As invasões biológicas são consideradas a segunda maior causa de perda de biodiversidade, pois podem causar diversos impactos ecológicos. Recentemente foi chamado à atenção um novo mecanismo pelo qual espécies invasoras podem afetar as nativas: através da interferência no nicho acústico. O mascaramento dos sinais a partir de fontes de ruídos pode afetar diretamente a fisiologia reprodutiva ou o consumo de energia. A vocalização está diretamente ligada com a seleção sexual em anfíbios, aves, insetos e muitos mamíferos, de maneira que a presença de novas fontes sonoras no ambiente pode afetar a aptidão das espécies. Uma das piores invasoras do mundo, a rã-touro Lithobates catesbeianus, nativa dos EUA, Canadá e México, está distribuída em mais de 40 países, e em pelo menos 130 municípios brasileiros. A rã-touro possui uma vocalização de amplo espectro de frequências, diferente do que é encontrado nas espécies nativas brasileiras. Seus cantos têm frequência dominante baixa, grande propagação e pouca degradação e atenuação no ambiente. A presença do canto da espécie pode causar alteração nos parâmetros dos cantos de uma espécie nativa da Mata Atlântica brasileira. Entretanto, não é sabido se outras espécies também podem ser afetadas e se a introdução da rã-touro é capaz de afetar o comportamento acústico das comunidades nativas. Neste estudo, nós avaliamos os efeitos da invasão de L. catesbeianus sobre o nicho acústico das espécies nativas, usando duas perspectivas, no nível de indivíduos e de comunidades. No Capítulo I nós avaliamos se o estímulo acústico da rã-touro pode alterar parâmetros temporais e espectrais dos cantos de espécies nativas com e sem sobreposição espectral com a espécie invasora. Nós testamos a hipótese de que espécies com sobreposição de frequências com a invasora apresentam mudanças maiores nos parâmetros de vocalização do que espécies que estejam fora desse espectro. Para isso realizamos um experimento em campo, introduzindo a vocalização da espécie em área onde não existe registro da sua ocorrência. Nós utilizamos mais dois estímulos, um ruído branco e uma vocalização de uma espécie nativa, Rhinella icterica. Todas as espécies testadas foram expostas aos três estímulos, onde comparamos com a sua atividade de vocalização espontânea. Nossos resultados mostram efeito de todos os tipos de ruídos nos cantos, mas todas as espécies responderam com mudanças maiores nos parâmetros para os dois anuros do que para o ruído. É provável que esse resultado esteja relacionado ao fato de que ambas as espécies de anuros utilizados nos experimentos tenham a frequência dominante baixa. Entretanto, os resultados reforçam que a introdução de novos sons no ambiente tem potencial de modificar os cantos. No Capítulo II, nós testamos se existe diferença na diversidade funcional acústica em ambientes com e sem a presença de L. catesbeianus em áreas invadidas da Mata Atlântica no sul do Brasil. Nós testamos a hipótese de que poças com a presença dos machos de rã-touro em atividade de vocalização e poças sem a presença dos mesmos apresentam distintos padrões de diversidade funcional acústica e composição funcional acústica mais homogênea. Para isso, nós utilizamos dados coletados em 15 pares de poças invadidas e não invadidas na Mata Atlântica no sul do Brasil e realizamos análises de diversidade e composição funcional utilizando atributos acústicos. Nossos resultados mostram que a composição funcional foi mais homogênea nas poças invadidas do que nas poças não invadidas. Nessas poças, há uma seleção de frequências, onde predominantemente as espécies com frequências dominantes mais altas vocalizam. Ou seja, as espécies que não possuem sobreposição de frequência dominante com a rã-touro. Este estudo é um dos primeiros a testar efeitos da invasão do nicho acústico sobre as comunidades nativas. Ambientes nativos podem ser vulneráveis à introdução de espécies generalistas que produzem sinais de baixa frequência e grande amplitude. Assim, a eficiência de propagação dos sinais confere à rã-touro vantagem adaptativa na comunicação e reprodução, favorecendo o processo de estabelecimento e dispersão em detrimento das espécies nativas. / Biological Invasions are considered the second leading cause of biodiversity loss, because they may cause many ecological impacts. Recently, was called attention to a new mechanism by which invasive species can affect native species: through interference in the acoustic niche. The masking of signals from noise sources can directly affect reproductive physiology or energy consumption. Vocalization is directly connected with the sexual selection in amphibians, birds, insects, and many mammals, so that the presence of new sound sources in the environment can affect the fitness of the species. One of the worst invasive species of the world, the bullfrog Lithobates catesbeianus, native from USA, Canada and Mexico, is distributed in over 40 countries around the world and, at least, 130 Brazilian municipalities. The bullfrog has a wide spectrum of frequencies vocalization, different from what is founded in Brazilian native species. Their calls have low dominant frequency, large propagation and little degradation and attenuation in the environment. The presence of the species’ call can cause changes in the parameters of the native species’ calls from Brazilian Atlantic Forest. However, it is not known if other species may also be affected and the introduction of bullfrogs can affect the acoustic behavior of the native communities. In this study, we evaluated the effects of L. catesbeianus invasion on the acoustic niche of native species, using two perspectives at the level of individuals and communities. In Chapter I we evaluate how the acoustic stimulus of bullfrogs can change temporal and spectral parameters of the native species’ calls with and without spectral overlap with invasive species. We tested the hypothesis that species with overlapping frequencies with the invading show greater change in vocalization parameters than species outside this spectrum. To make this we conducted a field experiment, introducing the vocalization of the invasion species in an area where there is no record of their occurrence. We used two stimuli, a white noise and calling of a native species, Rhinella icterica. All species tested were exposed to the three stimuli, which compared with its spontaneous vocalization activity. Our results show the effect of all kinds of noise at the calls, but all the species responded with large changes in the parameters for the two frogs than for noise. It is probable that this result is related to the fact that both species of frogs used in the experiments have low dominant frequency. However, the results reinforce the introduction of new sounds in the environment has the potential to modify the native calls. In Chapter II, we tested if exist differences in the acoustic functional diversity in environment with and without the presence of L. catesbeianus in invaded areas of the Atlantic Forest in southern Brazil. We tested the hypothesis that the ponds with presence of bullfrog males in vocalization activity and ponds without their presence have distinct patterns of acoustic functional diversity and acoustic functional composition more homogeneous. For this, we used data collected from 15 pairs of ponds invaded and not invaded by bullfrog in the Atlantic Forest in southern Brazil and realized analysis of diversity and functional composition using acoustic traits. Our results show that the functional composition was more homogeneous in ponds invaded than in ponds not invaded. In these ponds, there is a selection of frequencies, where predominantly the species that vocalize at highest frequencies are calling, that don´t have dominant frequency overlap with the bullfrog. This study is one of the first to test effects of acoustic niche invasion on native communities. Native environments may be vulnerable to the introduction of generalist species that produces low frequency and large amplitude signals. Therefore, signal propagation efficiency gives the bullfrog adaptive advantage in communication and reproduction, favoring the process of establishment and dispersion at the expense of native species.

As invasões biológicas são consideradas a segunda maior causa de perda de biodiversidade, pois podem causar diversos impactos ecológicos. Recentemente foi chamado à atenção um novo mecanismo pelo qual espécies invasoras podem afetar as nativas: através da interferência no nicho acústico. O mascaramento dos sinais a partir de fontes de ruídos pode afetar diretamente a fisiologia reprodutiva ou o consumo de energia. A vocalização está diretamente ligada com a seleção sexual em anfíbios, aves, insetos e muitos mamíferos, de maneira que a presença de novas fontes sonoras no ambiente pode afetar a aptidão das espécies. Uma das piores invasoras do mundo, a rã-touro Lithobates catesbeianus, nativa dos EUA, Canadá e México, está distribuída em mais de 40 países, e em pelo menos 130 municípios brasileiros. A rã-touro possui uma vocalização de amplo espectro de frequências, diferente do que é encontrado nas espécies nativas brasileiras. Seus cantos têm frequência dominante baixa, grande propagação e pouca degradação e atenuação no ambiente. A presença do canto da espécie pode causar alteração nos parâmetros dos cantos de uma espécie nativa da Mata Atlântica brasileira. Entretanto, não é sabido se outras espécies também podem ser afetadas e se a introdução da rã-touro é capaz de afetar o comportamento acústico das comunidades nativas. Neste estudo, nós avaliamos os efeitos da invasão de L. catesbeianus sobre o nicho acústico das espécies nativas, usando duas perspectivas, no nível de indivíduos e de comunidades. No Capítulo I nós avaliamos se o estímulo acústico da rã-touro pode alterar parâmetros temporais e espectrais dos cantos de espécies nativas com e sem sobreposição espectral com a espécie invasora. Nós testamos a hipótese de que espécies com sobreposição de frequências com a invasora apresentam mudanças maiores nos parâmetros de vocalização do que espécies que estejam fora desse espectro. Para isso realizamos um experimento em campo, introduzindo a vocalização da espécie em área onde não existe registro da sua ocorrência. Nós utilizamos mais dois estímulos, um ruído branco e uma vocalização de uma espécie nativa, Rhinella icterica. Todas as espécies testadas foram expostas aos três estímulos, onde comparamos com a sua atividade de vocalização espontânea. Nossos resultados mostram efeito de todos os tipos de ruídos nos cantos, mas todas as espécies responderam com mudanças maiores nos parâmetros para os dois anuros do que para o ruído. É provável que esse resultado esteja relacionado ao fato de que ambas as espécies de anuros utilizados nos experimentos tenham a frequência dominante baixa. Entretanto, os resultados reforçam que a introdução de novos sons no ambiente tem potencial de modificar os cantos. No Capítulo II, nós testamos se existe diferença na diversidade funcional acústica em ambientes com e sem a presença de L. catesbeianus em áreas invadidas da Mata Atlântica no sul do Brasil. Nós testamos a hipótese de que poças com a presença dos machos de rã-touro em atividade de vocalização e poças sem a presença dos mesmos apresentam distintos padrões de diversidade funcional acústica e composição funcional acústica mais homogênea. Para isso, nós utilizamos dados coletados em 15 pares de poças invadidas e não invadidas na Mata Atlântica no sul do Brasil e realizamos análises de diversidade e composição funcional utilizando atributos acústicos. Nossos resultados mostram que a composição funcional foi mais homogênea nas poças invadidas do que nas poças não invadidas. Nessas poças, há uma seleção de frequências, onde predominantemente as espécies com frequências dominantes mais altas vocalizam. Ou seja, as espécies que não possuem sobreposição de frequência dominante com a rã-touro. Este estudo é um dos primeiros a testar efeitos da invasão do nicho acústico sobre as comunidades nativas. Ambientes nativos podem ser vulneráveis à introdução de espécies generalistas que produzem sinais de baixa frequência e grande amplitude. Assim, a eficiência de propagação dos sinais confere à rã-touro vantagem adaptativa na comunicação e reprodução, favorecendo o processo de estabelecimento e dispersão em detrimento das espécies nativas. / Biological Invasions are considered the second leading cause of biodiversity loss, because they may cause many ecological impacts. Recently, was called attention to a new mechanism by which invasive species can affect native species: through interference in the acoustic niche. The masking of signals from noise sources can directly affect reproductive physiology or energy consumption. Vocalization is directly connected with the sexual selection in amphibians, birds, insects, and many mammals, so that the presence of new sound sources in the environment can affect the fitness of the species. One of the worst invasive species of the world, the bullfrog Lithobates catesbeianus, native from USA, Canada and Mexico, is distributed in over 40 countries around the world and, at least, 130 Brazilian municipalities. The bullfrog has a wide spectrum of frequencies vocalization, different from what is founded in Brazilian native species. Their calls have low dominant frequency, large propagation and little degradation and attenuation in the environment. The presence of the species’ call can cause changes in the parameters of the native species’ calls from Brazilian Atlantic Forest. However, it is not known if other species may also be affected and the introduction of bullfrogs can affect the acoustic behavior of the native communities. In this study, we evaluated the effects of L. catesbeianus invasion on the acoustic niche of native species, using two perspectives at the level of individuals and communities. In Chapter I we evaluate how the acoustic stimulus of bullfrogs can change temporal and spectral parameters of the native species’ calls with and without spectral overlap with invasive species. We tested the hypothesis that species with overlapping frequencies with the invading show greater change in vocalization parameters than species outside this spectrum. To make this we conducted a field experiment, introducing the vocalization of the invasion species in an area where there is no record of their occurrence. We used two stimuli, a white noise and calling of a native species, Rhinella icterica. All species tested were exposed to the three stimuli, which compared with its spontaneous vocalization activity. Our results show the effect of all kinds of noise at the calls, but all the species responded with large changes in the parameters for the two frogs than for noise. It is probable that this result is related to the fact that both species of frogs used in the experiments have low dominant frequency. However, the results reinforce the introduction of new sounds in the environment has the potential to modify the native calls. In Chapter II, we tested if exist differences in the acoustic functional diversity in environment with and without the presence of L. catesbeianus in invaded areas of the Atlantic Forest in southern Brazil. We tested the hypothesis that the ponds with presence of bullfrog males in vocalization activity and ponds without their presence have distinct patterns of acoustic functional diversity and acoustic functional composition more homogeneous. For this, we used data collected from 15 pairs of ponds invaded and not invaded by bullfrog in the Atlantic Forest in southern Brazil and realized analysis of diversity and functional composition using acoustic traits. Our results show that the functional composition was more homogeneous in ponds invaded than in ponds not invaded. In these ponds, there is a selection of frequencies, where predominantly the species that vocalize at highest frequencies are calling, that don´t have dominant frequency overlap with the bullfrog. This study is one of the first to test effects of acoustic niche invasion on native communities. Native environments may be vulnerable to the introduction of generalist species that produces low frequency and large amplitude signals. Therefore, signal propagation efficiency gives the bullfrog adaptive advantage in communication and reproduction, favoring the process of establishment and dispersion at the expense of native species.

Amphibians develop in aquatic environments where they are very susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are widely used and have been shown to affect survival and development of tadpoles under laboratory conditions. The goal my thesis is to determine if agriculturally relevant exposure to Roundup WeatherMax®, a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the survival and development of wood frogs tadpoles (Lithobates sylvaticus) under both laboratory and field conditions. In the field, experimental wetlands were divided in half using an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and predicted environmental concentrations (PEC, 2.89 mg a.e./L), after which survival, growth, development, and expression of genes involved in metamorphosis were measured. Results indicate that exposure to the PEC is extremely toxic to tadpoles under laboratory conditions but not under field conditions. Results from both experimental conditions show sublethal effects on growth and development, and demonstrate that ERC of glyphosate-based herbicides have the potential to alter hormonal responses during metamorphosis. My secondary objectives were to compare the effects of Roundup WeatherMax® to the well-studied Vision® formulation (containing the isopropylamine (IPA) salt of glyphosate and POEA), and to determine which ingredient(s) are responsible for the sublethal effects on development. Survival, growth and gene expression results indicate that Roundup WeatherMax® has greater toxicity than Vision® formulation. Contrary to my prediction, results suggest that, under realistic exposure scenarios, POEA is not the sole ingredient responsible for the observed developmental effects. However, my results demonstrate that chronic exposure to the POEA surfactant at the PEC (1.43 mg/L) is extremely toxic to wood frog tadpoles in laboratory. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research contributes to overall knowledge of the impacts of glyphosate-based herbicides on aquatic communities.

Submitted by (edna.saturno@ufrpe.br) on 2017-02-21T17:09:23Z No. of bitstreams: 1 Sandro Ricardo da Costa.pdf: 720379 bytes, checksum: 054515c8a5c3b3fc9ff3150fc0b86e14 (MD5) / Made available in DSpace on 2017-02-21T17:09:23Z (GMT). No. of bitstreams: 1 Sandro Ricardo da Costa.pdf: 720379 bytes, checksum: 054515c8a5c3b3fc9ff3150fc0b86e14 (MD5) Previous issue date: 2012-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The technique of use of gonadotrophin releasing hormone (GnRH) in the reproduction of amphibians can be used to control spawning throughout the year, avoiding production delays resulting from the seasonality of the reproductive cycle. The objective was to study the induced breeding of bullfrog Lithobates catesbeianus using different types and dosages of GnRH to the optimization of the hormone and the consequent reduction in operating costs in commercial production. Breeders (average weight 650 g) were previously prepared, and conditioned to an average temperature of 26°C, submitted to a photoperiod of 15 hours of light. We tested three different hormone treatments; treatment 1 (T1) with 5 μg Etilamid hydrated acetate, treatment (T2) 2.5 μg Etilamid hydrated acetate and treatment 3 (T3) with 0.8 μg of Buserelin acetate, divided in three experimental groups. For each treatment, three applications were made at intervals of 12 hours hormones application in females and a single one for males. After the hormone treatment was done the extrusion of eggs in females and spermiation in males fertilized eggs hydrated with water at neutral pH. All males responded to the hormones applied resulting in a "pool of sperm" used to fertilize the females. Females of treatments T1 and T2 ovulated in its entirety and there was a failure only in T3. From the first and second trials, it could be detected that the dosages of 2.5 μg Etilamid hydrated acetate and 0.8 μg Buserelin acetate are more technically and economic viable for induced breeding of bullfrog. The average weight of spawning was 92.8 and 93.7 g respectively. The fertilization rate was estimated from the percentage of eclosion, with rates of fertilization rates average with 84.2 % (T2) and 60.3 % (T3). Based on the results, we conclude that the reduction in usual dosages for Etilamid hydrated acetate and the use of Buserelin acetate were efficient on ovulation and spermiation in bullfrogs, but it is necessary to adjust the dosage of Buserelin acetate as well to get a better improvement through environmental control and breeder maturation conditions in further experiments. / A técnica de uso de hormônios liberadores da gonadotrofina (GnRH) na reprodução de anfíbios pode ser usada para controlar as desovas ao longo do ano, evitando atrasos na produção decorrente da sazonalidade do ciclo reprodutivo. Objetivou-se estudar a reprodução induzida da rã touro Lithobates catesbeianus, utilizando diferentes tipos e dosagens do GnRH visando à otimização do hormônio e conseqüente redução nos custos operacionais na produção comercial. Os reprodutores (peso médio de 650 g) foram previamente preparados, sendo climatizados à temperatura média de 26° C, e submetidos a fotoperíodo de 15h luz. Foram testados três diferentes tratamentos hormonais, sendo o tratamento um (T1) com 5 μg/rã de acetato de Etilamida hidratado, o tratamento dois (T2) 2,5 μg/rã de acetato de Etilamida hidratado e o tratamento três (T3) com 0,8 μg/rã de acetato de Busserelina, divididos em três fases experimentais. Em cada tratamento foram feitas três aplicações de hormônios com intervalo de 12 h nas fêmeas, e apenas uma aplicação nos machos. Após o tratamento hormonal foi feita a extrusão dos óvulos nas fêmeas e espermiação nos machos, fecundando e hidratando os ovos com água de pH neutro. Todos os machos responderam aos hormônios aplicados resultando em um ―pool espermático‖, usado na fertilização das fêmeas. As fêmeas dos Tratamentos T1 e T2 ovularam em sua totalidade havendo uma abstenção apenas no T3. Nas primeira e segunda fase experimental, foi detectado que as dosagens de 2,5 μg/rã de acetato de Etilamida hidratado e 0,8 μg/rã de acetato de Busserelina foram mais tecnicamente e economicamente viáveis para indução à reprodução da rã touro. A média no peso das desovas foi de 92,8 e 93,7 g, respectivamente. A taxa de fecundação foi estimada na porcentagem de eclosão das larvas, com índices de fecundação com medias no T2 com 84,2 % e 60,3 % no T3. Com base nos resultados, é possível concluir que houve eficiência com a redução das dosagens usuais para o acetato de Etilamida hidratado e também houve eficiência com o uso do acetato de Busserelina na ovulação e espermiação de rãs, porém se faz necessário ajuste de dosagens para acetato de Busserelina bem como uma melhor adequação entre as condições de controle ambiental e maturação dos reprodutores, afim de melhorar os índices de fecundação em novos experimentos.

Amphibians develop in aquatic environments where they are very susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are widely used and have been shown to affect survival and development of tadpoles under laboratory conditions. The goal my thesis is to determine if agriculturally relevant exposure to Roundup WeatherMax®, a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the survival and development of wood frogs tadpoles (Lithobates sylvaticus) under both laboratory and field conditions. In the field, experimental wetlands were divided in half using an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and predicted environmental concentrations (PEC, 2.89 mg a.e./L), after which survival, growth, development, and expression of genes involved in metamorphosis were measured. Results indicate that exposure to the PEC is extremely toxic to tadpoles under laboratory conditions but not under field conditions. Results from both experimental conditions show sublethal effects on growth and development, and demonstrate that ERC of glyphosate-based herbicides have the potential to alter hormonal responses during metamorphosis. My secondary objectives were to compare the effects of Roundup WeatherMax® to the well-studied Vision® formulation (containing the isopropylamine (IPA) salt of glyphosate and POEA), and to determine which ingredient(s) are responsible for the sublethal effects on development. Survival, growth and gene expression results indicate that Roundup WeatherMax® has greater toxicity than Vision® formulation. Contrary to my prediction, results suggest that, under realistic exposure scenarios, POEA is not the sole ingredient responsible for the observed developmental effects. However, my results demonstrate that chronic exposure to the POEA surfactant at the PEC (1.43 mg/L) is extremely toxic to wood frog tadpoles in laboratory. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research contributes to overall knowledge of the impacts of glyphosate-based herbicides on aquatic communities.

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

This study devised a method to detect Ribeiroia ondatrae (class Trematoda) in water-bodies using environmental DNA (eDNA) collected from filtered water samples from selected ponds in the USA and Canada. Species-specific PCR primers were designed to target the Internal Transcribed Spacer-2 (ITS-2) region of the parasite’s genome. The qualitative PCR method was 70% (n=10) accurate in detecting R. ondatrae in ponds previously found to contain the parasite, while the qPCR method was 88.9% (n=9). To examine how the retinoic acid (RA) pathway gene expression may be perturbed during R. ondatrae infections, leading to limb development abnormalities in the wood frog (Lithobates sylvaticus). Multiple sequence alignments were used to design degenerate PCR primers to eight RA biosynthesis genes, but only two gene fragments were identified using this approach. Without effective primer sets it was not possible to measure changes in gene expression in infected frogs.

This study devised a method to detect Ribeiroia ondatrae (class Trematoda) in water-bodies using environmental DNA (eDNA) collected from filtered water samples from selected ponds in the USA and Canada. Species-specific PCR primers were designed to target the Internal Transcribed Spacer-2 (ITS-2) region of the parasite’s genome. The qualitative PCR method was 70% (n=10) accurate in detecting R. ondatrae in ponds previously found to contain the parasite, while the qPCR method was 88.9% (n=9). To examine how the retinoic acid (RA) pathway gene expression may be perturbed during R. ondatrae infections, leading to limb development abnormalities in the wood frog (Lithobates sylvaticus). Multiple sequence alignments were used to design degenerate PCR primers to eight RA biosynthesis genes, but only two gene fragments were identified using this approach. Without effective primer sets it was not possible to measure changes in gene expression in infected frogs.

Amphibians develop in aquatic environments where they are very susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are widely used and have been shown to affect survival and development of tadpoles under laboratory conditions. The goal my thesis is to determine if agriculturally relevant exposure to Roundup WeatherMax®, a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the survival and development of wood frogs tadpoles (Lithobates sylvaticus) under both laboratory and field conditions. In the field, experimental wetlands were divided in half using an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and predicted environmental concentrations (PEC, 2.89 mg a.e./L), after which survival, growth, development, and expression of genes involved in metamorphosis were measured. Results indicate that exposure to the PEC is extremely toxic to tadpoles under laboratory conditions but not under field conditions. Results from both experimental conditions show sublethal effects on growth and development, and demonstrate that ERC of glyphosate-based herbicides have the potential to alter hormonal responses during metamorphosis. My secondary objectives were to compare the effects of Roundup WeatherMax® to the well-studied Vision® formulation (containing the isopropylamine (IPA) salt of glyphosate and POEA), and to determine which ingredient(s) are responsible for the sublethal effects on development. Survival, growth and gene expression results indicate that Roundup WeatherMax® has greater toxicity than Vision® formulation. Contrary to my prediction, results suggest that, under realistic exposure scenarios, POEA is not the sole ingredient responsible for the observed developmental effects. However, my results demonstrate that chronic exposure to the POEA surfactant at the PEC (1.43 mg/L) is extremely toxic to wood frog tadpoles in laboratory. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research contributes to overall knowledge of the impacts of glyphosate-based herbicides on aquatic communities.

Amphibian populations are declining globally due to a litany of factors including pollution, disease, climate change, and most importantly, habitat destruction. As most amphibian life histories involve their populations being recruitment limited, focusing on the mechanism behind breeding habitat selection will reveal useful cues that managers may use to increase abundance and breeding success. Though there are many theoretical models that describe the distribution of animals in response to a resource, the ideal free distribution (IFD) theory has not yet been applied to amphibian settling decisions. Through this application of the IFD, I have found that a population of wood frogs (Lithobates sylvaticus) in Patuxent National Wildlife Refuge select vernal pools that are large, deep, and hold water into the summer months to breed from 2010-2015. This information will provide managers with the ability to predict sites where wood frogs will breed in the future, as well as describe the cues that wood frogs are cueing in on so we can protect, alter, or create ideal breeding habitat.