) to characterize the the trophic structure and energy flow in this system. I observed the values of δ15N were higher for tadpoles than algal material (t-test, t= -8.60, df= 34, p< 0.01), mayfly nymphs (t-test, t= 5.25, df= 30, p< 0.01), and predatory aquatic invertebrates (t-test, t= -4.18, df= 47, p< 0.01). In regard to the δ15N values of tadpoles and frogs, tadpoles had a lower value (t-test, t= -3.0, df= 31, p< 0.01). Values of δ15N in tadpoles were relatively high, signaling the presence of animal tissue in their diet. I also investigated changes in tadpole diet associated with mouthpart deformities caused by the fungal pathogen Bd. There was a positive association between the extent of mouthpart deformity and Bd infection (Fisher’s Exact test, p<0.001). The relative proportions of diatom morphotaxa groups found in the foregut of T. intermedius tadpoles varied in association with the degree of mouthpart deformity, as indicated by an ANOSIM analysis (R=0.875, p<0.001). Finally, in addition to Bd prevalence in adult aquatic frogs, I investigated whether Alpaca Water Frogs and other Andean Water Frogs tested positive for the antifungal bacterium Janthinobacterium lividum (Jliv). My results show that 57% of the sampled frogs were infected with Bd, 12.5% of frogs hosted both Jliv and Bd, while 7.2% hosted just Jliv. We found that the probability of an individual being infected with Bd was independent of the presence of Jliv; however, we did detect a protective effect of Jliv with respect to intensity of infection. My findings demonstrate that the fungal pathogen Bd influences the trophic ecology of tadpoles of Andean water frogs.

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

La declinación de los anfibios a nivel mundial, e incluso la desaparición de algunas especies, ha sido un fenómeno de especial importancia para muchos científicos en los últimos años. Del total de las especies de anfibios que se conocen en el mundo hasta el momento, un tercio se encuentra amenazada y 165 especies, parecen haberse extinguido a partir de 1980. Los factores que influyen en esta declinación son muchos, y hasta el momento se siguen discutiendo; pero las enfermedades, han sido reconocidas como uno de los factores que más han contribuido a esta declinación. La quitridiomicosis es una enfermedad infecciosa emergente que, en los últimos años, ha sido vinculada a la muerte masiva de algunas especies de anuros. Esta enfermedad es causada por el hongo acuático zoospórico Batrachochytrium dendrobatidis- Bd- (Orden Rhyzophydiales) que se encuentra actualmente distribuido en todos los continentes con excepción de la Antártida. Bd infecta la epidermis estratificada de la piel de los anfibios anuros y urodelos y sus talos se desarrollan dentro de las células epidérmicas principalmente en los dedos y la parte ventral del cuerpo y en el disco oral queratinizado de las larvas. La infección afecta muchas funciones de la piel de los anfibios, como la osmoregulación y la respiración a través de este órgano. Entre las poblaciones de anfibios es posible encontrar un gran rango de susceptibilidades a la infección por Bd. Las larvas y algunos estadios post-larvales de anuros y urodelos parecen desarrollar una infección subclínica por lo que actúan como reservorios de Bd y esparcen el hongo por diferentes ambientes y especies. Teniendo en cuenta lo anteriormente expuesto, en este trabajo de tesis se plantearon los siguientes objetivos: *Analizar la presencia de Bd en distintas especies de anuros nativas en el área de estudio seleccionada y en anuros provenientes de otras áreas. *Aislar la cepa nativa de Bd a partir de especies de anuros autóctonos de Argentina. *Analizar genéticamente la cepa autóctona y evaluar su similitud con cepas de Bd de otros países. *Analizar la susceptibilidad y sensibilidad de las distintas especies de anuros a la infección por Bd en bioensayos de laboratorio.

Thesis (Ph. D.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 115-128). Also available on the World Wide Web.

Anurans are declining worldwide because of the spread of Batachochytrium dendrobatidis (Bd), the fungus that causes chytridiomycosis. However, some frogs are resistant to this disease, and understanding why may be critical to saving those that are susceptible. In Peru, Gastrotheca excubitor is resistant to chytridiomycosis while Gastrotheca nebulanastes is susceptible. Two anuran skin defenses, symbiotic bacteria and antimicrobial peptides (AMPs), have demonstrated the ability to inhibit Bd in vitro when isolated from certain frogs. We tested if these defenses can explain the difference in susceptibility between the two Gastrotheca species. The cutaneous bacteria and AMPs of both species were collected, tested for their abilities to inhibit the growth of Bd, and analyzed for their compositions. Results indicate that 34%of the strains of skin bacteria from G. excubitor were able to inhibit the growth of Bd whereas only 10% isolated from G. nebulanastes were effective. Gastrotheca excubitor also has stronger anti-Bd skin bacteria. Neither frog species has peptide mixtures capable of completely inhibiting Bd, and overall species did not differ in the anti-Bd abilities of their peptides. These results suggest that the chytridiomycosis resistance experienced by G. excubitor may be attributed to its skin bacteria.