Embed Size (px)
Citation preview
UNIVERSIDADE ESTADUAL DE CAMPINAS
INSTITUTO DE BIOLOGIA
CAROLINA LAMBERTINI
OCCURRENCE PATTERNS OF Batrachochytrium dendrobatidis IN
AMPHIBIANS FROM BRAZILIAN ATLANTIC FOREST AND
AMAZONIA
PADRÕES DE OCORRÊNCIA DE Batrachochytrium dendrobatidis EM
ANFÍBIOS DA MATA ATLÂNTICA E AMAZÔNIA BRASILEIRAS
CAMPINAS
2019
CAROLINA LAMBERTINI
OCCURRENCE PATTERNS OF Batrachochytrium dendrobatidis IN
AMPHIBIANS FROM BRAZILIAN ATLANTIC FOREST AND AMAZONIA
PADRÕES DE OCORRÊNCIA DE Batrachochytrium dendrobatidis EM
ANFÍBIOS DA MATA ATLÂNTICA E AMAZÔNIA BRASILEIRAS
Thesis presented to the Institute of
Biology of the University of Campinas
in partial fulfillment of the
requirements for the degree of Doctor,
in the area of Animal Biology, specific
area of Animal Biodiversity
Tese apresentada ao Instituto de
Biologia da Universidade Estadual de
Campinas como parte dos requisitos
exigidos para a obtenção do título de
Doutora em Biologia Animal, na área
de Biodiversidade Animal
ESTE EXEMPLAR CORRESPONDE À
VERSÃO FINAL DA TESE DEFENDIDA
PELA ALUNA CAROLINA LAMBERTINI,
E ORIENTADA PELO PROF. DR. LUIS
FELIPE DE TOLEDO RAMOS PEREIRA
Orientador: Prof. Dr. Luis Felipe de Toledo Ramos Pereira
CAMPINAS
2019
Campinas, 28 de Maio de 2019.
COMISSÃO EXAMINADORA
Prof.(a) Dr.(a). Luis Felipe de Toledo Ramos Pereira
Prof.(a). Dr.(a) Cinthia Aguirre Brasileiro
Prof.(a) Dr(a). Rodrigo Lingnau
Prof.(a) Dr(a). Mariana Lucio Lyra
Prof.(a) Dr(a). Marcelo José Sturaro
Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se
encontra no processo de vida acadêmica do aluno.
Dedico este trabalho à minha família, que esteve sempre ao meu lado em
momentos difíceis e felizes, me incentivando e me dando forças para seguir em
frente.
“And those who were seen dancing were thought to be insane by those who could not
hear the music”
FRIEDRICH NIETZSCHE
AGRADECIMENTOS
Ao Programa de Pós-graduação em Biologia Animal da UNICAMP.
O presente trabalho foi realizado com apoio da Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) - Código de
Financiamento 001.
À Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP
#2011/51694-7; #2014/23388-7; #2016/25358-3) e Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq #405285/2013-2; #312895-2014-3)
pelo financiamento do presente trabalho.
Ao meu orientador Prof. Dr. Luís Felipe Toledo, por estar ao meu lado durante
nove anos de orientação, por todo seu tempo e dedicação em me ensinar e me formar
intelectualmente, e pelas inúmeras oportunidades de crescimento profissional que me
concedeu. Além da orientação, pela amizade, confiança e parceria que desenvolvemos
durante todos esses anos, e por me ajudar a superar alguns dos momentos mais difíceis
que tive de passar em meu caminho. Meu eterno carinho e gratidão.
Ao Prof. Dr. Domingos da Silva Leite, que me abriu as portas da carreira
científica, sempre com muita paciência e atenção ao me ensinar diversas técnicas
laboratoriais. Muito obrigada por toda dedicação.
Aos membros da banca de avaliação prévia, Prof. Dr. Rodrigo Lingnau, Prof.
Dr. André Rinaldo Senna Garrafoni, Prof. Dra. Elaine Maria Lucas Gonsales, e aos
membros da banca examinadora Prof. Dr. Rodrigo Lingnau, Prof. Dra. Cinthia
Brasileiro, Prof. Dr. Marcelo José Sturaro, Dra. Mariana Lyra, pela disponibilidade e
auxílio para grandes melhorias na qualidade do presente trabalho.
Aos Professores Doutores Danilo Ciccone Miguel, André Rinaldo Senna
Garrafoni, Martin Francisco Pareja e Adriano Cappellazzo Coelho por toda a ajuda,
paciência e compreensão no processo de qualificação.
Ao Instituto Chico Mendes de Biodiversidade e ao Instituto Florestal pela
concessão das autorizações necessárias para a elaboração do presente trabalho.
À minha família, Nuno Lambertini, Divina Felício de Souza Lambertini, Nuno
Lambertini Jr., Marlene Soares Lambertini, Bruno Lambertini, Keylla Mara Campos
Lambertini, Pedro Campos Lambertini, Fernanda Lambertini Piva, Adailton Renato
Piva, e aos dois novos membros Vinicius Lambertini Piva e Felipe Lambertini Piva,
pelo amor incondicional, parceria, compreensão e motivação, por acreditarem em mim e
serem meus maiores exemplos de vida.
À minha família científica, Camila Zornosa Torres, Simone Dena, Mariane de
Oliveira, Guilherme Augusto Alves, Raoni Rebouças, Ronaldo M. N. Santos, Mariana
Rettuci Pontes, Diego Moura, Tamilie Carvalho, Luisa de Pontes Ribeiro, Anat Belasen,
Carlos Henrique L. N. Almeida, Victor Fávaro Augusto, Janaina Serrano, Raquel Salla
Jacob, Joice Ruggeri Gomes, Daniel Christofer Medina López, Thomas S. Jenkinson,
Lucas Forti, Prof. Dr. C. Guilherme Becker, Prof. Dr. Timothy Y. James, Prof. Dr.
David Rodriguez, Prof. Dra. Kelly R. Zamudio, muito amor e gratidão a todos.
Aos meus amores da vida, Anita C. Presotto, Fernanda C. O. Fernandes, Marjori
Laporte, Carolina Marcucci, Luís Fernando Moreno de Lima, Lucas Beraldo Martins,
por estarem sempre ao meu lado, mesmo eu estando ausente em diversos momentos,
obrigada pela compreensão, amizade e parceria eterna.
À minha família de Barão Geraldo, Bruno Polato Sanches, Eduardo Teodoro
Fernandes, Mariane de Oliveira Freitas, Lucas Madureira e Júlia Lombardi, por
compartilharmos a mesma casa, nos unirmos nas situações difíceis que já enfrentamos,
pelo suporte que damos uns aos outros em diversos momentos, por sermos família!
À Sandra Borsetti, pela enorme ajuda no início de tudo. Um anjo que caiu no
meu caminho, sem você nada disso seria possível.
À Dra. Ana Paula José e Dr. Mário Fernando Oliveira Rocha pelo
acompanhamento e ajuda excepcional no meu processo de formação. Muito carinho e
eterna gratidão por vocês!
RESUMO
Doenças infecciosas emergentes são consideradas uma das principais ameaças à
biodiversidade mundial, e sua ocorrência é determinada através da influência do
ambiente nas interações entre patógenos e hospedeiros. A quitridiomicose, causada pelo
fungo Batrachochytrium dendrobatidis (Bd), é uma doença infecciosa emergente que
acomete populações de anfíbios em todo o mundo, e sua dinâmica de infecção já foi
diretamente associada à variação ambiental, direcionando condições para o
desenvolvimento do patógeno e/ou interferindo na susceptibilidade do hospedeiro. O Bd
é um patógeno generalista, infectando em sua maioria Anura, porém já detectado em
Caudata e Gymnophiona, mas até então não existem informações sobre os padrões de
ocorrência de Bd em cecílias no mundo. No Brasil, o bioma Amazônia foi considerado
como região de baixa adequabilidade ambiental para o desenvolvimento do Bd, e dois
estudos retrospectivos corroboraram esse padrão. A Mata Atlântica, por sua vez, é uma
região que apresenta alta adequabilidade ambiental para o Bd, na qual o patógeno já foi
amplamente detectado, principalmente na porção sul. Contudo, não existem estudos até
então que analisaram padrões de ocorrência do Bd em populações naturais de anfíbios
da Amazônia, e abrangendo a Mata Atlântica como um todo. Com isso, realizamos uma
ampla amostragem de espécimes fixados da ordem Gymnophiona ao longo da América
do Sul, para verificar se existe variação nos padrões de ocorrência do Bd nesse grupo.
Realizamos também uma amostragem de populações naturais de anfíbios na região
amazônica, abrangendo não somente espécies da ordem Anura, mas também das ordens
Gymnophiona e Caudata, verificando potenciais associações entre variáveis bióticas e
abióticas com as taxas de infecção, e testando experimentalmente o efeito da potencial
chegada de cepas exóticas de Bd em hospedeiros endêmicos da região. Finalmente,
realizamos uma ampla amostragem de anuros ao longo de um transecto latitudinal na
Mata Atlântica, identificando padrões de ocorrência do Bd, e potenciais associações
entre variáveis bióticas e abióticas nas taxas de infecção dos hospedeiros. Descrevemos
aqui o primeiro registro de infecção por Bd no Brasil em espécies da ordem Caudata e
Gymnophiona, propondo que as últimas podem servir como reservatório patógeno,
devido à história de vida dos hospedeiros. Na região amazônica, detectamos altas taxas
de prevalência de infecção, padrão oposto ao esperado para região, onde as variáveis
ambientais não foram boas preditoras de ocorrência do patógeno, e detectamos
experimentalmente variação em resposta dos hospedeiros à infecção por diferentes
cepas de Bd. Detectamos uma associação positiva entra as taxas de infecção por Bd e
variação latitudinal ao longo do transecto na Mata Atlântica; identificamos variáveis
abióticas e bióticas associadas às taxas de infecção encontradas, e detectamos um efeito
de amplificação da riqueza de espécies de hospedeiros nas taxas de infecção por Bd. O
presente trabalho traz informações inéditas sobre os padrões de ocorrência do Bd nos
biomas Mata Atlântica e Amazônia.
ABSTRACT
Emerging infectious diseases are one of the main threats to worldwide biodiversity,
being determined by the environmental influence on host-pathogen interactions.
Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is an
emerging infectious disease that affects amphibian populations worldwide, and its
infectious dynamics has been already directly associated to environmental variation, by
basically driving conditions for the pathogen development and/or interfering on host’s
susceptibility. Bd is a generalist pathogen, infecting mostly Anuran species, but also
detected in Gymnophiona and Caudata species. However, there is no information until
now on Bd occurrence patterns in Gymnophiona species in the world. In Brazil,
Amazonia was considered a region with low environmental suitability for Bd, and two
retrospective studies detected low Bd infection prevalence rates. On the other hand,
Atlantic Forest is a region considered environmentally suitable for Bd, and it has been
already widely detected, mainly in the south portion of the biome. But, no studies
attempted to analyze Bd occurrence patterns on amphibian natural populations from
Amazonia and in Atlantic Forest as a whole. Given this, we broadly sampled
Gymnophiona museum specimens across South America, to verify if Bd occurrence
patterns vary in this group. We also sampled wild amphibian populations across
Brazilian Amazon, including Gymnophiona and Caudata species, to test for potential
associations between biotic and abiotic variables and infection rates, and we
experimentally tested the effects of exotic Bd strains on host species from the region.
Finally, we broadly sampled anuran species along a latitudinal transect across Brazilian
Atlantic Forest, identifying Bd occurrence patterns and potential associations between
biotic and abiotic variables with Bd infections. We describe here the first report of Bd
infections in Caudata and Gymnophiona species from Brazil, and propose that
individuals from the last order may serve as pathogen reservoir because of host’s life
history. In Amazonia, we detected higher Bd infection prevalence, which is the opposite
pattern expected for the region, where environmental variables were not good predictors
for Bd occurrence, and we experimentally detected variation in host response to the
infection by exotic Bd strains. We detected a positive relationship between Bd infection
rates and latitudinal variation across the transect in Atlantic Forest. We also identified
biotic and abiotic variables associated to Bd infections, and an amplification effect of
species richness on Bd infections. The present study provides new information on Bd
occurrence patterns in Brazilian Atlantic Forest and Amazon.
LISTA DE ILUSTRAÇÕES
Introdução Geral
Figura 1. Modelo conceitual do triângulo da doença, representando os três vértices e
suas interações: impacto do ambiente, susceptibilidade dos hospedeiros e virulência
do patógeno. Fonte: Scholthof 2007.......................................................................... 20
Figura 2. Distribuição mundial dos fungos Batrachochytrium dendrobatidis e
Batrachochytrium salamandrivorans. Fonte: Bower et al. 2017............................... 22
Figura 3. Esquema simplificado representando o ciclo de vida do Bd. Zoósporo livre
(A), após penetrar na epiderme do hospedeiro inicia-se o desenvolvimento do
zoosporângio (B), seguido do processo de maturação e produção de zoósporos, e
formação de papilas de descarga (C), com a liberação dos zoósporos recém-formados
(D). Fotos: Luís Felipe Toledo................................................................................... 23
Capítulo 1. Spatial Distribution of Batrachochytrium dendrobatidis in South American
Caecilians
Figura 1. Amostragem de Batrachochytrium dendrobatidis em espécimes fixados de
cecílias do Brazil e Uruguai (amostras positivas: círculos vermelhos; amostras
negativas: círculos brancos), e cecílias coletadas na natureza (amostras positivas:
cruz vermelha; amostras negativas: cruzes brancas; Vásquez-Ochoa et al. 2012,
Gower et al. 2013, Rendle et al. 2015, presente trabalho). Siphonops paulensis é uma
espécie terrestre fossorial encontrada na porção leste do Brazil, e Chthonerpeton
indistinctum é uma espécie aquática, e representa a única espécie de cecília
encontrada no Uruguai. Créditos: Dr. Daniel Loebmann (fotos), US National Park
Service (layer).......................................................................................................... 41
Capítulo 2. The Killing-Chytrid Fungus in Amphibian Populations of the Brazilian
Amazon
Figura 1. Pontos de amostragem de Batrachochytrium dendrobatidis (pontos pretos)
ao longo da Amazônia brasileira. O tamanho dos pontos representa o valor da
prevalência de infecção para cada localidade amostrada........................................... 57
Figura 2. Associações entre prevalência (A) e carga de infecção (B) por
Batrachochytrium dendrobatidis e tipo de habitat das espécies de hospedeiros. As
letras minúsculas (a, b e c) representam diferenças entre cada categoria.................. 58
Figura 3. Curvas de sobrevivência de Atelopus aff. spumarius. Grupos infectados
por Batrachochytrium dendrobatidis são representados pela linha marrom (CLFT
156), linha amarela (CLFT 102), e grupo controle pela linha azul (A). Diferenças
entre carga de infecção dos grupos experimentais no meio (ou morte) e ao final do
experimento (B)......................................................................................................... 59
Figura S1. Ponto de coleta de Atelopus hoogmoedi (azul: Manaus, Amazonas,
Amazon), e localidades de isolamento de cepas de Batrachochytrium dendrobatidis
(marrom = CLFT 156: Morretes, Paraná; amarelo = CLFT 102: Camacan, Bahia;
ambos na Mata Atlântica).......................................................................................... 60
Figura S2. Dendrograma da análise de cluster utilizando variáveis bioclimáticas de
temperatura e precipitação, mostrando similaridade climática entre localidades de
isolamento de cepas de Batrachochytrium dendrobatidis (CLFT 156 e CLFT 102), e
localidade de coleta de Atelopus aff. spumarius (Manaus). O eixo y representa
distâncias Euclidianas................................................................................................ 61
Capítulo 3. Latitudinal Distribution of the Frog-Killing Fungus across the Brazilian
Atlantic Forest
Figura 1. Pontos de amostragem de Batrachochytrium dendrobatidis (pontos pretos)
ao longo de um transecto latitudinal na Mata Atlântica. Pontos vermelhos a azuis são
extremos de um contínuo de prevalência e carga de infecção para cada ponto de
amostragem. A linha pontilhada no topo do Rio Doce indica o limite entre as porções
sul e norte da Mata Atlântica, delimitando também os dados utilizados nos gráficos
inseridos na figura...................................................................................................... 75
Figura 2. Associação entre prevalência e carga de infecção de Batrachochytrium
dendrobatidis com tipo de hábito (A, B) e habitat dos hospedeiros (C,
D)............................................................................................................................... 76
LISTA DE TABELAS
Capítulo 1. Spatial Distribution of Batrachochytrium dendrobatidis in South American
Caecilians
Tabela 1. Famílias, espécies tamanho amostral, tipo de ambiente e país nos quais
indivíduos da família Gymnophiona foram capturados, e prevalência de infecção por
Batrachochytrium dendrobatidis (número de positivos/total)................................... 39
Tabela 2. Resultados da análise de model averaging, com o ranqueamento de
variáveis ambientais que explicam a ocorrência de Batrachochytrium dendrobatidis
em cecílias da América do Sul................................................................................... 40
Tabela S1. Contempla os dados brutos das análises referentes ao presente capítulo.
Material em formato de mídia eletrônica com acesso disponível em: <
https://www.int-res.com/articles/suppl/d124p109_supp.pdf >.
Tabela S2. Seleção de modelo com variáveis bioclimáticas associadas à infecção por
Batrachochytrium dendrobatidis............................................................................... 42
Capítulo 2. The Killing-Chytrid Fungus in Amphibian Populations from the Brazilian
Amazon
Tabela 1. Prevalência (positivos/total) e carga de infecção (g.e.) por
Batrachochytrium dendrobatidis (valores representam média ± desvio padrão) por
local de coleta na bacia Amazônica Brasileira.......................................................... 55
Tabela 2. Prevalência [porcentagem (positivo/total)] e carga de infecção (valores são
média ± desvio padrão) de Batrachochytrium dendrobatidis por local de coleta na
bacia Amazônica Brasileira....................................................................................... 56
Tabela S1. Contempla os dados brutos das análises referentes ao presente capítulo.
Este material será disponibilizado em formato de mídia eletrônica no momento da
publicação.
Capítulo 3. Latitudinal Distribution of the Frog-Killing Fungus across the Brazilian
Atlantic Forest
Tabela 1. Prevalência (positive/total) e carga de infecção de Batrachochytrium
dendrobatidis (rounded without decimals) por família e modo reprodutivo. Valores
de intensidade de infecção (equivalentes genômicos de zoósporos - g.e.) são média ±
desvio padrão (variação)............................................................................................ 72
Tabela 2. Localidades de coleta e detecção de Batrachochytrium dendrobatidis ao
longo do transecto latitudinal na Mata Atlântica....................................................... 73
Tabela 3. Melhor modelo para prevalência de infecção por Batrachochytrium
dendrobatidis ao longo do transecto latitudinal......................................................... 74
Tabela S1. Contempla os dados brutos das análises referentes ao presente capítulo.
Este material será disponibilizado em formato de mídia eletrônica no momento da
publicação.
Tabela S2. Cinco melhores modelos para prevalência e carga de infecção por
Batrachochytrium dendrobatidis. Bio 1 = Temperatura Média Anual; Bio 2 =
Variação Média Diurna; Bio 4 = Sazonalidade de Temperatura; Bio 5 = Máxima
Temperatura do Mês Mais Quente; Bio 12 = Precipitação Anual; Bio 18 =
Precipitação do Quarto Mais Quente......................................................................... 77
SUMÁRIO
INTRODUÇÃO .............................................................................................................. 19
Capítulo 1. SPATIAL DISTRIBUTION OF Batrachochytrium dendrobatidis IN
SOUTH AMERICAN CAECILIANS ........................................................................ 30
Capítulo 2. THE KILLING-CHYTRID FUNGUS IN AMPHIBIAN
POPULATIONS OF THE BRAZILIAN AMAZONIA ............................................. 44
Capítulo 3. LATITUDINAL DISTRIBUTION OF THE FROG-KILLING FUNGUS
ACROSS THE BRAZILIAN ATLANTIC FOREST ................................................. 62
SÍNTESE GERAL .......................................................................................................... 78
REFERÊNCIAS ............................................................................................................. 81
ANEXOS ........................................................................................................................ 99
19
INTRODUÇÃO
Doenças infecciosas emergentes (DIE) são definidas como aquelas que aparecem
pela primeira vez em uma população, aumentando em incidência, patogenicidade, impacto,
distribuição geográfica e número de hospedeiros afetados em um curto período de tempo
(Morse 1995, revisado em Williams et al. 2002, Daszak et al. 2003). Eventos de DIE’s
aumentam substancialmente ao longo do tempo, afetando tanto seres humanos quanto
diversos outros táxons na natureza (Daszak et al. 2001, Daszak et al. 2003, Jones et al. 2008,
Tompkins et al. 2015). As DIE’s são causadas por uma grande diversidade de patógenos
como bactérias, vírus/príons, fungos, protozoários e helmintos (Daszak et al. 2000, Jones et
al. 2008). Em seres humanos, a maioria dos agentes infecciosos que causam DIE’s são
bactérias (principalmente estirpes que apresentam resistência a antibióticos) e, pelo menos
60% dos eventos de DIE’s são representados por zoonoses (doenças infecciosas de origem
animal), sendo a grande maioria de origem selvagem (Jones et al. 2008). Em outros
vertebrados, vírus representam a maioria dos agentes infecciosos responsáveis por surtos de
doenças, seguidos de bactérias e fungos (Dobson e Foufopoulos 2001, Tompkins et al. 2015).
A origem e emergência de doenças infecciosas representam um risco crescente
tanto para seres humanos, quanto para a biodiversidade como um todo (Daszak et al. 2000,
Cunningham et al. 2017), e são diretamente associadas ao declínio de populações de diversas
espécies no mundo (Lips et al. 2006, Skerrat et al. 2007, Blehert et al. 2009, Vredenburg et al.
2010, Tompkins et al. 2011, Lorch et al. 2016). Duas hipóteses competem pela explicação da
origem de uma doença infecciosa. A Novel Pathogen Hypothesis (NPH) sugere que, em um
período recente, determinado patógeno se espalhou afetando novas espécies ou espécies
altamente susceptíveis. A Endemic Pathogen Hypothesis (EPH) por sua vez, sugere que um
patógeno já estabelecido e disseminado em determinado ambiente encontra novos hospedeiros
ou apresenta aumento em patogenicidade (Laurance et al. 1996, Alford 2001, Rachowicz et al.
2005, Skerrat et al. 2007). A origem de diversas doenças pode ser explicada tanto pela NPH
quanto pela EPH e, a classificação dentre essas duas hipóteses, pode influenciar diretamente
em medidas de conservação que podem interferir na disseminação de um dado patógeno
novo, ou prevenir surtos de um patógeno endêmico (Rachowicz et al. 2005).
Dentre os potenciais fatores que direcionam a emergência de doenças infecciosas
em vertebrados, além de alterações ecossistêmicas de origem natural ou antropogênica e
movimentação de patógenos ou vetores (revisado em Willians et al. 2002), podemos citar
fatores relacionados aos hospedeiros como instabilidade e baixa diversidade genética,
20
Figura 1. Modelo conceitual do triângulo da doença, representando os três vértices e suas interações: impacto
do ambiente, susceptibilidade do hospedeiro e virulência do patógeno. Fonte: Sholthof (2007).
deficiência nutritiva e pouca variação de suplementos alimentares, exposição a agentes
infecciosos provenientes de outras populações naturais ou domésticas, estresse relacionado à
alteração de habitats e alterações na resposta imunológica, estresse térmico e mudanças
climáticas (Daszak et al. 2003, Tompkins et al. 2015). Em resumo, as inter-relações entre
ambiente e características dos patógenos e hospedeiros determinam diretamente a emergência
de doenças infecciosas na natureza (Scholthof et al. 2007, Engering et al. 2013).
Para uma melhor compreensão de tais interações, foi desenvolvido um modelo
conceitual denominado triângulo da doença (McNew 1960), que descreve como fatores
ambientais afetando a susceptibilidade do hospedeiro e a virulência do patógeno, podem
resultar em potenciais surtos de doenças (Figura 1) e, como estas podem ser previstas,
limitadas ou controladas (Keane and Keer 1997, Scholthof 2007).
Por exemplo, sabe-se que a variação em temperatura e precipitação impactam as
interações patógeno-hospedeiro em diferentes sistemas de doenças (Altizer et al. 2006),
resultando em uma alta incidência do patógeno em uma população e potencial morte dos
hospedeiros (Altizer et al. 2006, Fisher et al. 2012, Flory et al. 2012, Ruggeri et al. 2018).
Diversos grupos de vertebrados são afetados por doenças infecciosas, resultando
em declínios de populações e extinções de espécies por todo o mundo (McMichals 2004,
Tompkins et al. 2015), com surtos de doenças ocorrendo principalmente durante modificações
21
ambientais (Anfíbios: Daszak et al. 2000; Peixes: Snieszko 1974; Humanos: Eisenberg et al.
2007). Um dos mecanismos pelos quais a variação ambiental pode influenciar o
desenvolvimento de uma determinada doença pode ser exemplificado com a White-Nose
Syndrom, uma doença infecciosa cutânea causada pelo fungo Geomyces destructans. Essa
doença afeta seriamente morcegos que hibernam em cavernas nos Estados Unidos e Canadá
(Blehert et al. 2009, Warnecke et al. 2012), e a variação de temperatura dentro dos
hibernáculos, pode influenciar tanto na severidade do patógeno quanto nas taxas de
sobrevivência dos hospedeiros afetados por essa doença (Boyles et al. 2010, Verant et al.
2012). Outro exemplo importante é a Snake Fungal Disease, uma doença infecciosa causada
pelo patógeno Ophidiomyces ophiodiicola, que afeta diversas espécies de serpentes
distribuídas ao leste dos Estados Unidos (Allender et al. 2016, Lorch et al. 2016), e foi
detectada uma correlação negativa entre a média de temperatura mensal e severidade dos
sinais clínicos apresentados pelos hospedeiros infectados (McCoy et al. 2017).
A quitridiomicose, mais um exemplo de DIE, afeta uma grande diversidade de
espécies de anfíbios e já teve sua dinâmica diretamente associada à variação ambiental em
diversas localidades no mundo (Kriger et al. 2007, Becker e Zamudio 2011, Bacigalupe et al.
2017, Carvalho et al. 2017, Greenberg et al. 2017). Esta doença é causada pelos fungos
patogênicos Batrachochytrium dendrobatidis (Bd) e Batrachochytrium salamandrivorans
(Bsal), que ameaçam de forma imperativa diversas populações de anfíbios no mundo (Martel
et al. 2013, James et al. 2015, Berger et al. 2016, Scheele et al. 2019).
Os anfíbios são classificados como o grupo de vertebrados mais ameaçado do
planeta (Stuart et al. 2004), apresentando o maior número de representantes dentro de
categorias de ameaça (pelo menos 40% das espécies) (IPBES 2019, IUCN 2019), e diversos
fatores como poluição ambiental, introdução de predadores e competidores (Daszak et al.
1999), contaminações químicas, comercialização de espécies, aumento na incidência de
radiação UV (Semlitsch 2003, McMenamin et al. 2008, Mann et al. 2009), mudanças
climáticas (Pounds et al. 2006), fragmentação e perda de habitats (Becker et al. 2007, Skerrat
et al. 2007), e doenças infecciosas (Daszak et al. 2000, Blehert et al. 2009, Allender et al.
2016, Scheele et al. 2019) exercem influência no declínio de populações. A fragmentação de
habitats atua de forma direta na ameaça aos anfíbios, como por exemplo, através do processo
de desconexão de habitats (Habitat split), que representa um grande risco para espécies de
anfíbios que necessitam realizar migrações reprodutivas (Becker et al. 2007). Em conjunto
com a fragmentação e perda de habitats, a quitridiomicose representa um impacto cada vez
mais significativo no declínio e extinção de anfíbios ao redor do mundo (Berger et al. 1998,
22
Figura 2: Distribuição mundial dos fungos Batrachochytrium dendrobatidis e Batrachochytrium
salamandrivorans. Fonte: Bower et al (2017).
Lips et al. 2006, Skerrat et al. 2007, Kriger & Hero 2008, Vredenburg et al. 2010, James et al.
2015, Berger et al. 2016, Scheele et al. 2019).
O Bd foi descoberto em 1998 (Berger et al. 1998), descrito no ano de 1999
(Longcore et al. 1999), e já foi registrado acometendo além de Anura, espécies de Caudata e
Gymnophiona (Longcore et al. 1999, Raffel et al. 2010, Doherty-Bone et al. 2013, Berger et
al. 2016). O Bsal foi descrito recentemente e, acreditava-se que acometia apenas espécies de
Caudata (Martel et al. 2013, Martel et al. 2014), mas atualmente sabe-se que também infecta
anfíbios anuros (Stegen et al. 2017). Além disso, foi descrito recentemente o primeiro caso de
coinfecção de Bd e Bsal em uma população de salamandras de fogo na Alemanha (Lötters et
al. 2018).
Enquanto Bsal possui registros de ocorrência apenas na Europa e Ásia (Martel
et al. 2013, Martel et al. 2014, Bower et al. 2017, Lötters et al. 2018), Bd foi amplamente
detectado em todo o mundo (Olson & Ronnenberg 2014, Bower et al. 2017, Scheele et al.
2019) (Figura 2) e associado a declínios substanciais de populações e extinções de espécies
em determinadas regiões na Austrália, América do Norte, América Central e América do Sul
(Berger et al. 1998, Lips et al. 2005, Lips et al. 2006, Vredenburg et al. 2010).
Cepas do Bd possuem faixa de temperatura de crescimento que em geral varia
entre 4 e 28ºC (Piotrowski et al. 2004). Porém, recentemente foi detectada uma variação em
tolerância térmica, e determinadas cepas apresentaram viabilidade de crescimento mesmo
23
Figura 3: Esquema simplificado representando o ciclo de vida do Bd. Zoósporo livre (A), após penetrar na
epiderme do hospedeiro inicia-se o desenvolvimento do zoosporângio (B), seguido do processo de maturação e
produção de zoósporos, e formação de papilas de descarga (C), com a liberação dos zoósporos recém-formados
(D). Fotos: Luís Felipe Toledo.
após choques de baixa (-12ºC) e alta (28ºC) temperatura, mas em geral apresentam faixa
ótima de crescimento entre 17 e 23ºC, o que influencia diretamente sua
virulência/patogenicidade (Piotrowsky et al. 2004, Voyles et al. 2017). O Bsal, por sua vez,
possui uma faixa ótima de crescimento que varia entre 10 e 15ºC, com cepas apresentando
mortalidade acima de 25ºC e, essa variação em preferência térmica em comparação com o Bd,
pode explicar sua distribuição geográfica mais restrita (Martel et al. 2013).
Ambos patógenos são aquáticos, apresentando ciclo de vida similar com fase
infectante representada pelo zoósporo móvel, e fase séssil representada pelo zoosporângio,
com formação em talo monocêntrico ou colonial (Longcore et al. 1999, Martel et al. 2013,
James et al. 2015). Os zoósporos colonizam a epiderme do hospedeiro (Figura 3A), formam
um tubo germinativo que penetra na membrana celular, transferem seu conteúdo para o
interior das células, e após esse processo ocorre o início da maturação dos zoosporângios
(Figura 3B) e formação de papilas de descarga (Figura 3C) que atravessam a epiderme para a
liberação dos zoósporos recém-formados (Figura 3D) (Berger et al. 2005, Greenspan et al.
2012). O mecanismo pelo qual o Bsal penetra e se desenvolve na epiderme de seus
hospedeiros provavelmente varia em comparação com o Bd, já que os sinais clínicos são mais
agressivos (Martel et al. 2013), porém não existem trabalhos que descrevem esse mecanismo
até então.
O desenvolvimento da quitridiomicose nos hospedeiros infectados pelo Bd afeta
processos fisiológicos de troca de água, gases e eletrólitos que ocorrem através da pele,
desencadeando um desequilíbrio nas concentrações de íons no plasma sanguíneo e morte
através de parada cardíaca em assistolia (Voyles et al. 2009). Além disso, ocorre a inibição da
resposta imune devido à produção de fatores tóxicos pelo patógeno (Fites et al. 2013).
Hospedeiros infectados pelo Bsal apresentam lesões erosivas multifocais e ulcerações
24
profundas, mostrando taxa de mortalidade rápida pós-infecção (Martel et al. 2013, Martel et
al. 2014).
O Bd é um patógeno generalista, tendo sido detectado em pelo menos 600
espécies de anuros ao redor do mundo (Olson e Ronnenberg et al. 2014, Scheele et al. 2019).
Em relação às outras ordens de anfíbios, infecções por Bd já foram relatadas em mais de 80
espécies de Caudata (Olson & Ronnemberg 2014), provenientes de diversas regiões nas
Américas (Davidson et al. 2003, Padgett-Flohr & Longcore 2007, Gaetner et al. 2009, Van
Rooij et al. 2011), Ásia (Parto et al. 2013) e Europa (Bovero et al. 2008). No Brasil não
existem registros, até então, de infecção por Bd em espécies de Caudata, que possuem
distribuição restrita à região Amazônica. Em relação à Gymnophiona, pelo menos sete
espécies testaram positivo para Bd (Olson & Ronnemberg 2014), com registros na África
(Doherty-Bone et al. 2013, Gower et al. 2013) e Guiana Francesa (Rendle et al. 2015).
Esforços amostrais foram realizados em outras regiões (e.g. Savage et al. 2011, Vasquez-
Ochoa et al. 2012, Penner et al. 2013, Labisko et al. 2015), porém todas as amostras foram
negativas. No Brasil, até o momento não existem registros de Bd infectando representantes
dessa ordem.
Aplicando o modelo conceitual do triangulo da doença à quitridiomicose, diversos
estudos correlacionaram cada vértice aos padrões de ocorrência da doença no mundo. Em
termos de variação ambiental, estudos demonstraram uma associação negativa entre
temperatura e taxas de infecção por Bd (Pushendorf et al. 2009, Ruggeri et al. 2015, Becker et
al. 2016, Carvalho et al. 2017, Lambertini et al. 2017). Precipitação, por sua vez, foi
positivamente associada às taxas de infecção (Pushendorf et al. 2009, Becker & Zamudio
2011, Ruggeri et al. 2015, Becker et al. 2016, Lambertini et al. 2017), além de densidade de
vegetação (Pushendorf et al. 2009, Becker et al. 2012, Becker et al. 2016), e elevação, que
frequentemente apresentam associação positiva com as taxas de infeção (Brem & Lips 2008,
Gründler et al. 2012, Catenazzi et al. 2013), e possivelmente a última apenas em faixas de
altitudes mais elevadas (Lambertini et al. 2016). Além desses fatores, grau de desmatamento
(Becker & Zamudio 2011), sazonalidade (Kriger & Hero 2007, Longo et al. 2010, Ruggeri et
al. 2015), complexidade topográfica (Becker et al. 2016) e grau de perturbação antrópica
(human footprint) também já foram associados às taxas de infecção por Bd (Becker et al.
2016, Carvalho et al. 2017).
Em relação ao patógeno, diversas características já foram diretamente associadas
aos padrões de infecção por Bd nos hospedeiros. Cepas de Bd isoladas de diversas localidades
do mundo apresentam variação genotípica, com o genótipo que apresenta distribuição global
25
denominado Global Pandemic Lineage (Bd-GPL) (Farrer et al. 2011, Schloegel et al. 2012),
considerado uma linhagem hipervirulenta, e previamente associado a surtos epizoóticos que
declinaram diversas populações de anfíbios em diversas regiões como Américas e Austrália
(Berger et al. 1998, Lips et al. 2005, Lips et al. 2006, Vredenburg et al. 2010, Farrer et al.
2011, James et al 2015). Outras linhagens com distribuição mais restrita foram descritas em
diferentes regiões, como Bd-CH (Europa), Bd-CAPE (África), e Bd-Brazil, e consideradas
como linhagens enzoóticas (Farrer et al. 2011, Schloegel et al. 2012), além de linhagens
genéticas altamente divergentes e endêmicas da Ásia (Bataille et al. 2013). Recentemente, o
sequenciamento genômico de diversos isolados de Bd no mundo, e a descoberta de uma
linhagem hiperdiversa na península Coreana, redefiniram essas linhagens e suas relações,
sendo então as principais linhagens divergentes de Bd definidas como: Bd-GPL, Bd-Asia-1,
Bd-CAPE, Bd-Asia-2/Bd-Brazil (O´Hanlon et al. 2018). No Brasil, além da linhagem Bd-
GPL, detectada por toda extensão da Mata Atlântica e recentemente na Amazônia, e da
linhagem endêmica Bd-Brazil, foi detectado pela primeira vez um genótipo híbrido entre as
linhagens Bd-GPL e Bd-Brazil, isolado na porção sul da Mata Atlântica, sendo a primeira
evidência de reprodução sexuada do patógeno (Schloegel et al. 2012, Rosenblum et al. 2013
Jenkinson et al. 2016).
A diversidade de genótipos de Bd e genótipos híbridos podem acarretar variação
em virulência do patógeno, afetando diretamente hospedeiros infectados (Jenkinson et al.
2016, Greenspan et al. 2018). Recentemente, um estudo avaliou a variação de virulência do
Bd utilizando cepas pertencentes aos três genótipos encontrados no Brasil, e detectou alta
virulência em cepas de genótipos híbridos, em comparação com hospedeiros infectados com
as linhagens Bd-GPL e Bd-Brazil, em um hospedeiro nativo (Brachycephalus ephippium),
evidenciando então o risco do processo de hibridização deste patógeno (Greenspan et al.
2018).
Além de variação genotípica, cepas de Bd também apresentam variações
fenotípicas. Inicialmente, foi detectada uma associação entre tamanho dos zoosporângios e
diferenciação genética de cepas isoladas na Europa (Fisher et al. 2009). Em seguida, o
tamanho dos zoósporos foi linearmente associado à variação de conteúdo de DNA de cepas
provenientes do Brasil, Panamá e Estados Unidos (Schloegel et al. 2012). Recentemente, a
variação fenotípica de cepas de Bd foi associada às taxas de infecção do patógeno na
natureza. Especificamente, o tamanho dos zoósporos e zoosporângios provenientes de um
transecto de elevação realizado na Mata Atlântica, foi positivamente associado às taxas de
prevalência e intensidade de infecção ao longo das áreas amostradas (Lambertini et al. 2016).
26
Além disso, foi detectado um efeito da variação de temperatura na plasticidade fenotípica de
cepas de Bd, para os três diferentes genótipos encontrados no Brasil, principalmente em
relação ao tamanho dos zoosporângios, que são maiores em temperaturas mais baixas
(Multez-Wolz et al. 2019).
Finalmente, diferentes espécies de hospedeiros apresentam variações em
susceptibilidade ou resistência às mesmas ou diferentes cepas do patógeno (Andre et al. 2008,
Gahl et al. 2012, Peterson et al. 2013, Greenberg et al. 2017). Fatores intrínsecos ao
hospedeiro podem mediar a variação em susceptibilidade apresentada por diferentes espécies,
com modificações temporais na intensidade de infecção por Bd espécie-específicas (Gervasi
et al. 2013). A riqueza de espécies dentro de uma comunidade é um fator que pode gerar
efeito tanto de diluição quanto de amplificação da doença, basicamente devido aos processos
pelos quais o patógeno é transmitido (Becker & Zamudio 2011, Searle et al. 2011, Becker et
al. 2014, Lambertini et al. em prep.). Além disso, o comportamento agregativo de
determinadas espécies, tanto para forrageamento quanto como defesa contra predadores (Han
et al. 2008), aumenta as taxas de infecção por Bd, pois representa uma maior fonte de
disseminação de zoósporos em uma dada área (Venesky et al. 2011) e, o tipo de
desenvolvimento de espécies de hospedeiros influencia taxas de infecção por Bd, que são
maiores em espécies de desenvolvimento direto devido à deficiência de respostas adaptativas
(Mesquita et al. 2017).
Inicialmente, acreditava-se que o Bd possuía a África como localidade de origem,
e que o comércio mundial de espécies africanas como Xenopus leavis e Xenopus tropicalis
contribuía para a disseminação do patógeno, o que suportava a hipótese de patógeno novo
(NPH) (Daszak et al. 1999, Weldon et al. 2004). Além disso, o comércio mundial da espécie
norte-americana Lithobates catesbeianus também foi reconhecido como parte fundamental do
mecanismo de disseminação do patógeno no mundo, por ser uma espécie tolerante à infecção
por Bd e comercializada para fins alimentícios (Fisher e Garner 2007, Schloegel et al. 2012).
Outras regiões já foram apontadas como potenciais localidades de origem do Bd, como
Américas do Sul e Norte (Rodriguez et al. 2014, Talley et al. 2015). Atualmente, a origem de
ambos os patógenos foi estabelecida para o continente asiático, com o Bsal apresentando uma
recente incursão em populações de salamandras na Europa (Martel et al. 2014), e com o
surgimento do Bd coincidindo com expansão do comércio global de anfíbios, sendo o leste da
Ásia considerado como hotspot de biodiversidade de linhagens genéticas de Bd (O’Hanlon et
al. 2018).
27
No Brasil, o primeiro registro de infecção por Bd foi realizado na Mata Atlântica,
na qual o patógeno foi detectado infectando uma espécie de riacho (Hylodes magalhaesi) de
altitude elevada no estado de Minas Gerais (Toledo et al. 2006). A partir deste registro,
muitos trabalhos foram realizados até então, principalmente na porção sul do bioma (Toledo
et al. 2006a, Toledo et al. 2006b, Vieira et al. 2012, Vieira et al. 2013, Lisboa et al. 2013,
Rodrigues et al. 2014, James et al. 2015, Preuss et al. 2015, Ruggeri et al. 2015, Valencia-
Aguilar et al. 2015, Jenkinson et al. 2016, Lambertini et al. 2016, Carvalho et al. 2017,
Lambertini et al. 2017, Lambertini et al. in prep).
Ao contrário de outras regiões nas Américas, declínios e extinções de espécies
não haviam sido associados a surtos epizoóticos de Bd no Brasil. Um estudo retrospectivo de
anuros, na porção sul da Mata Atlântica, detectou prevalência de infecção por Bd
relativamente constante ao longo do tempo, indicando um padrão enzoótico de dinâmica do
patógeno (Rodriguez et al. 2014). Porém, declínios enigmáticos de espécies de anfíbios que
ocorreram entre as décadas de 80 e 90 foram recentemente associados à quitridiomicose
(Carvalho et al. 2017), com comunidades de anfíbios apresentando uma proporção alta de
declínio, similar às comunidades afetadas pelo Bd em locais onde epizootias foram
acompanhadas e documentadas, como na América Central (Lips et al. 2008, Carvalho et al.
2017).
A Amazônia brasileira, por sua vez, é uma região pouco explorada em termos de
detecção do Bd. Trabalhos de modelagem de nicho fundamental consideraram este bioma
como uma região com probabilidade de ocorrência do Bd extremamente baixa ou nula, de
acordo com suas condições de adequabilidade para o patógeno (Ron et al. 2005, Rödder et al.
2010). O primeiro registro de Bd contou com a detecção do patógeno em um indivíduo da
espécie Adelphobates galactonotus (Valencia-Aguilar et al. 2015), apresentando carga de
infecção baixa em comparação com a média conhecida para a Mata Atlântica (Rodriguez et
al. 2014). Outro estudo realizou uma amostragem mais ampla na região, analisado a
distribuição histórica do Bd e variáveis ambientais que explicavam a prevalência de infecção
(Becker et al. 2016). No entanto, as análises foram restritas a espécimes depositados em
coleções científicas e pertencentes à família Leptodactylidae, cujos representantes apresentam
hábito altamente aquático e desenvolvimento indireto (Haddad et al. 2013). Carvalho et al.
(2017) analisaram a ocorrência do Bd com o diagnóstico de infecção pela análise do aparato
bucal de girinos, também depositados em coleções científicas. Esses estudos nos trazem uma
compreensão sobre determinados padrões de ocorrência do Bd, porém não existem estudos
analisando a dinâmica de infecção do Bd em populações naturais de anfíbios (incluindo
28
cecílias e salamandras), abrangendo uma maior diversidade de espécies e ocupando diferentes
microhabitats.
Além de ser uma região de ocorrência exclusiva de espécies da ordem Caudata,
outra particularidade da Amazônia brasileira é a presença de anuros do gênero Atelopus. Em
outras regiões das Américas, representantes deste gênero foram classificados como em
condição crítica, com suas populações sofrendo um declínio dramático, sendo que pelo menos
81% das espécies do gênero mostraram evidências de declínio e desaparecimento, e pelo
menos 30 espécies já foram extintas na natureza (LaMarca & Lötters 1997, Ron et al. 2003,
LaMarca et al. 2005). Declínios associados à infecção por Bd foram registrados em algumas
regiões como Costa Rica, Panamá, Equador, Venezuela e Peru (Berger et al. 1998, Lips et al.
2008, LaMarca et al. 2005, Lampo et al. 2006, Rodríguez-Contreras et al. 2008), e um estudo
recente identificou Atelopus como o gênero mais afetado pelo Bd no mundo (Scheele et al.
2019). No entanto, não se sabe se no Brasil as espécies do gênero presentes na Amazônia
estão infectadas pelo Bd, e qual seria a resposta dos indivíduos frente a potenciais eventos de
infecção por cepas exóticas, já que diferentes cepas de Bd podem ser amplamente
disseminadas por vias naturais e antropogênicas (Kilburn et al 2011, Garmyn et al. 2012,
Pontes et al. 2018).
Desde o primeiro registro de ocorrência do Bd na Mata Atlântica, muitos
trabalhos que exploram diferentes aspectos relacionados à dinâmica de infecção foram
desenvolvidos, mas a grande maioria se concentra na porção sul do bioma (James et al. 2015,
Jenkinson et al. 2016, Lambertini et al. 2016). Até então, não existem trabalhos que
exploraram a variação dos padrões de infecção por Bd ao longo do bioma por inteiro. Além
disso, os trabalhos desenvolvidos na região amazônica até então, são restritos a espécimes
depositados em coleções científicas, o que obviamente nos trazem informações importantes e
possibilitam uma ampla amostragem ao longo do bioma, mas não refletem com acurácia a
dinâmica de infecção do Bd em populações naturais.
Frente a todos os fatores acima citados, este trabalho analisou os padrões de
ocorrência do Bd nos biomas Amazônia e Mata Atlântica, explorando variáveis relacionadas
aos hospedeiros e ao ambiente dentro deste contexto. Levando em conta a escassez de
trabalhos que abordam a dinâmica de infecção por Bd espécies de Gymnophiona, realizamos
uma ampla amostragem de espécimes depositados em coleções científicas, e identificamos os
padrões de ocorrência do patógeno em cecílias ao longo da América do Sul. Além disso,
realizamos uma amostragem na região amazônica, identificando padrões de ocorrência do Bd
em populações naturais de anfíbios, buscando associações entre variáveis bióticas e abióticas
29
com as taxas de infecção detectadas. Também amostramos espécies de Caudata, que ocorrem
exclusivamente na região amazônica e, não existem até então, registros de Bd para tais
espécies no Brasil. Avaliamos através de experimentação em laboratório, os efeitos de
infecção por cepas exóticas de Bd em hospedeiros endêmicos da Amazônia, evidenciando
assim os riscos da disseminação de cepas do patógeno. Finalmente, realizamos uma ampla
amostragem de anfíbios anuros ao longo de um transecto latitudinal por toda extensão da
Mata Atlântica. Essa amplitude de amostragem nos possibilitou analisar a variação dos
padrões de infecção por Bd ao longo do bioma, e detectar associações entre variáveis bióticas
e abióticas e as taxas de infecção detectadas. O presente trabalho foi dividido em três
capítulos, os quais estão completamente detalhados a seguir, em formato de publicações
científicas.
30
Capítulo 1. SPATIAL DISTRIBUTION OF Batrachochytrium dendrobatidis IN SOUTH
AMERICAN CAECILIANS
** Artigo publicado no periódico
Diseases of Aquatic Organisms (2017)
Carolina Lambertini*,1
, C. Guilherme Becker2, Cecilia Bardier
1,3,
Domingos da Silva Leite4, Luís Felipe Toledo
1
1Laboratório de História Natural de Anfíbios Brasileiros (LaHNAB), Departamento de
Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, São
Paulo, 13083-862, Brazil.
2Departamento de Zoologia, Universidade Estadual Paulista, Rio Claro, São Paulo, 13506-
900, Brazil.
3Laboratorio de Sistemática e Historia Natural de Vertebrados, Instituto de Ecología y
Ciencias ambientales, Universidad de la República, Montevideo, 11400, Uruguay.
4Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade
Estadual de Campinas, Campinas, São Paulo, 13083-862, Brazil.
*Corresponding author:
Carolina Lambertini
E-mail: [email protected]
31
Abstract
The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) is linked to
population declines in anurans and salamanders globally. To date, however, few studies
attempted to screen Bd in live caecilians; Bd-positive caecilians were only reported in
Africa and French Guiana. Here, we performed a retrospective survey of museum
preserved specimens to (i) describe spatial patterns of Bd infection in Gymnophiona
across South America and (ii) test whether areas of low climatic suitability for Bd in
anurans predict Bd spatial epidemiology in caecilians. We used quantitative PCR to
detect Bd in preserved caecilians collected over a 109-year period, and performed
autologistic regressions to test the effect of bioclimatic metrics of temperature and
precipitation, vegetation density, and elevation on the likelihood of Bd occurrence. We
detected an overall Bd prevalence of 12.4%, with positive samples spanning across the
Uruguayan savanna, Brazilian Atlantic Forest, and the Amazon basin. Our Autologistic
models detected a strong effect of macroclimate, a weaker effect of vegetation density,
and no effect of elevation on the likelihood of Bd occurrence. Although most of our Bd-
positive records overlapped with reported areas of high climatic suitability for the
fungus in the Neotropics, many our new Bd-positive samples extend far into areas of
poor suitability for Bd in anurans. Our results highlight an important gap in the study of
amphibian chytridiomycosis: the potential negative impact of Bd on Neotropical
caecilians and the hypothetical role of caecilians as Bd reservoirs.
Keywords: Chytrid infection dynamics, Gymnophiona, Life history, Environmental
variables
32
Introduction
Amphibian fungal pathogens of the genus Batrachochytrium are linked to
population declines in anurans and salamanders globally through the infectious disease
chytridiomycosis (Berger et al. 1998, Lips et al. 2008, Martel et al. 2013). The
amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has been acknowledged
as one of the most destructive wildlife pathogens to wildlife (Fisher & Garner 2007,
Skerratt et al. 2007), causing population declines in a large fraction of infected species.
Bd is generalist among amphibians (Valencia-Aguilar et al. 2015), infecting anurans
and salamanders from tropical and temperate regions (Lips et al. 2006, Lips et al. 2008,
Vredenburg et al. 2010, Cheng et al. 2011). Reports of Bd infection in caecilians
(Gymnophiona), however, are rare in the literature. To our knowledge, only four reports
of Bd from wild-caught caecilians are available to date (Doherty-Bone et al. 2013,
Gower et al. 2013, Hydeman et al. 2013, Rendle et al. 2015).
The first study screening for Bd in caecilians reported 53 Bd-positive
caecilians out of 85 tested individuals from Cameroon (Doherty-Bone et al. 2013). In
the same year, Gower et al (2013) reported the first case of lethal chytridiomycosis in
caecilians from Cameroon and Tanzania. They reported that wild-caught specimens
from the genus Geotrypetes seraphini that tested positive for Bd died in captivity with
signs of chytridiomycosis. Bd infections were also confirmed in an endemic caecilian
(Schistometopum thomense) from an island of the archipelago of São Tomé and
Príncipe (Hydeman et al. 2013). The only record of Bd infecting caecilians outside
Africa was recently published for a wild-caught specimen from French Guiana (Rendle
et al. 2015). Despite the observed high Bd prevalence in African caecilians, sampling
efforts to detect Bd in Gymnophiona have been made in other regions but without any
Bd-positive samples from specimens collected in the wild in peninsular Malaysia (n=2;
Savage et al. 2011), Colombia (n=1; Vasquez-Ochoa et al. 2012), West Africa (n=6;
Penner et al. 2013), and the Seychelles archipelago (n=78; Labisko et al. 2015). The
small sample sizes of these published reports reflect the difficulty and/or lack of effort
in working with this generally inconspicuous taxon (e.g. Gower & Wilkinson 2005), it
cannot be ruled out that Gymnophiona suffers with Bd as extensively as do anurans.
Furthermore, conspicuous die-offs due to chytridiomycosis, such as those observed in
anurans, are relatively unlikely to be observed in caecilians (Gower et al. 2013: p.180)
due to their fossorial or fully-aquatic life styles (Wells 2007, Vitt & Caldwell 2014).
Caecilians are broadly distributed in the tropics (Taylor 1968, Frost 2016), often co-
33
occurring with anuran populations heavily impacted by chytridiomycosis (James et al.
2015, Seimon et al. 2007, Gower et al. 2012, Bataille et al. 2013), and inhabiting
microhabitats within Bd’s optimal growth conditions of temperature and humidity
(Piotrowski et al. 2004). Determining whether caecilians are suffering with
chytridiomycosis as do anurans and salamanders, and if they are serving as pathogen
reservoirs, is relevant for amphibian conservation (Gower & Wilkinson 2005).
Several environmental factors influence Bd infection in anurans. Infection
prevalence and zoospore loads are often positively correlated with elevation (Brem &
Lips 2008, Gründler et al. 2012, Catenazzi et al. 2013), vegetation density (Puschendorf
et al. 2009, Becker & Zamudio 2011, Becker et al. 2015), precipitation (Becker &
Zamudio 2011), and negatively correlated with temperature (Becker & Zamudio 2011,
Becker et al. 2015, Ruggeri et al. 2015). Because most caecilians are fossorial, they are
likely exposed to lower microclimatic fluctuations dictated by land cover, insolation,
and humidity. Therefore, microclimatic optimum/average of caecilians might fall within
Bd’s optimal growth conditions, allowing Bd to persist in areas where it would
otherwise not endure year-round.
Here, we performed a retrospective survey of museum preserved specimens
to (i) describe spatial patterns of Bd infection in Gymnophiona in South America and
(ii) test whether areas of low climatic suitability for Bd in anurans predict Bd spatial
epidemiology in caecilians. We used quantitative PCR to detect Bd in preserved
caecilians collected over a 109 year period, and performed autologistic regressions to
test the effect of bioclimatic metrics of temperature and precipitation, vegetation
density, and elevation on the likelihood of Bd occurrence. Our results provide novel
information on Bd spatial epidemiology and suggest that caecilians could be potentially
threatened or serve as a Bd reservoir in regions where anurans are not infected during
most of the year.
Methods
Species sampling
We sampled 193 museum-preserved specimens of Gymnophiona; 160 from
Brazil (Caeciliidae, Siphonopidae, and Typhlonectidae) and 33 from Uruguay
(Typhlonectidae; Table 1). We screened specimens from three our of four South
American caecilian families housed at the following herpetological collections: Museu
Paraense Emílio Goeldi (MPEG), Museu de Zoologia “prof. Adão José Cardoso”,
34
Universidade Estadual de Campinas (ZUEC), Museu Nacional, Universidade Federal do
Rio de Janeiro (MNRJ), Coleção de Anfíbios Célio F. B. Haddad, Universidade
Estadual Paulista (CFBH), and Colección de Vertebrados de la Universidad de La
Republica, Montevideo (ZVCB) (Table S1). For standardization purposes, we did not
include in our analyses published Bd data from wild caught caecilians from French
Guiana, Guyana and Colombia (Vásquez-Ochoa et al. 2012, Gower et al. 2013, Rendle
et al. 2015). We gathered GPS coordinates in decimal degrees for each sampled
specimen based on museum data. We used the geographic centroid of municipalities as
an approximation when precise geographic coordinates were not available. We did not
consider records of Bd from captive and live specimens for methodological consistency
(e.g. Raphel and Pramuk 2007, Churgin et al. 2013).
Bd detection
Retrospective sampling of museum specimens has been used widely to
determine Bd-historical dynamics across space and time (Weldon et al. 2004, Ouellet et
al. 2005, Soto-Azat et al. 2010, Cheng et al. 2011, Vredenburg et al. 2013, Rodriguez et
al. 2014, Becker et al. 2015, Courtois et al. 2015, Talley et al. 2015). We swabbed
individual specimens on their head, anal disc, dorsal, and ventral surfaces with a single
swab per individual, following Rendle et al (2015), and stored each sample in a 1.5 mL
dry sterile tube. We extracted DNA from each swab using 50 µL de PrepMan
ULTRA®, and proceeded with the molecular detection with TaqMan® qPCR assay
(Life Technologies), using the strain CLFT 023 as a quantitative standard for the
reactions diluted from 103 to 10
-1zoospore genomic equivalents (g.e.) (Boyle et al. 2004,
Lambertini et al. 2013). We considered Bd-positive samples with g.e. ≥ 1 (Kriger et al.
2007).
Statistical Analyses
We described patterns of Bd infection in Caecilians from Brazil and
Uruguay (proportion of Bd-infected individuals ± binomial 95% CI). We classified
species based on their predominant life history (aquatic or fossorial) and reported the
proportion (± binomial 95% CI) of infected individuals for each life-history category
and ecoregion. We also reported on spatiotemporal patterns of Bd infections from 1905-
2014; 28 specimens lacking information of collection year were excluded from these
calculations.
35
Furthermore, we conducted a multi-model inference using Autologistic
regressions to test the effect of bioclimatic variables, vegetation density, and elevation
on the likelihood of Bd infection while accounting for the effects of spatial
autocorrelation (Rangel et al. 2010). For each sampling location we extracted 19
bioclimatic variables of temperature and precipitation averaged over a period of 50
years (Hijmans et al. 2005), vegetation density (FAO 2010) and elevation, using Arc
Map v.10.1 (ESRI 2012). We used a model averaging procedure, including Bd as the
response variable (presence vs. absence) and the aforementioned environmental factors
as explanatory variables. Our model averaging ranked all possible models based on AIC
and averaged beta coefficients of variables present in 90% of models within ΔAIC < 2.
We reported the strength and the direction that each environmental variable influenced
Bd. We used SAM v4.0 to perform statistical analyses (Rangel et al. 2010).
Results
Our qPCR reactions detected Bd in 24 out of 193 screened specimens
(12.4%, 95% CI = 0.08-0.17; Fig. 1). Infected individuals belonged to the families
Siphonopidae (eight individuals) and Typhlonectidae (16 individuals) distributed across
the Uruguayan savanna (proportion of infected individuals = 12.1%, 95% CI = 0.03 -
0.28, n = 33), the Amazon Basin (16%, 95% CI = 0.08 - 0.26, n = 75), and Brazilian
Atlantic Forest (13.5%, 95% CI = 0.06 - 0.24, n = 59). We did not detect Bd in
individuals of Caeciliidae and also in samples from the Brazilian Cerrado, Caatinga, and
Pantanal, though our sampling size in these ecoregions was small (n = 2, n = 22 and n =
2, respectively). We detected a proportion of infected individuals of 11.7% in aquatic
species (95% CI = 0.06 - 0.18, n = 136) and 14% in terrestrial (typically fossorial)
species (95% CI = 0.06 - 0.25, n = 57).
Although our sampling spanned 109 years, most of our Bd positive
specimens (n = 16) were collected after 1994. Only five samples before this period
tested positive for Bd, and were collected from the wild between 1965 and 1994.
Twenty-one samples collected prior to 1971 were screened, and they all tested negative
for Bd.
Our spatial regression models indicated a significant effect of macroclimate
on the likelihood of Bd occurrence (Table 2). Our Autologistic model averaging showed
a negative effect of maximum temperature of warmest month and precipitation of
wettest quarter, and a positive effect of annual precipitation on Bd infection likelihood;
36
full set of significant variables found on Table 2. Vegetation density had a weak
negative effect on Bd occurrence, and elevation was not a significant variable in our
models (Table 2; Table S2).
Discussion
Seasonal variations in temperature and precipitation strongly mediate Bd
infections by changing optimal physiological conditions of hosts and pathogen
(Piotrowski et al. 2004, Becker & Zamudio 2011, Ruggeri et al. 2015). These
environmental constraints are revealed in several environmental niche models,
indicating that much of South America is unsuitable for Bd during at least for part of the
year (Rödder et al. 2009, Liu et al. 2013, Becker et al. 2015, James et al. 2015).
Although most of the Bd-positive records overlapped with reported areas of high
climatic suitability for the fungus in the Neotropics (Rödder et al. 2009, Liu et al. 2013,
Becker et al. 2015, James et al. 2015), many new Bd-positive records extend far into
areas of poor suitability for Bd in anurans (e.g., central Amazon: see Becker et al.
2015). Our data points to widespread Bd infections in Neotropical caecilians, and that
this taxon may serve as an environmental reservoir, perhaps because hosts are able to
avoid harsh seasonal extremes where Bd would otherwise not persist year-round. These
results, combined with the recent report of lethal chytridiomycosis in wild-caught
caecilians (Gower et al. 2013), indicate that Gymnophiona are potentially experiencing
silent population declines in the wild due to Bd.
Spatial regressions are also consistent with the observed associations
between macroclimate and Bd infection in anuran species (Becker & Zamudio 2011,
Becker et al. 2015, James et al. 2015). Specifically, we detected a positive effect of
precipitation and a negative effect of temperature variables on the likelihood of Bd
infection in caecilian hosts. Vegetation density, which is often positively associated
with Bd infection in anurans (Raffel et al. 2010, Becker & Zamudio 2011, Becker et al.
2012), showed a weak negative effect on Bd in caecilians. This finding might be due to
the high degree of fossoriality of terrestrial caecilian species, which spares them from
the direct or indirect effects of habitat quality, with downstream shifts in both macro-
and micro-climates. Elevation, which is often positively associated with Bd infection in
anurans (Walker et al. 2010, Piovia-Scott et al. 2011, Gründler et al. 2012) due to
optimal growth conditions in highlands (Piotrowski et al. 2004), showed no effect on Bd
in Caecilians. Although our sampling spanned 1000 meters in elevation, most of our
37
samples were collected at lower altitudes. This uneven sampling across the elevation
gradient may have thus impacted our ability to detect a significant effect of elevation in
our analyses. Although large-scale climate may play a role in Bd epizootiology of
caecilians, these results indicate that infection dynamics in caecilians and anurans might
be different.
Although results suggest that caecilians could act as pathogen reservoirs in
environments or periods of harsh microclimatic conditions, there is limited natural
history information for most caecilian species (Gower & Wilkinson 2005, Vitt &
Caldwell 2014). Basic information on foraging behavior, population densities, and
breeding habits that would be key to quantify transmission dynamics between
Gymnophiona and Anura are typically lacking. It is known that five out of 10 families
of caecilians are found in South America (Wilkinson et al. 2011, Frost 2016), and that
these five families span from completely fossorial to aquatic (Haddad et al. 2013, Vitt &
Caldwell 2014). We predict that fossorial and fully aquatic species will be less likely
exposed to environmental and climatic fluctuation than terrestrial anurans because they
spend longer periods of time underground or underwater; future studies of caecilians’
foraging behavior and habitat use may help test the link between habitat use and
temperature variability. Lower temperature extremes and variability are linked to higher
Bd growth and persistence in amphibian hosts both in the wild and in the laboratory
(Pounds et al. 2006, Raffel et al. 2013, 2015). Because Bd is a waterborne fungus
(Longcore et al. 1999, Kilpatrick et al. 2009), we also expect fully aquatic caecilians to
be exposed to the pathogen not only during early life stages. Therefore, fossorial and
aquatic life styles observed in caecilians are two life history traits that likely make an
efficient host reservoir, especially in areas where Bd does not persist in anuran hosts
year-round.
In areas of low predicted suitability for Bd such as the Amazon basin (Ron
et al. 2005, Becker et al. 2015, James et al. 2015), we detected an infection prevalence
of 16%, which is surprisingly high compared to the observed ~3% in museum-preserved
anurans in this region (Becker et al. 2015). In contrast, the proportion of infected
caecilians in the Atlantic Forest was slightly lower than what has been observed for
preserved anurans (~23%) in this ecoregion (Rodriguez et al. 2014). Nonetheless,
limited sample size for caecilians prior to the 1970s precludes us from making any
concrete spatio-temporal comparison between Bd in caecilians and in anurans from both
the Atlantic Forest (Rodriguez et al. 2014) and the Amazon basin (Becker et al. 2015).
38
Although most aquatic caecilians included in this study were sampled from the Amazon
basin and most terrestrial caecilians from the Atlantic Forest, we did not detect a
significant effect of host life style (aquatic vs. terrestrial) on the likelihood of Bd
infection. These results further indicate that Bd infection dynamics in Gymnophiona
might experience a lower pressure from macroclimate than Anura.
Our results highlight an important gap in the study of amphibian
chytridiomycosis: the possible impact of Bd on Neotropical caecilians and the
hypothetical role of caecilians as Bd reservoirs. To date, information on susceptibility of
caecilian hosts to Bd infection is still lacking. Therefore, Bd genotypes detected in
caecilians may present different adaptations to host histophysiology or microclimates,
which provides a key opportunity to isolate and genotype new Bd isolates from live
caecilians and test the virulence of these new isolates in anurans. Because Bd has a
disproportionately higher impact in tropical amphibians, a better understanding of Bd
infection dynamics in Gymnophiona may increase our knowledge about the
chytridiomycosis pandemic and advance our conservation efforts in the wild.
Acknowledgements
We thank Adriano O. Maciel, Alexandre F.R. Missassi, Manoela Woitovicz Cardoso,
Nadya C. Pupin and Tamilie Carvalho for help with swabbing and providing museum
specimens. Ana L.C. Prudente (MPEG), Célio F.B. Haddad (CFBH), José P. Pombal Jr.
(MNRJ) and Raúl Maneyro (ZVCB) allowed access to museum specimens. David J.
Gower and two anonymous reviewers for constructive feedback on our manuscript. Our
work was funded by Coordination for the Improvement of Higher Education Personnel
(CAPES) and the National Council of Technological and Scientific Development
(CNPq #405285/2013-2; #312895/2014-3) and Sao Paulo Research Foundation
(FAPESP #2014/23388-7).
39
Table 1. Families, species, sample size, environment and country where individual
caecilians were captured, and prevalence (as the number of positives/total screened).
Family n Habit Habitat Country Prevalence
Caeciliidae
Caecilia gracilis 01 Fossorial Rainforest Brazil 0/1
Siphonopidae
Luetkenotyphlus brasiliensis 09 Fossorial Rainforest Brazil 3/9
Siphonops annulatus 12 Fossorial Rainforest Brazil 1/12
Siphonops cf. annulatus 01 Fossorial Rainforest Brazil 1/1
Siphonops paulensis 08 Fossorial Rainforest Brazil 2/8
Siphonops cf. paulensis 15 Fossorial Rainforest
Grassland
Brazil 0/15
Siphonops sp. (aff. paulensis) 02 Fossorial Grassland Brazil 0/2
Siphonops hardyi 02 Fossorial Rainforest Brazil 0/2
Siphonops sp. 07 Fossorial Rainforest
Grassland
Brazil 1/7
Typhlonectidae
Atretochoana eiselti 04 Aquatic Rainforest Brazil 0/4
Chthonerpeton braestrupi 06 Aquatic Rainforest Brazil 0/6
Chthonerpeton indistinctum 33 Aquatic Grassland Uruguay 4/33
Chthonerpeton indistinctum 01 Aquatic Rainforest Brazil 0/1
Chthonerpeton noctinetes 08 Aquatic Rainforest Brazil 0/8
Chthonerpeton sp. 01 Aquatic Rainforest Brazil 0/1
Chthonerpeton tremembe 04 Aquatic Grassland Brazil 0/4
Chthonerpeton viviparum 08 Aquatic Rainforest Brazil 0/8
Potamotyphlus kaupii 39 Aquatic Rainforest Brazil 9/39
Typhlonectes compressicauda 32 Aquatic Rainforest Brazil 3/32
40
Table 2. Model averaging results ranking significant environmental variables
explaining Batrachochytrium dendrobatidis occurrence in South American caecilians.
Variable Rank Importance Beta coefficient 95% CI
Bio 5 43 -0.038 0.002
Bio 12 32 0.003 0.001
Bio 16 22 -0.009 0.004
Bio 3 12 0.076 0.032
Bio 13 11 0.039 0.011
Bio 17 10 0.002 0.008
Bio 15 9 -0.029 0.006
Bio 14 9 -0.027 0.023
Bio 18 7 0.003 0.001
Bio 19 7 0.002 0.001
Vegetation Density 6 -0.011 0.003
Bio 10 4 -0.034 0.003
Bio 9 3 -0.011 0.006 Rank importance corresponds to the number of models that each variable was present. CI stands for confidence
interval. Description of bioclimatic variables: Bio 3 = Isothermality, Bio 5 = Temperature of Warmest Month, Bio
9 = Mean Temperature of Driest Quarter, Bio 10 = Mean Temperature of Warmest Quarter, Bio 12 = Annual
Precipitation, Bio 13 = Precipitation of Wettest Month, Bio 14 = Precipitation of Driest Month, Bio 15 =
Precipitation Seasonality, Bio 16 = Precipitation of Wettest Quarter, Bio 17 = Precipitation of Driest Quarter, Bio
18 = Precipitation of Warmest Quarter, Bio 19 = Precipitation of Coldest Quarter
41
Figure 1
42
Table S2. Model selection showing bioclimatic variables associated to
Batrachochytrium dendrobatidis infection.
Model No. of
variables
AIC ΔAIC
Bio 15, Bio 3, Bio 5 3 139.356 0
Bio 12, Bio 5 2 139.596 0.240
Bio 12, Bio 13, Bio 14, Bio 16, Bio 5 5 139.707 0.351
Bio 12, Bio 13, Bio 16, Bio 5 4 139.752 0.396
Bio 12, bio 16, Bio 5 3 139.878 0.522
Bio 15, Bio 3, Bio 5, Bio 9 4 140.012 0.656
Bio 12, Bio 15, Bio 5 3 140.035 0.679
Bio 13, Bio 15, Bio 16, Bio 3n, Bio 5n 5 140.106 0.750
Bio 16, Bio 18, Bio 19, Bio 5n 4 140.382 1.026
Bio 14, Bio 17, Bio 5 3 140.421 1.065
Bio 12, Bio 13, Bio 5 3 140.422 1.066
Vegetation density, Bio 12, Bio 14, Bio 16, Bio 10 5 140.466 1.067
Bio 12, Bio 13, Bio 16, Bio 17, Bio 5 5 140.466 1.110
Bio 11, Bio 18, Bio 2, Bio 3 4 140.495 1.139
Bio 12, Bio 14, Bio 16, Bio 19, Bio 5 5 140.505 1.149
Bio 13, Bio 15, Bio 16, Bio 5 4 140.559 1.203
Bio 17, Bio 3, Bio 5 3 140.561 1.205
Bio 12, Bio 17, Bio 5 3 140.627 1.271
Bio 17, Bio 5 2 140.638 1.282
Bio 12, Bio 14, Bio 16, Bio 5 4 140.642 1.286
Vegetation density, Bio 12, Bio 14, Bio 16, Bio 5 5 140.642 1.286
Bio 15, Bio 18, Bio 3, Bio 5 4 140.709 1.353
Bio 12, Bio 5, Bio 8 3 140.715 1.359
Bio 15, Bio 18, Bio 19, Bio 5 4 140.725 1.369
Bio 11, Bio 15, Bio 2, Bio 3, Bio 6 5 140.821 1.465
Vegetation density, Bio 12, Bio 16, Bio 5 4 140.829 1.473
Elevation, Bio 12, Bio 13, Bio 16, Bio 5 5 140.848 1.492
Bio 12, Bio 14, Bio 5 3 140.853 1.497
Bio 18, Bio 19, Bio 5 3 140.947 1.591
Bio 12, Bio 4, Bio 5 3 141.003 1.647
Bio 18, Bio 2, Bio 3, Bio 6 4 141.008 1.652
Bio 12, Bio 16, Bio 19, Bio 5 4 141.010 1.654
Bio 12, Bio 5, Bio 9 3 141.030 1.674
Bio 12, Bio 10 2 141.049 1.693
Bio 11, Bio 12, Bio 5 3 141.052 1.696
Bio 14, Bio 3, Bio 5 3 141.061 1.705
Vegetation density, Bio 12, Bio 13, Bio 16, Bio 5 5 141.066 1.710
Bio 13, Bio 17, Bio 5 3 141.091 1.735
Bio 12, Bio 5, Bio 1 3 141.155 1.799
Bio 12, Bio 6, Bio 7 3 141.160 1.804
Bio 12, Bio 6,Bio5 3 141.176 1.820
Bio12, Bio5, Bio7 3 141.178 1.822
Bio12, Bio16, Bio17, Bio5 4 141.254 1.898
Vegetation density, Bio12, Bio16, Bio17, Bio5 5 141.254 1.898
Bio 12, Bio 13, Bio 16, Bio 3, Bio 5 5 141.259 1.903
Bio 15, Bio 3, Bio 9 3 141.295 1.939
Bio 12,Bio 16, Bio 3, Bio 5 4 141.305 1.949
Bio12, Bio 13, Bio 16, Bio 19, Bio 5 5 141.310 1.954
Vegetation density, Bio 12, Bio 16, Bio 17, Bio 10 5 141.310 1.954
Bio 14, Bio 5 2 141.311 1.955
43
Bio 18, Bio 19, Bio10 3 141.352 1.996
Bio 16,Bio 17, Bio 5 3 141.360 2.004 Bio 1 = Annual Mean Temperature, Bio 2 = Mean Diurnal Range, Bio 3: Isothermality, Bio 4 = Temperature
Seasonality, Bio 5 = Maximum Temperature of Warmest Month, Bio 6 = Minimum Temperature of Coldest Month,
Bio 7 = Temperature Annual Range, Bio 8 = Mean Temperature of Wettest Quarter, Bio 9 = Mean Temperature of
Driest Quarter, Bio 10 = Mean Temperature of Warmest Quarter, Bio 11 = Mean Temperature of Coldest Quarter,
Bio 12 = Annual Precipitation, Bio 13 = Precipitation of Wettest Month, Bio14 = Precipitation of Driest Month,
Bio15 = Precipitation Seasonality, Bio16 = Precipitation of Wettest Quarter, Bio17 = Precipitation of Driest Quarter,
Bio 18 = Precipitation of Warmest Quarter, Bio 19 = Precipitation of Coldest Quarter.
44
Capítulo 2. THE KILLING-CHYTRID FUNGUS IN AMPHIBIAN POPULATIONS
OF THE BRAZILIAN AMAZONIA
Carolina Lambertini1, Alexandre F. R. Missassi
2, Rafael F. Jorge
3, Domingos da Silva
Leite4, Albertina P. Lima
3, Luís Felipe Toledo
1
1Laboratório de História Natural de Anfíbios Brasileiros (LaHNAB), Departamento de
Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas,
São Paulo, 13083-862, Brazil
2Programa de Pós-graduação em Biodiversidade e Evolução, Departamento de
Zoologia, Museu Paraense Emílio Goeldi, Belém, Pará, 66077-830, Brazil
3Instituto Nacional de Pesquisas da Amazônia, Manaus, Amazonas, 69067-375, Brazil
4Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade
Estadual de Campinas, Campinas, São Paulo, 13083-862, Brazil.
*Corresponding author:
Carolina Lambertini
E-mail: [email protected]
45
Abstract
Infectious diseases pose one of the main threats to biodiversity. Chytridiomycosis,
caused by the fungus Batrachochytrium dendrobatidis (Bd), was responsible for
amphibian massive losses all over the globe and Bd establishment and development
may be dictated by environmental variation over areas that can be or not suitable for the
pathogen. The Brazilian Amazonia was considered climatic unsuitable for Bd, and
retrospective surveys detected low infection rates, but we lack information of Bd
infection dynamics in current wild amphibian populations. We sampled 462 amphibians
in seven sites in the Brazilian Amazonia and quantified Bd infections by qPCR. We
tested whether abiotic variables explained Bd infections. We also tested for
relationships between species reproductive biology and type of habitat with Bd
infections. Finally, we experimentally tested the effect of Bd infections on Atelopus cf.
spumarius with two different strains (CLFT 156 and CLFT 102 – isolated from the
south and north Atlantic forest, respectively). We detected high Bd infection rates in
Amazonia, and infection in all three orders of amphibians. Only annual mean
temperature explained Bd infection prevalence, and none of the examined variables
explained infection load. Host’s reproductive biology was not related with Bd
infections, but we detected a positive relationship between species’ type of habitat and
Bd infections. Besides, we detected higher mortality rate on the group of Atelopus aff.
spumarius infected with CLFT 156, probably because this strain was isolated from a
site more environmentally distinct than the strain isolated in the northern Atlantic forest.
Contrary to ecological niche modellings, neither climate nor the other abiotic variables
we tested explained Bd occurrence in our small scale sampling, and we found a much
higher prevalence than previously modeled. Based on our study, it is clear that the
Amazon is still underexplored and different disease dynamics could be described after
future studies targeting all amphibian orders in this region.
Keywords. Batrachochytrium dendrobatidis, disease ecology, tropical forest,
susceptible species, Anura, Gymnophiona, Caudata
46
Introduction
Infectious diseases are one of the main drivers of population declines and
species extinctions globally (Daszak et al. 2000, Skerrat et al. 2007, Jones et al. 2008,
Scheele et al. 2019), caused by a diversity of pathogenic agents and affecting several
taxa (Tompkins et al. 2015). For example, fungal infectious disease outbreaks have
caused remarkable population declines in coral species (Aspergillus sydowii, Kim and
Harvell 2004), bats (Geomyces destructans, Blehert et al. 2009), snakes (Ophidiomyces
ophiodiicola, Allender et al. 2016), salamanders (Batrachochytrium salamandrivorans,
Martel et al. 2014) and anurans (Batrachochytrium dendrobatidis, Longcore et al.
1999). Given the biodiversity crisis and its current threat by spreading of infectious
diseases (Stuart et al. 2004, Tompkins et al. 2015), characterizing factors that influences
and drives the dispersion and establishment of a particular pathogenic agent in a given
area, is imperative to development of conservation actions targeting populations at risk.
Environment has a key role on host-pathogen interactions (Scholthof 2007)
and spreading of diseases, basically because its variation (e.g., climate) may dictate
whether a given area is suitable or not for the establishment and development of a
pathogen (Ron 2005, Rödder et al. 2010), which may be characterized by using
ecological niche modelling analysis (Elith and Leathwick 2009). This approach has
been applied in widely spread diseases with huge impact on biodiversity, such as the
chytridiomycosis, an emerging infectious disease that caused the declines of amphibian
populations all over the globe (Scheele et al. 2019).
This disease is caused by the water-borne fungus Batrachochytrium
dendrobatidis (hereafter Bd) (Longcore et al. 1999). While Bd is enzootic in some
regions, epizootics has already occurred causing amphibian population massive losses
(Scheele et al. 2019). For example, Bd rapidly emerged and spread in a wave-like
pattern throughout Central America, which seriously affected several amphibian
populations (Lips et al. 2006). In Brazil, a retrospective study showed that Bd was
potentially enzootic at Atlantic forest (Rodriguez et al. 2014), but enigmatic amphibian
declines that occurred about three decades ago were associated to chytridiomycosis
(Carvalho et al. 2017). Therefore, both the arrival of the pathogen and some changes in
its local dynamics can lead to permanent amphibian extinctions (Toledo 2017).
On the other hand, the Brazilian Amazonia has been considered a region
with low suitability for Bd, due to its climate that generally is inadequate for the
pathogen development (Ron et al. 2005, Rödder et al. 2010, Liu et al. 2013, Becker et
47
al. 2016, Voyles et al. 2017). Two retrospective studies has already been conducted in
Brazilian Amazonia, analyzing museum preserved anuran species, and both studies
detected lower Bd infection prevalence (i.e., less than 4 %: Becker et al. 2016, Carvalho
et al. 2017). However, at the same time that samplings of wild amphibian populations
(Bd detection and quantification) in several other regions accurately reflected Bd
environmental suitability or unsuitability by distribution models (Murray et al. 2011,
Flechas et al. 2017), some studies detected the opposite, where distribution models
detected suitable areas for Bd development, but Bd detection in wild populations did not
reflect modelling results (e.g., Asia, Swei et al. 2011). For the Brazilian Amazonia, we
lack information on whether Bd infections in wild populations are in agreement with
predicted environmental suitable areas for Bd. We also lack information on general
patterns of Bd infections, as which biotic and abiotic factors may explain potential Bd
infection variation. Finally, we also don’t know which Bd lineage are in the Amazon,
since at least three highly divergent strains occurs in the Brazilian Atlantic forest
(Jenkinson et al. 2016).
The Brazilian Amazonia harbors at least three species of the genus Atelopus
(A. spumarius, A. hoogmoedi, A. flavescens) (Frost, 2018). Populations of several
species of Atelopus from Central and South America are suffering dramatic declines,
with at least 80 % of the species showing evidences of declines, and at least 30 species
are considered to be extinct from the wild (Scheele et al. 2019). Atelopus spp. declines
were associated to Bd infections in Costa Rica, Ecuador, Panama, Venezuela and Peru,
being considered a genus highly susceptible to Bd infections (Berger et al. 1998, Lips et
al. 2008, LaMarca et al. 2005, Lampo et al. 2006, Rodríguez-Contreras et al. 2008,
Scheele et al. 2019). Atelopus spp. occurrence in Brazil is restricted to Amazon forest,
and since we have no information on Bd infections in wild populations, we lack
information on whether or not Atelopus are susceptible to Bd infection, and if the
patterns of host response to infection vary with different Bd strains.
Therefore, we sampled 462 wild-caught amphibian (Anura, Caudata and
Gymnophiona) individuals in seven sites, and tested whether abiotic and biotic variables
were related to Bd infection prevalence and load across the Brazilian Amazonia. We
also experimentally infected individuals of Atelopus aff. spumarius with two Bd strains
isolated from different regions to verify if the strain isolated in more similar climatic
conditions to the site where Atelopus aff. spumarius was found induced higher mortality
rates than the infection with the other, more distinct, strain. We show different patterns
48
of Bd infection dynamics at Brazilian Amazonia when compared to other Neotropical
rainforest, as the Brazilian Atlantic forest and discuss the implications of such
divergence.
Methods
Study site and species samplings
We sampled amphibians in the Brazilian Amazonia, in seven sites within
five municipalities from the states of Amapá, Amazonas and Pará (Figure 1, Table 2).
We also sampled salamander specimens housed at the Coleção Herpetológica Osvaldo
Rodriguez da Cunha, Museu Paraense Emílio Goeldi (MPEG), municipality of Belém,
state of Pará, and Museu de Zoologia, Universidade Federal do Acre (UFAC),
municipality of Rio Branco, state of Acre, Brazil.
We conducted field samplings for amphibians (Anuran, Caudata and
Gymnophiona) by active and acoustic searches, also by using pitfall traps, between
2014 and 2018. We sampled 462 individuals, allocated into 13 families, 24 genera and
57 species, covering all three classes of amphibians (Table 1, Table S1). We recorded
GPS coordinates in decimal degrees for each sampling location, and used the
municipality geographic centroid when GPS coordinates were unavailable. Given the
small sample size of salamander specimens, we also sampled 56 museum preserved
Bolitoglossa spp. specimens, housed at the herpetological collections Museu Paraense
Emílio Goeldi (MPEG) and Universidade Federal do Acre (UFAC) (Table 1).
Experimental design and infection
We collected 23 individuals of Atelopus aff. spumarius, at the Reserva
Florestal Adolpho Ducke, municipality of Manaus, state of Amazonas. Each individual
was placed into plastic boxes containing Sphagnum sp. and water, simulating terrestrial
and aquatic environments. We then proceeded with the infection of individuals, using
two different strains of the fungus Batrachochytrium dendrobatidis, one of those
isolated from municipality of Morretes, state of Paraná, south Atlantic Forest (CLFT
156), and the other from municipality of Camacan, state of Bahia, north Atlantic Forest
(CLFT 102). Both strains belong to the Global Pandemic Lineage (GPL), and were
reactivated from liquid stock in petri dishes containing 1% Triptone, incubated at 21 ºC
during seven days. After that, plates were flooded with distilled water, and the total
number of zoospores in the suspension was defined with a hemocytometer (1 x 106
49
zoospores) (Lambertini et al. 2013). Before experimental infection, all individuals were
tested for Bd presence by qPCR, and they were all uninfected. The individuals were
then infected individually, each one in a clean Petri plate in direct contact with the
zoospore suspension (or distilled water for the control group) during 50 minutes and,
right after allocated in their respective boxes, where they were kept until the end of
experiment. We defined three experimental groups: i) CLFT 156 group (n = 8), ii)
CLFT 102 group (n = 8) and iii) Control group (n = 7). We fed individuals with
Drosophila sp. ad libitum and they were daily monitored during all experimental
procedure. Infected individuals that survived until the end of experiment (day 48) were
euthanized with Lidocaine 5% and deposited at the Museu de Zoologia prof. Adão José
Cardoso (ZUEC), UNICAMP, Campinas, Brazil.
Pathogen detection
During field samplings, we placed the specimens individually in plastic
bags, to avoid cross contamination. We then swabbed each individual on their skin,
following standard protocols (Hyatt et al. 2007, Lambertini et al. 2013). For museum
specimens, we followed the same swabbing protocol, but each specimen was rinsed
with 70% EtOH before, avoiding cross contamination from the storage (Rodriguez et al.
2014). Swabs were placed individually in 2.0 mL cryotubes and stored at -20 ºC until
molecular analyses were performed. We then extracted DNA from the samples using
PrepMan ULTRA® and proceeded with molecular detection and quantification by using
TaqMan® qPCR Assay (Life Technologies) (Boyle et al. 2004, Lambertini et al. 2013).
We used the strain CLFT 159 to generate the qPCR standard curve, serially diluted from
103 to 10
-1 zoospore genomic equivalents (g.e.). We considered Bd-positive samples
with at least one g.e. (Kriger et al. 2007). All infection load results were rounded to
integer numbers.
Abiotic variables
We extracted seven bioclimatic metrics for temperature and precipitation –
Annual Mean Temperature (Bio1), Mean Diurnal Range (Bio 2), Temperature
Seasonality (Bio 4), Maximum Temperature of Warmest Month (Bio 5), Annual
Precipitation (Bio 12), Precipitation Seasonality (Bio 15), Precipitation of Warmest
Quarter (Bio 18), for each sampling site (Hijmans et al. 2005). We also extracted data
50
on human footprint, elevation and topographic complexity using Arc Map v.10.1 (ESRI
2012).
Statistical analyses
In order to verify if there were associations between abiotic variables and
Bd infection rate, we performed a model selection [parameters: GLM with binomial
distribution (prevalence) and Gaussian distribution (infection load)], testing whether
bioclimatic variables, elevation, topographic complexity and human footprint could
better explain Bd infection prevalence and load across the Brazilian Amazon.
To test for biotic variables and Bd infections, we classified the species by
their type of habitat (Forested, Open/Forested and Open areas) and simplified
reproductive mode, according to their reproductive biology [aquatic (aquatic larvae) and
terrestrial (direct and indirect development)] (Table 1; Table S1). We performed
analysis of variance (ANOVA), to test for differences between type of habitat and
infections rates, and t test for differences between species reproductive modes and
infections rates, with a Tukey test a posteriori to verify where the differences were.
For the experiment result analyses, we performed a logrank-test, using the
package survival (Therneau 2012), to detect differences between mortality curves
among the treatments. We then performed a t test to verify if there were differences on
Bd infection load between both treatments. Finally, to test for bioclimatic similarity
between the strains isolating sites and Atelopus aff. spumarius collecting site, we
performed a hierarchical clustering using the algorithm (UPGMA) based on Euclidean
similarity index. The statistical analyses were performed in R (R Core Team, 2012) and
Past v.2.16.
Results
We detected an overall infection prevalence of 14.5 %, and a mean infection
load of 95 zoospore g.e. at the Brazilian Amazonia. Infection prevalence varied from
2.6 to 28.8 %, and the mean infection load from 2 to 221 g.e. across sampling sites
(Figure 1, Table 2). The highest infection prevalence was detected at the municipality of
Altamira, state of Pará (28.8 %), and the highest infection load was detected in an
individual of the species Allobates cf. hodlii (1,672 g.e.), also from municipality of
Altamira. Besides in anurans, we detected Bd in two wild-caught salamanders and two
51
caecilians (Table 1, Table S1). These individuals were from the municipality of
Altamira. We did not detect Bd in museum preserved salamanders (Table 1).
Our model selection did not detect influence of bioclimatic variables,
elevation, topographic complexity, and human footprint on Bd infection load. However,
our best model showed that annual mean temperature explained Bd infection prevalence
(β = -0.152396; P = 0.0006). For the reproductive biology, we did not detect differences
on Bd infection prevalence and load between aquatic and terrestrial species (t = -1.031;
P = 0.302 and t = 0.735; P = 0.462, respectively). For species type of habitat, we
detected higher Bd infection prevalence and load in forested areas than open/forested
and open areas (F[2,380] = 9.1840; P < 0.0001 and F[2,380] = 5.4086; P < 0.0001,
respectively, Figure 2 A,B).
According to our logrank-test, infected individuals from the treatment CLFT
156 showed higher mortality rate than infected ones from the treatment CLFT 102 (P <
0.0001, Figure 3 A), in which all individuals survived until the end of experiment. The
infection load of individuals from the treatment CLFT 156 was higher than infection
load from the treatment CLFT 102, both in the middle and at the end of experiment (t =
2.3077; P = 0.0464, and t = 4.1809; P = 0.0009, respectively, Figure 3 B). The strain
CLFT 102 showed a higher climatic similarity with the A. hoogmoedi collecting site
than the strain CLFT 156 (cophenetic correlation coeficient = -0.9311) (Figure S2).
Discussion
Our samplings across the Brazilian Amazonia showed unexpected Bd
infection rates. Specifically, we detected higher Bd infection prevalence, compared with
was previously detected in retrospective analyses (Becker et al. 2016, Carvalho et al.
2017), and with what was predicted by ecological niche modellings done for the region,
which classified the Brazilian Amazon as environmentally unsuitable for Bd (Ron 2005,
Rödder et al. 2010, Liu et al. 2013, Becker et al. 2016). In terms of Bd infection load,
we have no previous data to make comparisons, as Bd molecular detection in museum
preserved specimens or visual detection only provides presence/absence data
(Rodriguez et al. 2014, Becker et al. 2016, Carvalho et al. 2017, Lambertini et al. 2017).
Nevertheless, we detected high infection loads across the regions and unexpected high
infection prevalence.
Other factors commonly associated to Bd infections in other regions, as
precipitation, elevation, topographic complexity, and human footprint (Brem & Lips
52
2008, Pushendorf et al. 2009, Becker & Zamudio 2011, Gründler et al. 2012, Ruggeri et
al. 2015, Becker et al. 2016), were also tested, but did not explain both Bd infection
prevalence and load. On the other hand, annual mean temperature explained Bd
infection prevalence in the Amazonia. Our sampling scale was smaller than other
retrospective studies developed in the same region (Becker et al. 2016, Carvalho et al.
2017). The effects of certain variables on infection patterns, in general, depends on the
scale in which data is sampled (Levin 1992, Cohen et al. 2016), and abiotic factors
normally explain Bd infections on large-scale samplings, where there is higher
environmental variability. In small scales, biotic factors play a larger role, and are more
likely to predict Bd occurrence (Cohen et al. 2016). Therefore, the relatively small
geographical range scale of our sampling could explain our results.
We detected a negative relationship between temperature and Bd infection
as previous studies (Becker & Zamudio 2011, Ruggeri et al. 2015, Becker et al. 2016).
However, this variable did not explain the high infection prevalence we detected.
Therefore, we suggest that in our small-scale sampling, other factors could be
influencing the measured variables, as biotic factors related to host species (Becker et
al. 2012, Cohen et al. 2016).
Analyzing host-related factors, both Bd infection prevalence and load were
not associated to species reproductive biology, contrary to our expectations. We
expected that aquatic species would show higher responses to Bd infections than
terrestrial species (as in Mesquita et al. 2017). We probably did not detect any
association because the number of terrestrial specimens sampled was small, only 12.5 %
of the samples. In spite of that, we detected higher Bd infection prevalence and load in
host species from forested areas, than species from intermediate and open areas, which
was expected given that areas with higher canopy density favors Bd development by
mechanisms related to microclimate regulation (Becker et al. 2012). Hence, the type of
habitat seems to be a good predictor of infection rates in local scales, where there is
more variation compared to abiotic factors (Cohen et al. 2016). Nevertheless, host-
related factors do explain variation on Bd infections as well, but still does not explain
the higher infection rates we detected.
We propose here two different scenarios to explain the higher infections we
detected. The first one would be a potential recent emergence of Bd strains, thermally
adapted to environmental conditions of some regions of the Amazonia. Bd strains were
thought to have a thermal optima restricted to a range between 17-23 ºC, but it was
53
recently described an increasing on strains thermal tolerance, where Bd strains showed
viability after heat shocks of 28 ºC (Piotrowski et al. 2004, Voyles et al. 2017, Muletz-
Wolz et al. 2019), which potentially allows Bd strains to have a successful development
over areas classified as unsuitable by the previous ecological niche modellings (Ron
2005, Rödder et al. 2010, Liu et al. 2013, Becker et al. 2016).
Another scenario would be a potential arrival of virulent strains in the
region. Bd strains may be widely dispersed by natural (Kilburn et al. 2011, Garmyn et
al. 2012, Pontes et al. 2018) and/or anthropogenic pathways (Schloegel et al. 2012,
James et al. 2015). Also, Bd isolates virulence varies with the size of zoospores and
zoosporangia (Fisher et al. 2009, Lambertini et al. 2016), among different genetic
lineages (Greenspan et al. 2018), different strains (Berger et al. 2005, O’Hanlon et al.
2018), and temperature (Voyles et al. 2012, Stevenson et al. 2013). As we expected, the
strains showed to different virulence in our experimental trails. We detected a higher
mortality rate on Atelopus.aff. hoogmoedi group infected with the strain CLFT 156.
This strain was isolated from south region, which is latitudinally farther from the host’s
original distributional range (Figure S1), showing lower climatic similarity to host’s
collecting site than the strain CLFT 102 (Figure S2). Therefore, we propose that CLFT
102 was less virulent by being isolated from a region closer to environmental conditions
from host’s distribution (Piotrowsky et al. 2004, Voyles et al. 2017, Lambertini et al. in
prep).
Recently, a study have associated larger Bd zoosporangia with lower
temperatures (Muletz-Wolz et al. 2019), and larger zoosporangia has already been
linked to higher infection rates than smaller ones, for example by potentially producing
and releasing more zoospores (Fisher et al. 2009, Lambertini et al. 2016). Therefore, Bd
strains from regions with lower overall mean temperatures might be more virulent by
potentially having higher diameters, and probably that is why individuals infected with
the strain CLFT 156 showed higher mortality rates.
Our study is the first report of Bd infection in in salamanders in Brazil and
the first for wild caecilians. In a previous study Bd was only reported in Brazilian
caecilians from museum preserved specimens (Lambertini et al. 2017). Besides this, this
is the first experiment providing data on differential tolerance to Bd of a Brazilian
species of Atelopus, recognized as a particularly susceptible Neotropical genus (Scheele
et al. 2019). Our findings advance in the understanding of Bd infections in wild
populations in the Amazon. As we found some contradictory patterns and unexpected
54
high prevalence and load, we highlight the need of further investigation in this region,
which could reveal different Bd dynamics than what is already reported. Also, the
proper identification of the Bd genetic lineages in the Amazon is fundamental for the
better understanding of Bd population genetics over a, both broader and finer-scale
geographic ranges. As a general, the Amazon remains underexplored in relation to Bd,
and future research must target this biome as well.
Legal and ethics statement
All samplings and experimental protocols were approved by Instituto Chico Mendes de
Conservação da Biodiversidade – SISBIO/ICMBio (Permit # 46876-6), local animal
care and use committee (Comissão de ética no uso de animais da Universidade Estadual
de Campinas – CEUA/UNICAMP #4440-1) and Conselho de Gestão do Patrimônio
Genético (SISGen #A1D66BF).
Acknowledgements
We thank Ana Paula Costa, Annelise D’Angiolella, Augusto Jarthe and Luisa Diele
Viegas for helping with field samplings. Daniel Medina and Raoni Rebouças for
helping with statistical analyses. This study was funded by Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior (CAPES Finance Code 001), Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq #300896/2016-6), and
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP #2016/25358-3).
55
Table 1. Batrachochytrium dendrobatidis infection prevalence (positive/total) and load
(zoospore g.e.) (values presented as mean ± standard deviation) per family, habit
(according to their reproductive biology) and habitat. An asterisk represents museum-
preserved specimens; all others were sampled in the wild.
Family n Prevalence Infection load Habit Habitat
Anura
Allophrynidae 8 0/8 0 Aquatic Forested Areas
Aromobatidae 45 15/45 119.22 ± 429.89
(1.19 – 1672.39) Aquatic Forested Areas
Bufonidae 42 5/42 350.98 ± 597.71
(2.55 – 1383.56) Aquatic Forested Areas
Centrolenidae 7 2/7 1.81 ± 0.72
(1.30 – 2.32) Aquatic Forested Areas
Craugastoridae 18 4/15 2.48 ± 1.04
(1.49 – 3.66) Terrestrial Forested Areas
Dendrobatidae 39 12/39 203.78 ± 494.29
(1.52 – 1593.36) Aquatic Forested Areas
Hylidae 195 16/195 4.86 ± 7.28
(1.05 – 29.31) Aquatic
Open, Open/Forested,
Forested Areas
Leptodactylidae 76 7/76 40.54 ± 45.63
(2.39 – 114.94)
Terrestrial/
Aquatic Open, Open/Forested
Phyllomedusidae 18 1/18 2.88 Aquatic Open, Forested Areas
Pipidae 1 0/1 0.00 Aquatic Forested Areas
Caudata
Plethodontidae 4 2/4 2.95 ± 1.26
(2.06 – 3.84) Terrestrial Forested Areas
Plethodontidae* 56 0/56 0.00 Terrestrial Forested Areas
Gymnophiona
Caeciliidae 1 1/1 2.58 Terrestrial Forested Areas
Siphonopidae 8 2/8 2.82 ± 1.54
(1.73 – 3.91) Terrestrial Forested Areas
56
Table 2. Batrachochytrium dendrobatidis infection prevalence [as: percentage
(positive/total)] and infection load (values as mean g.e. ± standard deviation) per
collecting locality across Brazilian Amazon basin.
State / Municipality Locality n Prevalence Infection load
Pará / Belém Parque Estadual do
UTINGA 115
2.6%
(3/115)
43.70 ± 62.08
(1.11 – 114.94)
Pará / Altamira UHE Belo Monte 146 28.8%
(42/146)
101.83 ± 367.95
(1.19 – 1672.39)
Pará / Acará Private property 87 3.4%
(3/87)
23.08 ± 29.56
(2.88 – 57.02)
Amapá / Macapá Floresta Nacional do
Amapá 18
5.5%
(1/18) 1.62
Amapá / Macapá Lontra da Pedreira 35 14.3%
(5/35)
1.91 ± 0.25
(1.61 – 2.29)
Amapá / Macapá Abacate da Pedreira 24 20.9%
(5/24)
24.54 ± 35.83
(1.46 – 85.23)
Amazonas / Manaus Reserva Florestal
Adolpho Ducke 37
21.6%
(8/37)
220.71± 486.29
(1.05 – 1383.56)
57
Figure 1
58
Figure 2
59
Figure 3
60
Figure S1
61
Figure S2
62
Capítulo 3. LATITUDINAL DISTRIBUTION OF THE FROG-KILLING FUNGUS
ACROSS THE BRAZILIAN ATLANTIC FOREST
Carolina Lambertini1, C. Guilherme Becker
2, Anat Belasen
3, Anyelet Valencia-
Aguilar4, Carlos Henrique L. N. de Almeida
1, Clarisse M. Betancourt-Roman
3, David
Rodriguez5, Domingos da Silva Leite
6, Igor S. Oliveira
7, João Luiz R. Gasparini
8, Joice
Ruggeri1, Tamí Mott
4, Thomas S. Jenkinson
3,9, Timothy Y. James
3, Kelly R. Zamudio
10,
Luís Felipe Toledo1
1Laboratório de História Natural de Anfíbios Brasileiros (LaHNAB), Departamento de
Biologia Animal, Instituto de Biologia, Universidade Estadual de Campinas, Campinas,
São Paulo, 13083-862, Brazil
2Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama,
35487, United States of America
3Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor,
Michigan, 48109, United States of America
4Setor de Biodiversidade, Instituto de Ciências Biológicas e da Saúde, Universidade
Federal de Alagoas, 57052-970, Maceió, Alagoas, Brazil
5Department of Biology, Texas State University, San Marcos, Texas, 78666, United
States of America
6Departamento de Genética, Evolução e Bioagentes, Instituto de Biologia, Universidade
Estadual de Campinas, Campinas, São Paulo, 13083-862, Brazil
7Campus Floresta, Universidade Federal do Acre, Cruzeiro do Sul, 69895-000, Acre,
Brazil
8Departamento de Ciências Agrárias e Biológicas, Universidade Federal do Espírito
Santo, 29932-540, São Mateus, Espírito Santo, Brazil
9Department of Environmental Science, Policy, and Management, University of
California, Berkeley, California, 94720, United States of America
10Department of Ecology and Evolutionary Biology, Cornell University, Ithaca, New
York, 14853-2701, United States of America
*Corresponding author:
Carolina Lambertini
E-mail: [email protected]
63
Abstract
Latitudinal variation directly reflects differences in disease outcomes mainly because of
non-uniformity of environmental variables that influences on infection rates of a given
pathogen. Chytridiomycosis, an infectious frog disease caused by the fungus
Batrachochytrium dendrobatidis (Bd), is strongly influenced by environmental
variations, and also by biotic factors, related to host species. Chytrid infections have
been documented all over south portion of Brazilian Atlantic forest, but there is no
information regarding Bd infection dynamics all over the biome. We sampled 2,554
anuran individuals from 21 field sites along a 3,600 km latitudinal transect across
Brazilian Atlantic forest. We quantified Bd infections by qPCR and performed a model
selection to verify whether abiotic variables explained Bd infections along the transect.
We also tested for associations between Bd infections and species richness, species
reproductive modes, and type of habitat. We detected a positive association between
infection prevalence and intensity with latitude; elevation, temperature and precipitation
better explained infection prevalence, and temperature best explained infection load
along the transect. We also detected an amplification effect between species richness
and Bd infections; and associations between Bd infections and host reproductive modes
and type of habitat. We characterized infection dynamics across a large scale latitudinal
transect and our results highlight the importance of taking into account the
environmental variability at different data sampling scales.
Keywords: Disease ecology, Environment, Species richness, Host life history, Tropical
forest, Batrachochytrium dendrobatidis
64
Introduction
Latitude impacts a number of biotic and abiotic conditions that in turn affect
host-pathogen interactions (Engering et al. 2013). For example, variation in temperature
and rainfall are known to influence host-pathogen interactions in several disease
systems, dictating population-level prevalence patterns and pathogen infection
intensities at the individual level (Altizer et al. 2006, Fisher et al. 2012, Flory et al.
2012, Ruggeri et al. 2018). In fact, the number emergent infectious diseases are
increasing rapidly, with sometimes severe consequences for species persistence
(Williams et al. 2002, Jones et al. 2008), due to population declines and species
extinctions across the globe (McMichals 2004, Tompkins et al. 2015), and important
infectious diseases caused by different taxa have been associated with latitudinal
variation. For instance, reports of influenza virus from several countries all over the
world had its epidemic time and duration associated with latitude (Bloom-Feshbach et
al. 2013). Also, the incidence of Lyme disease all over United States increased across a
latitudinal range (Tuite et al. 2013).
Chytridiomycosis is one of the emerging infectious diseases that have had
catastrophic consequences for amphibian diversity (James et al. 2015, Berger et al.
2016). This disease is caused by the chytrid fungus Batrachochytrium dendrobatidis
(hereafter Bd), a generalist pathogen of amphibians that has caused declines in over 500
species globally (Scheele et al. 2019). In the amphibian-Bd system, a number of
environmental factors have a marked influence on variation of pathogen occurrence and
disease outcome of infected hosts (Garner et al. 2011, Ruggeri et al. 2015), and it has
been already associated to latitude in a tropical forest in Australia, for instance (Kriger
et al. 2007).
The most common abiotic factors driving spatial variation in Bd infections
are temperature, precipitation, elevation and vegetation density. Bd infections are
negatively associated with temperature (Puschendorf et al. 2009, Ruggeri et al. 2015,
Becker et al. 2016, Carvalho et al. 2017, Lambertini et al. 2017), and positively
associated with precipitation, elevation and vegetation density (Brem & Lips 2008,
Pushendorf et al. 2009, Becker & Zamudio 2011, Gründler et al. 2012, Ruggeri et al.
2015, Becker et al. 2016, Carvalho et al. 2017, Lambertini et al. 2017). These are basic
known patters for the Bd-host system; however, these relationships vary in direction of
response (e.g. Lambertini et al. 2016, Ruggeri et al. 2018), possibly due to data
65
sampling in different geographic regions or at different spatial scales (Cohen et al.
2016).
A recent study showed no effect of elevation on Bd prevalence and infection
load at a local sampling scale (200 – 700 m above sea level) in Brazilian Atlantic forest
(Lambertini et al. 2016), but the opposite pattern was true at a regional scale (Walker et
al. 2010, Gründler et al. 2012). Similarly, a latitudinal sampling transect over 2,300 km
in the Australian coast revealed that both Bd infection prevalence and intensity were
higher with increased rainfall and colder temperatures (Kriger et al. 2007), whereas a
local scale study in Brazil revealed higher infection intensities during cooler periods but
lower intensities during the rainy season (Ruggeri et al. 2018). In terms of biotic factors,
host species richness is known to be negatively associated to Bd infections, due to the
dilution effect (Searle et al. 2011, Becker et al. 2014), but the opposite was detected in a
large scale study performed in Costa Rica and Australia, where species richness was
positively associated to Bd infections (Becker and Zamudio 2011). Host species
richness may dilute or amplify pathogen transmission through different mechanisms
(Luis et al. 2018). In general, it seems likely that several factors influence Bd infection
in different ways, and there are many different mechanisms where biotic and abiotic
factors might influence Bd infections. Given these contradictory results, we could infer
that data sampled at the local scale do not provide sufficient resolution to analyze
environmental variables, whereas regional scales include greater environmental
variability, and potentially more power for analysis of relationships between abiotic
variables and Bd infections. Indeed, abiotic factors such as climate, for instance, are
more variable and significantly predict Bd infections at regional scales, whereas biotic
factors are more variable and significantly predict Bd dynamics at local scales (Cohen et
al. 2016).
Here our goal is to disentangle the mechanisms responsible for Bd infection
variation at a regional scale, by focusing on biotic and abiotic variables. Specifically, we
performed anuran samplings over a 3,600 km latitudinal transect at the Brazilian
Atlantic forest, to test whether and how potential structuring factors as temperature,
precipitation, elevation, vegetation density and topographic complexity influences Bd
infections. We also tested the associations between species richness, reproductive mode,
and type of habitat with Bd infections. Our results shed light on large scale Bd infection
dynamics in natural landscapes.
66
Methods
Study sites and Species Sampling
We sampled 21 field sites along a north to south 3,600 km latitudinal transect across the
Brazilian Atlantic Forest. Sampling sites were in the states of Santa Catarina, Paraná,
São Paulo, Rio de Janeiro, Espírito Santo, Bahia, Sergipe, Alagoas, Pernambuco,
Paraíba and Rio Grande do Norte (Figure 1). Anuran sampling permits were approved
by Chico Mendes national institute for biodiversity conservation (SISBio #27745-13)
Conselho de Gestão do Patrimônio Genético (SISGen #A1D66BF).
We field sampled anurans during the breeding season for each region:
between June and July in the northern region (north of the Rio Doce River, from
northern Bahia to Rio Grande do Norte) and between December and February in the
southern region (south of the Rio Doce River, from southern Bahia to Santa Catarina)
during the years of 2011-2015. We sampled 2,554 individual frogs across the latitudinal
transect. For each site, we aimed to sample the greatest diversity of species possible,
totaling 14 families, 43 genera, and 148 anuran species (Table S1). We recorded GPS
coordinates in decimal degrees for each sampling location. When GPS coordinates were
not available for data that had been previously collected, we used the geographic
centroid of municipalities for geographic analyses.
Bd prevalence and infection load
We swabbed the skin of each field-captured frog following established
protocols (Hyatt et al. 2007). Swabs were placed individually in 1.5 mL cryovials, and
stored at -20ºC until molecular diagnosis in the lab. We extracted DNA from each swab
using 50µL of PrepMan ULTRA® (Life Technologies) and then proceeded with
molecular detection and quantification using TaqMan® qPCR Assay (Life
Technologies) (Boyle et al. 2004, Lambertini et al. 2013). To generate the qPCR
standard curve, we used the Atlantic Forest collected strain CLFT 023 as a quantitative
standard, serially diluted from 103 to 10
-1 zoospore genomic equivalents (g.e.). We
considered samples with at least one g.e. positive for Bd (Kriger et al. 2007).
Abiotic variables
For each sampling location, we extracted seven bioclimatic metrics for
temperature and precipitation (BioClim, WorldClim) – Annual Mean Temperature
(Bio1), Mean Diurnal Range (Bio 2), Temperature Seasonality (Bio 4), Maximum
67
Temperature of Warmest Month (Bio 5), Annual Precipitation (Bio 12), Precipitation
Seasonality (Bio 15), Precipitation of Warmest Quarter (Bio 18), at a scale of 1 km for
each metric (Hijmans et al. 2005). We also extracted data on vegetation density (FAO
2010), elevation, topographic complexity and IUCN species richness using Arc Map
v.10.1 (ESRI 2012).
Statistical Analysis
We performed a Generalized Linear Model (GLM), with binomial
distribution and logit link, and a Generalized Regression (GR), with a zero inflated (ZI)
negative binomial distribution, to test whether latitude is associated to prevalence and
infection load, and to test whether bioclimatic metrics, as well as vegetation density,
elevation, topographic complexity and IUCN species richness explain prevalence and
infection load along our latitudinal transect.
To test for the influence of host species richness on Bd infections, we
performed a model selection for Bd prevalence and infection load, using IUCN species
richness data for each sampling locality as a fixed factor. We classified all the species
by their habitat (Forested, Open/Forested and Open areas) and performed analysis of
variance (ANOVA) and Tukey test a posteriori, to verify if there were differences
between both prevalence and infection load and species habitat. We also classified all
the species by their habit, according to their reproductive biology [aquatic (aquatic
larvae) and terrestrial (direct and indirect development)] and performed a t test to verify
if there were differences between both prevalence and infection load and species habit.
We used the software JMP-SAS v.10. to perform statistical analyses (SAS 2010).
Results
We observed an overall prevalence of 24.7%, and a mean infection load of
713.5 zoospore genomic equivalents (g.e.) along our latitudinal transect. We detected
the highest infection prevalence in the family Hylodidae and the highest infection load
in the family Craugastoridae. Individuals from only two out of the 14 sampled families
(Centrolenidae and Eleutherodactylidae) did not test positive for Bd (Table 1; Table
S1).
We detected Bd at 95% of our sampling sites (Table 2). From the 11 states
sampled across our latitudinal transect, we detected the lowest infection prevalence in
the northeast region, in the state of Rio Grande do Norte (0.8%), and the highest
68
infection prevalence in the south region, state of Paraná (51.7%). For the infection load,
we detected the lowest rate in the southeast region, state of São Paulo (1.00 g.e.) and the
highest rate in the northeast region, state of Alagoas (158,772 g.e.). Our GLM analysis
indicated that both infection prevalence (X2
= 194.0014; P < 0.0001) and infection load
(X2
= 99.1909; P < 0.0001) were positively associated with latitude (Fig. 1).
We performed model selection analysis including environmental variables
and IUCN species richness for each sampling locality, and the best model showed that
elevation, mean diurnal range, temperature seasonality and annual precipitation best
explain infection prevalence (Table 3), and that annual mean temperature best explained
the infection load (β = -0.022387; P = 0.0009). We also report the 5 best models for
prevalence and infection load (Table S2).
When analyzing IUCN species richness for each sampling locality in our
model selection, we detected a positive relationship between this parameter and both
prevalence (P < 0.0001) and infection load (P = 0.0093) throughout the transect.
For species reproductive mode, we detected higher infection prevalence for
aquatic species than for terrestrial species (t = 3.2160; P = 0.0013, Fig. 2A), but the
opposite for infection load, where higher intensity was observed for terrestrial species (t
= -2.9433; P = 0.0032, Fig. 2B). We also detected higher infection prevalence and
intensity in forested areas, than for species from open/forested and open areas (F[2,2551] =
16.9224; P < 0.0001, F[2,2551] = 19.3534; P < 0.0001, respectively, Fig. 2C, D).
Discussion
Our large-scale sampling throughout Brazilian Atlantic forest revealed
latitudinal variation in Bd prevalence and infection load, with a positive association
between infection rates and latitude (Fig. 1). This same pattern was described in a
similar study with broad latitudinal sampling along the Australian coast, where Bd
prevalence showed a trend toward increasing with latitude, and infection load were
positively associated with latitude (Kriger et al. 2007). The decrease in Bd in northern
Atlantic forest is likely because environmental conditions become less suitable for Bd
development as latitude decrease. Our results clearly show higher infection rates in
southern portions (Fig 1) where environmental conditions as annual mean temperature
are more suitable for Bd development. In contrast, a recent study detected a negative
trend between Bd prevalence and latitude, in a latitudinal sampling throughout a
Chilean tropical forest (Bacigalupe et al. 2017). But, this pattern can be partly explained
69
by the ongoing spread of Bd in the region, due to a recent introduction of Xenopus
leavis (Bacigalupe et al. 2017). This means that different mechanisms might influence
different patterns of Bd infections in samples collected over large spatial scales, but
environmental variation is in fact a strong predictor of this variance.
Based on our model selection, the variables that better explained Bd
infection prevalence and intensity were macroclimate and elevation.
Environmental conditions are highly heterogeneous throughout the forest, and probably
this explains the patterns we detected, given that in large geographic scales we may
detect higher environmental variability. Our results are in agreement with previous
studies performed at large geographic scales. For example, positive relationships
between elevation, topographic complexity and rainfall, and negative relationships
between temperature and the likelihood of Bd occurrence, were reported in tadpoles
from all Brazilian biomes (Carvalho et al. 2017). In the Amazon Basin, which shows
different patterns of environmental conditions compared to Brazilian Atlantic forest,
milder temperatures and higher precipitation and vegetation density were significant
predictors of Bd occurrence in anurans (Becker et al. 2016). Besides anuran hosts,
temperature and precipitation also had a negative and positive effect, respectively, on
the likelihood of Bd infections in South American caecilians (Lambertini et al. 2017).
Both prevalence and infection load were negatively correlated with the mean
temperature of the warmest quarter along the sampling transect on east coast of
Australia, but positively correlated to rainfall (mm) metrics of each sampling locations
(Kriger et al. 2007).
At smaller geographic scales patterns of Bd infection rates may vary
significantly. For example, several studies detected a positive correlation between
elevation and infection rates (Brem & Lips 2008, Gründler et al. 2012, Catenazzi et al.
2013), but at a small spatial scale, this correlation was not detected (Lambertini et al.
2016). Given that one of the proposed mechanisms by which elevation influences Bd
infections is that cooler temperatures at higher elevations would provide better
conditions for the pathogen growth (Longcore et al. 1999, Piotrowski et al. 2004,
Voyles et al. 2017), we hypothesize that there must be a threshold on the effect of
elevation on Bd infections. The influence of elevation was detected when there was a
high elevational variation (in this case, from 10 to 1,400 m a.s.l.), but not detected in a
small elevational variation, from 200 to 700 m a.s.l. (Lambertini et al. 2016), probably
due to low variation in temperature.
70
Cohen et al (2016) proposed that abiotic factors were significant and most
important for Bd infections only at regional scales, whereas biotic factors were more
important at local scales. For instance, species richness was important and negatively
correlated to Bd prevalence only at local scales and its relative importance decreases at
larger scales of analysis. Here, we detected an amplification effect on Bd infection
prevalence and intensity along our transect. Specifically, infection rates increased with
increasing species richness, which is the opposite of what has been reported in
controlled laboratory experiments, where for both larval and adult amphibians,
increased species richness reduced Bd infections (Searle et al. 2011, Becker et al. 2014).
On the other hand, the amplification effect on Bd infections has also been detected in
large scale analysis of wild amphibian populations in Costa Rica and Australia, and this
pattern could be explained by a greater diversity of species enhancing Bd transmission
due to the expanded availability of susceptible hosts (Becker & Zamudio 2011). These
different patterns indicate that the complexity of interactions in natural communities is
much greater than those in controlled laboratory experiments, and the difficulty of
capturing these community level mechanisms may bias experimental results. Also, even
though the dilution effect has been detected in plant and animal diseases almost seven
times more than the amplification effect (Cardinale et al. 2012), and in general the effect
of diversity and density of host population is stronger, the dilution and amplification
effects might occur at the same time in the same host-pathogen system through different
mechanisms (Luis et al. 2018), which means that the role of species diversity on Bd
infections has to be better elucidated.
In accordance with our expectations, both reproductive modes and type of
habitat from host species were related to Bd infection prevalence and intensity.
Prevalence was higher for aquatic species, which is expected given that Bd is a
waterborne fungus (Longcore et al. 1999; Berger et al. 2005). However, terrestrial
species harbored higher Bd infection intensities, and this might happen because
terrestrial species are less exposed to Bd and therefore they lack adaptive responses to
this pathogen (Mesquita et al. 2017). Our results reinforce that host life history is an
important predictor of Bd infections. For species habitat, we detected higher Bd
infection prevalence and intensity in species from forested areas. This pattern was also
expected, given that canopy density modulates temperature in natural vegetation, which
is directly associated to disease risk (Becker et al. 2012).
71
In this study, we identified several critical factors affecting Bd infections in
natural landscapes, and characterized infection dynamics across a large scale latitudinal
transect with significant environmental gradients. Our results highlight the importance
of taking into account the environmental variability at different data sampling scales,
and the need for studies focusing on a better understanding of the relations between host
species diversity and Bd infections in the wild.
Acknowledgements
We thank M. J. M. Dubeux, L. R. Lima, U. G. da Silva, C. N. S. Palmeira, D. Santana,
A. de Padua Almeida for field assistance, and Paula P. Morão for qPCR assistance. This
work was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES, Financial Code 001), Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq #300896/2016-6) and Sao Paulo Research Foundation (FAPESP
#2016/25358-3).
72
Table 1. Batrachochytrium dendrobatidis prevalence (positive/total) and infection load
(rounded without decimals) per family, and habit (according to their reproductive
biology). Infection load (g.e.) values are mean ± standard deviation (range).
Family N Habit Prevalence Infection load
Brachycephalidae 237 Terrestrial 50/237 1,741 ± 6,113
(1 – 38,999)
Bufonidae 102 Aquatic 17/102 576 ± 1,681
(1 – 6,599)
Centrolenidae 9 Aquatic 0/9 0.0
Craugastoridae 119 Terrestrial 17/119 9,522 ± 38,463
(2 – 158,771)
Cycloramphidae 103 Aquatic 38/103 79 ± 392
(1 – 2,424)
Eleutherodactylidae 1 Terrestrial 0/1 0.0
Hemiphractidae 14 Terrestrial 6/14 30 ± 32
(6 – 91)
Hylidae 1,361 Aquatic 312/1361 131 ± 400
(1 – 2,428)
Hylodidae 183 Aquatic 122/183 494 ± 1,321
(1 – 6,633)
Leptodactylidae 284 Aquatic and
Terrestrial
43/284 82 ± 216
(1 – 1,225)
Microhylidae 5 Aquatic 1/5 11
Odontophrynidae 24 Aquatic 7/24 13 ± 12
(3 – 39)
Phyllomedusidae 45 Aquatic 6/45 57 ± 73
(2 – 158)
Ranidae 67 Aquatic 12/67 165 ± 511
(2 – 3)
73
Table 2. Sampling localities and Batrachochytrium dendrobatidis detection along the
Atlantic Forest latitudinal transect.
State Municipality Bd detection
Rio Grande do Norte Mata Estrela +
Paraíba João Pessoa +
Pernambuco Paulista +
Alagoas Murici +
Sergipe Areia Branca +
Bahia Mata de São João –
Bahia Camacan +
Espírito Santo Linhares +
Espírito Santo Santa Tereza +
Espírito Santo Vargem Alta +
Rio de Janeiro Sana +
São Paulo Bertioga +
São Paulo Biritiba-Mirim +
São Paulo Iporanga +
São Paulo Pedro de Toledo +
São Paulo São Luiz do Paraitinga +
Paraná Morretes +
Santa Catarina Pomerode +
Santa Catarina Rancho Queimado +
74
Table 3. Best model for Batrachochytrium dendrobatidis infection prevalence.
Term Estimate dF X2
P
Intercept -7.151 1 117.719 <0.0001
Elevation (m) < 0.001 1 27.380 <0.0001
Bio 2 0.0238806 1 10.897 0.0001
Bio 4 0.0006103 1 45.974 <0.0001
Bio 12 0.0011922 1 108.086 <0.0001
Bio 2 = Mean Diurnal Range; Bio 4 = Temperature Seasonality; Bio 12 = Annual
Precipitation.
75
Figure 1
76
Figure 2
77
Table S2. Five best models for prevalence and infection load. Bio 1 = Annual Mean
Temperature; Bio 2 = Mean Diurnal Range; Bio 4 = Temperature Seasonality; Bio 5 =
Max Temperature of Warmest Month; Bio 12 = Annual Precipitation; Bio 18 =
Precipitation of Warmest Quarter.
Term AIC Δ AIC # Variables
Prevalence
Elevation (m), Bio 2, Bio 4, Bio 12 259.161 0 4
Vegetation density, Bio 1, Bio 2, Bio 12 263.875 4.714 4
Elevation (m), Bio 4, Bio 12 266.558 7.397 3
Vegetation density, Elevation (m), Bio 4, Bio 12 266.871 7.710 4
Elevation (m), Bio 1, Bio 5, Bio 12 267.231 8.070 4
Infection load
Bio 1 521.619 0 1
Elevation (m) 527.208 5.590 1
Bio 5 547.512 25.893 1
Species richness 569.260 47.641 1
Bio 18 597.242 75.623 1
78
SÍNTESE GERAL
Florestas tropicais representam um ecossistema que abriga parte relevante
da biodiversidade terrestre mundial. Dentre as principais, a Floresta Amazônica
apresenta a maior riqueza de espécies do mundo, e a Mata Atlântica representa um
bioma com ampla diversidade de espécies e alto grau de endemismo, sendo considerado
um dos mais importantes hotspots de biodiversidade do mundo. Das mais de 1000
espécies de anfíbios que compõem a biodiversidade brasileira, pelo menos 600 espécies
são encontradas na Mata Atlântica. Levando em consideração o grande e preocupante
impacto da quitridiomicose sobre a diversidade mundial de anfíbios, estudos que
buscam um melhor entendimento sobre os padrões de ocorrência do patógeno
Batrachochytrium dendrobatidis (Bd) em ambos os biomas se tornam essenciais.
No presente trabalho, apresentamos dados inéditos em relação aos padrões
de ocorrência do Bd em ambos os biomas. Realizamos na Mata Atlântica a amostragem
de maior extensão em transecto já desenvolvida, abrangendo mais de 2500 indivíduos
pertencentes a 148 espécies de anfíbios anuros. De acordo com nosso transecto
latitudinal, o Bd está amplamente distribuído ao longo da Mata Atlântica, com as taxas
de prevalência e carga de infecção positivamente associadas com a latitude. Este padrão
era esperado já que, basicamente, as condições ambientais para o desenvolvimento do
patógeno se tornam menos favoráveis com a diminuição da latitude. Além disso,
detectamos e descrevemos associações entre fatores abióticos e bióticos e taxas de
infecção por Bd em nossa amostragem em larga escala.
Elevação e variáveis bioclimáticas de temperatura e precipitação explicaram
as taxas de infecção encontradas ao longo do transecto, o que era esperado já que
amostragens em larga escala refletem variação ambiental normalmente associada à
variação de infecção por Bd. Propomos aqui que exista um limiar no qual a elevação
está associada às taxas de infecção, já que quanto maior a variação de elevação, maior a
variação de temperatura que pode prover condições mais favoráveis para o
desenvolvimento do Bd. Além disso, detectamos um efeito de amplificação da riqueza
de espécies nas taxas de prevalência e carga de infecção por Bd ao longo do transecto,
que representa o oposto já detectado em estudos experimentais. Sabe-se atualmente que
tanto o efeito de diluição quanto de amplificação podem ocorrer concomitantemente na
natureza, através de diferentes mecanismos. Sendo assim, o papel da diversidade de
espécies nos padrões de infecção por Bd deve ser melhor explorado.
79
Diversos estudos realizaram a detecção do Bd na Mata Atlântica. Porém, a
maioria dos trabalhos está concentrada na porção sul do bioma, que já foi classificada
como uma região de alta adequabilidade ambiental para o estabelecimento e
desenvolvimento do patógeno, através de modelagens de nicho climático. O presente
trabalho é o primeiro em larga escala a ser realizado no bioma e, corroboramos
resultados de modelagem de nicho climático, já que encontramos cargas de infecção
mais altas na porção sul, que diminuem conforme diminuição da latitude.
Por outro lado, as amostragens realizadas na Floresta Amazônica não
corroboram previsões de modelagens de nicho climático, já que encontramos altas taxas
de infeção em localidades consideradas de baixa adequabilidade para o estabelecimento
de desenvolvimento do Bd. As únicas variáveis em comum que foram associadas às
taxas de infecção encontradas em ambos os biomas são temperatura média anual e tipo
de habitat dos hospedeiros. Variáveis como elevação, complexidade topográfica, outras
variáveis bioclimáticas e até footprint humano não explicaram as taxas de infecção
encontradas no presente trabalho (Capítulo 2), mas foram associadas previamente às
taxas encontradas ao longo do transecto latitudinal realizado na Mata Atlântica, também
no presente trabalho (Capítulo 1), e em outros dois estudos retrospectivos de larga
escala desenvolvidos na região Amazônica. Propomos então, que outros fatores que não
ambientais estão influenciando a variação nas taxas de infecção, como a emergência
recente de cepas do Bd, ou até a chegada de cepas virulentas na região, através da
disseminação tanto por vias antropogênicas quanto naturais.
Demostramos experimentalmente os riscos do fluxo de diferentes cepas de
Bd em hospedeiros da espécie Atelopus hoogmoedi, cujos congêneres de outras regiões
são considerados altamente susceptíveis à infecção por Bd e já sofreram declínios
severos de populações. Evidenciamos a variação em virulência de cepas pertencentes à
mesma linhagem genética do patógeno – Global Pandemic Lineage (Bd-GPL), porém
de regiões geográficas que variam em similaridade climática em comparação com a
distribuição geográfica dos hospedeiros.
Apresentamos também o primeiro registro de infecção por Bd em
membros da ordem Caudata no Brasil (Capítulo 2), e o primeiro estudo que analisa os
padrões de ocorrência de Bd em espécies da ordem Gymnophiona no mundo (Capítulo
3), sugerindo que espécies dessa ordem podem agir como reservatórios do Bd, devido
aos hábitos completamente fossoriais ou aquáticos, pelos quais os indivíduos são menos
expostos a variações ambientais.
80
O presente trabalho apresenta diferentes padrões de ocorrência do fungo Bd
na Mata Atlântica e Amazônia brasileiras, e abre portas para o desenvolvimento de
diversos estudos como: um melhor entendimento da influência da diversidade de
espécies nas taxas de infecção por Bd na natureza; o papel da escala de amostragem nos
fatores bióticos e abióticos que influenciam padrões de ocorrência de Bd; maior
amplitude de amostragem de populações naturais de anfíbios na região Amazônica,
focando tanto em anuros quanto em salamandras e cecílias; verificar se espécies de
gênero Atelopus da Amazônia brasileira são de fato mais susceptíveis à infecção por Bd;
quais genótipos de Bd podem estar distribuídos na região Amazônica e nos membros
das ordens Caudata e Gymnophiona. Quanto maior a compreensão dos padrões de
ocorrência do Bd, maiores as possibilidades de desenvolvimento de ações de
conservação direcionadas às espécies de anfíbios no Brasil e no mundo.
81
REFERÊNCIAS
Alford RA (2001) Testing the novel pathogen hypothesis. Developing management
strategies to control amphibian diseases: decreasing the risks due to
communicable diseases. School of Public Health and Tropical Medicine, James
Cook University, Townsville, Australia, p.20
Allender MC, Hileman ET, Moore J, Tetzlaff S (2016). Detection of Ophidiomyces, the
causative agent of snake fungal disease, in the Eastern Massasauga (Sistrurus
catenatus) in Michigan, USA, 2014. Journal of Wildlife Diseases 52:694–698
Altizer S, Dobson A, Hosseini P, Hudson P, Pascual M, Rohani P (2006) Seasonality
and the dynamics of infectious diseases. Ecology Letters 9:467–48
Andre SE, Parker J, Briggs CJ (2008) Effect of temperature on host response to
Batrachochytrium dendrobatidis infection in the mountain yellow-legged frog
(Rana muscosa). Journal of Wildlife Diseases 44:716–720
Ávila-Pires TCS; Hoogmoed MS; Rocha, WA (2010) Notes on Vertebrates of Northern
Pará, Brazil: a forgotten part of the Guianan Region, I. Herpetofauna. Boletim do
Museu Paraense Emílio Goeldi Ciências Naturais 5:13–112
Bacigalupe LD, Soto‐Azat C, García‐Vera C, Barría‐Oyarzo I, Rezende EL (2017)
Effects of amphibian phylogeny, climate and human impact on the occurrence of
the amphibian‐killing chytrid fungus. Global Change Biology 23:3543–3553
Bataille A, Fong JJ, Cha M, Wogan GO and others (2013) Genetic evidence for a high
diversity and wide distribution of endemic strains of the pathogenic chytrid fungus
Batrachochytrium dendrobatidis in wild Asian amphibians. Molecular Ecology
22:4196–4209
Becker, C. G., Fonseca, C. R., Haddad, C. F. B., Batista, R. F. & Prado, P. I. 2007.
Habitat split and the global decline of amphibians. Science 318:1775–1777
Becker CG, Zamudio KR (2011) Tropical amphibian populations experience higher
disease risk in natural habitats. Proceedings of the National Academy of Sciences
108:9893–9898
Becker CG, Rodriguez D, Longo AV, Talaba AL, Zamudio KR (2012) Disease risk in
temperate amphibian populations is higher at closed-canopy sites. PLoS ONE
7:e48205
Becker CG, Rodriguez D, Toledo LF, Longo AV, Lambertini C, Corrêa DT, Leite DS,
Haddad CFB, Zamudio KR (2014) Partitioning the net effect of host diversity on an
emerging amphibian pathogen. Proceedings of the Royal Society B 281:20141796
82
Becker CG, Rodriguez D, Lambertini C, Toledo LF, Haddad CFB (2016) Historical
dynamics of Batrachochytrium dendrobatidis in Amazonia. Ecography 39:954–960
Berger L, Marantelli G, Skerratt LF Speare R (2005) Virulence of the amphibian chytrid
fungus Batrachochytrium dendrobatidis varies with the strain. Diseases of
Aquatic Organisms 68:47–50
Berger L, Speare R, Daszak P, Green DE, and others (1998) Chytridiomycosis causes
amphibian mortality associated with population declines in the rain forests of
Australia and Central America. Proceedings of the National Academy of Science
95:9031–9036
Berger L, Speare R, Daszak P, Green DE, Cunningham AA, Goggin CL, Slocombe R,
Ragan AM, Hyatt AD, McDonald KR, Hines HB, Lips KR, Marantelli G, Parkes H
(1998). Chytridiomycosis causes amphibian mortality associated with population
declines in the rain forests of Australia and Central America. Proceedings of the
National Academy of Sciences 95:9031–9036
Berger L, Roberts AA, Voyles J, Longcore JE, Murray KA, Skerratt LF (2016) History
and recent progress on chytridiomycosis in amphibians. Fungal Ecology 19:89–99
Blehert DS, Hicks AC, Behr M, Meteyer CU, Berlowski-Zier BM, Buckles EL,
Coleman JTH, Darling SR, Gargas A, Niver R, Okoniewski JC, Rudd J, Stone
WB (2009) Bat white-nose syndrome: an emerging fungal
pathogen? Science 323:227–227
Bloom-Feshbach K, Alonso WJ, Charu V, Tamerius J, Simonsen L, Miller MA, Viboud
C (2013) Latitudinal variations in seasonal activity of influenza and respiratory
syncytial virus (RSV): a global comparative review. PloS ONE 8:e54445
Bovero S, Sotgiu G, Angelini C, Doglio S, Gazzaniga E, Cunningham AA, Garner
TWJ. (2008) Detection of chytridiomycosis caused by Batrachochytrium
dendrobatidis in the endangered Sardinian newt (Euproctus platycephalus) in
southern Sardinia, Italy. Journal of Wildlife Diseases 44:712–715
Bower DS, Lips KR, Schwarzkopf L, Georges A, Clulow S (2017) Amphibians on the
brink. Science 357:454–455
Boyle DG, Boyle DB, Olsen V, Morgan JAT, Hyatt AD (2004) Rapid quantitative
detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian
samples using real-time Taqman PCR assay. Diseases of Aquatic Organisms 60:141–
148
83
Boyles JG, Willis CK (2010) Could localized warm areas inside cold caves reduce
mortality of hibernating bats affected by white‐nose syndrome? Frontiers in
Ecology and the Environment 8:92–98
Brem FM, Lips KR (2008) Batrachochytrium dendrobatidis infection patterns among
Panamanian amphibian species, habitats and elevations during epizootic and
enzootic stages. Diseases of Aquatic Organisms 81:189–202
Cardinale BJ, Duffy JE, Gonzalez A, Hooper DU, Perrings C, Venail P, Narwani A,
Mace GM, Tilman D, Wardle DA, Kinzig AP, Daily GC, Loreau M, Grace JB,
Larigauderie A, Srivastata DS, Naeem S (2012) Biodiversity loss and its impact on
humanity. Nature 486:59–67
Carvalho T, Becker CG, Toledo LF (2017) Historical amphibian declines and
extinctions in Brazil linked to chytridiomycosis. Proceedings of the Royal Society B
284:20162254
Catenazzi A, Lehr E, Rodriguez LO, Vredenburg VT (2011) Batrachochytrium
dendrobatidis and the collapse of anuran species richness and abundance in the upper
Manu National Park, southeastern Peru. Conservation Biology 25:382–391
Catenazzi A, von May R, Vredenburg VT (2013) High prevalence of infection in
tadpoles increases vulnerability to fungal pathogen in high-Andean amphibians.
Biological Conservation 159:413–421
Cheng TL, Rovito SM, Wake DB, Vredenburg VT (2011) Coincident mass extirpation
of neotropical amphibians with the emergence of the infectious fungal pathogen
Batrachochytrium dendrobatidis. Proceedings of the National Academy of Science
108:9502–9507
Churgin SM, Raphael BL, Pramuk JB, Trupkiewicz JG, West G (2013)
Batrachochytrium dendrobatidis in aquatic caecilians (Typhlonectes natans): a
series of cases from two institutions. Journal of Zoo and Wildlife Medicine
44:1002–1009
Cohen JM, Civitello DJ, Brace AJ, Feichtinger EM, Ortega CN, Richardson JC, Sauer
EL, Liu X, Rohr JR (2016) Spatial scale modulates the strength of ecological
processes driving disease distributions. Proceedings of the National Academy of
Sciences 113:3359–3364
Courtois EA, Gaucher P, Chave J, Schmeller DS. (2015) Widespread occurrence of Bd
in French Guiana, South America. PLoS ONE 10: e0125128
84
Cunningham AA, Daszak P, Wood JL (2017) One Health, emerging infectious diseases
and wildlife: two decades of progress? Philosophical Transactions of the Royal
Society B 372:20160167
Daszak P, Berger L, Cunningham AA, Hyatt AD, Green DE Speare R (1999) Emerging
infectious diseases and amphibian population declines. Emerging Infectious
Diseases 5: 735–748
Daszak P, Cunningham AA, Hyatt AD (2000). Emerging infectious diseases of wildlife-
-threats to biodiversity and human health. Science 287:443–449
Daszak P, Cunningham AA, Hyatt AD (2001) Anthropogenic environmental change
and the emergence of infectious diseases in wildlife. Acta Tropica 78:103–116
Daszak P, Cunningham AA, Hyatt AD (2003) Infectious disease and amphibian
population declines. Diversity and Distributions 9:141–150
Davidson EW, Parris M, Collins JP, Longcore JE, Pessier AP, Brunner J (2003)
Pathogenicity and transmission of chytridiomycosis in tiger salamanders
(Ambystoma tigrinum). Copeia 2003:601–607
Dobson A, Foufopoulos J (2001) Emerging infectious pathogens of
wildlife. Philosophical Transactions of the Royal Society of London B 356:1001–
1012
Doherty-Bone TM, Gonwouo NL, Hirschfeld M, Ohst T, Weldon C (2013)
Batrachochytrium dendrobatidis in amphibians of Cameroon, including first
records for caecilians. Diseases of Aquatic Organisms 102:187–194
Eisenberg JN, Desai MA, Levy K, Bates SJ, Liang S, Naumoff K, Scott JC (2007)
Environmental determinants of infectious disease: a framework for tracking
causal links and guiding public health research. Environmental Health
Perspectives 115:1216–1223
Elith J and Leathwick JR (2009) Species distribution models: ecological explanation
and prediction across space and time. Annual review of ecology, evolution, and
systematics 40:667–697
Engering A, Hogerwerf L, Singenbergh J (2013) Pathogen-host-environment interplay
and disease emergence. Emerging Microbes and Infections 2:1–7
ESRI (2012) Arcview 10.1. – Redlands, CA
FAO (2010) Global Forest Resources Assessment 2010, Main Report. Food and
Agriculture Organization of the United Nations, Rome, 1997
85
Farrer RA, Weinert LA, Bielby J, Garner TW, Balloux F, Clare F, Bosch J,
Cunningtham AA, Weldon C, Preez AH, Anderson L, Pond SLK, Shahar-Golan
R, Henk DA, Fisher MC. 2011. Multiple emergences of genetically diverse
amphibian-infecting chytrids include a globalized hypervirulent recombinant
lineage. Proceedings of the National Academy of Sciences 108:18732–18736
Fisher MC, Garner TWJ (2007) The relationship between the emergence of
Batrachochytrium dendrobatidis, the international trade in amphibians and
introduced species. Fungal Biological Reviews 21:2–9
Fisher MC, Bosch J, Yin Z, Stead DA, Walker J, Selway L, Brown AJP, Walker LA,
Gow NAR, Stajich JE, Garner TW (2009) Proteomic and phenotypic profiling of the
amphibian pathogen Batrachochytrium dendrobatidis shows that genotype is linked
to virulence. Molecular Ecology 18:415–429
Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL, Gurr SJ
(2012) Emerging fungal threats to animal, plant and ecosystem health. Nature
484:186–194
Fites JS, Ramsey JP, Holden WM, Collier SP, Sutherland DM, Reinert LK, Rollins-
Smith LA (2013) The invasive chytrid fungus of amphibians paralyzes
lymphocyte responses. Science 342:366–369
Flechas SV, Paz A, Crawford AJ, Sarmiento C, Acevedo AA, Arboleda A, Bolívar-
Garcia W, Echevery-Sandoval CL, Franco R, Mojica C, Muñoz A, Palacios-
Rodriguez P, Posso-Terranova AM, Quintero-Marin P, Rueda-Solano L, Castro-
Herrera F, Amézquita A (2017) Current and predicted distribution of the pathogenic
fungus Batrachochytrium dendrobatidis in Colombia, a hotspot of amphibian
biodiversity. Biotropica 49:685–694
Flory AR, Kumar S, Stohlgren TJ, Cryan PM (2012) Environmental conditions
associated with bat white‐nose syndrome mortality in the north‐eastern United
States. Journal of Applied Ecology 49:680–689
Frost D R (2016) Amphibian Species of the World: an Online Reference. Version 6.0
(accessed 23 Aug 2016). Electronic Database accessible at
http://research.amnh.org/herpetology/amphibia/index.html. American Museum of
Natural History, New York, USA
Gaertner JP, Forstner MR, O’Donnell L, Hahn D. 2009. Detection of Batrachochytrium
dendrobatidis in endemic salamander species from Central Texas. EcoHealth
6:20–26
86
Gahl MK, Longcore JE, Houlahan JE (2012). Varying responses of northeastern North
American amphibians to the chytrid pathogen Batrachochytrium dendrobatidis.
Conservation Biology 26:135–141
Garmyn A, Van Rooij P, Pasmans F, Hellebuyck T, Van Den Broeck W, Haesebrouck
F, Martel A (2012) Waterfowl: potential environmental reservoirs of the chytrid
fungus Batrachochytrium dendrobatidis. PLoS One 7:e35038
Gervasi S, Gondhalekar C, Olson DH, Blaustein AR (2013) Host identity matters in the
amphibian-Batrachochytrium dendrobatidis system: fine-scale patterns of
variation in responses to a multi-host pathogen. PLoS One 8:e54490
Gower DJ, Wilkinson M (2005) Conservation biology of caecilian amphibians.
Conservation Biology 19:45–55
Gower DJ, Doherty-Bone T, Loader SP, Wilkinson M, Kouete MT, Tapley B, Orton F,
Daniel OZ, Wynne F, Flach E, Müller H, Menegon M, Stephen I, Browne RK,
Fisher MC, Cunningham AA, Garner TWJ (2013) Batrachochytrium
dendrobatidis infection and lethal chytridiomycosis in caecilian amphibians
(Gymnophiona). EcoHealth 10:173–183
Gower DJ, Doherty-Bone TM, Loader SP, Wilkinson M and others (2013)
Batrachochytrium dendrobatidis infection and lethal chytridiomycosis in caecilian
amphibians (Gymnophiona). EcoHealth 10:173–183
Greenberg DA, Palen WJ, Mooers AØ (2017) Amphibian species traits, evolutionary
history and environment predict Batrachochytrium dendrobatidis infection
patterns, but not extinction risk. Evolutionary applications 10:1130–1145
Greenspan SE, Longcore JE Calhoun AJ (2012) Host invasion by Batrachochytrium
dendrobatidis: fungal and epidermal ultrastructure in model anurans. Diseases of
Aquatic Organisms 100:201–210
Greenspan SE, Lambertini C, Carvalho T, James TY, Toledo LF, Haddad CFB, Becker
CG (2018) Hybrids of amphibian chytrid show high virulence in native hosts.
Scientific Reports 8:1–10
Gründler MC, Toledo LF, Parra-Olea G, Haddad CF, Giasson LO, Sawaya RJ, Prado,
CPA, Araujo OGS, Zara FJ, Centeno FC, Zamudio KR (2012) Interaction
between breeding habitat and elevation affects prevalence but not infection
intensity of Batrachochytrium dendrobatidis in Brazilian anuran assemblages.
Diseases of Aquatic Organisms 97:173–184
87
Haddad CFB, Toledo LF, Prado CPA, Loebmann D, Gasparini JL, Sazima I (2013).
Guia dos anfíbios da Mata Atlântica: diversidade e biologia. Anolis Books
Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A (2005) Very high resolution
interpolated climate surfaces for global land areas. International Journal of
Climatology 25:1965–1978
Hyatt, A.D., Boyle, D.G., Olsen, V., Boyle, D.B., Berger, L., Obendorf, D.,Colling, A.,
(2007) Diagnostic assays and sampling protocols for the detection of
Batrachochytrium dendrobatidis. Diseases of Aquatic Organisms 73:175–192
Hydeman ME, Bell RC, Drewes RC, Zamudio KR (2013) Amphibian chytrid fungus
confirmed in endemic frogs and caecilians on the island of São Tomé, Africa.
Herpetological Review 44:254–257
James TY, Toledo LF, Rödder D, Leite DS and others (2015) Disentangling host,
pathogen, and environmental determinants of a recently emerged wildlife disease:
lessons from the first 15 years of amphibian chytridiomycosis research. Ecology
and Evolution 5:4079–4097
Jenkinson TS, Bettancourt-Román CM, Lambertini C, Valencia‐Aguilar A, Rodriguez
D, Nunes‐de‐Almeida CHL, Ruggeri J, Belasen AM, Leite DS, Zamudio KR,
Longcore JE, Toledo LF, James TY (2016) Amphibian‐killing chytrid in Brazil
comprises both locally endemic and globally expanding populations. Molecular
Ecology 25:2978–2996
Jones KE, Patel NG, Levy MA, Storeygard A, Balk D, Gittleman JL, Daszak P (2008)
Global trends in emerging infectious diseases. Nature 451:990–994
Keane P, Kerr A (1997) Factors affecting disease development. Plant Pathogens and
Plant Diseases, 287–298
Keesing F, Holt RD, Ostfeld RS (2006) Effects of species diversity on disease risk.
Ecology Letters 9:485–498
Kilburn VL, Ibáñez R, Green DM (2011) Reptiles as potential vectors and hosts of the
amphibian pathogen Batrachochytrium dendrobatidis in Panama. Diseases of
Aquatic Organisms 97:127–134
Kilpatrick AM, Briggs CJ, Daszak P (2009) The ecology and impact of
chytridiomycosis: an emerging disease of amphibians. Trends in Ecology and
Evolution 25:109–118
Kim K, Harvell CD (2004). The rise and fall of a six-year coral-fungal epizootic. The
American Naturalist 164:S52–S63
88
Kriger KM, Ashton KJ, Hines HB, Hero JM (2007) On the biological relevance of a
single Batrachochytrium dendrobatidis zoospore: a reply to Smith (2007).
Diseases of Aquatic Organisms 73:257–260
Kriger KM, Hero JM (2008) Altitudinal distribution of chytrid (Batrachochytrium
dendrobatidis) infection in subtropical Australian frogs. Austral Ecology
33:1022–1032
La Marca E, Lips KR, Lötters S, Puschendorf R, Ibáñez R, Rueda‐Almonacid JV,
Schulte R, Marty C, Castro F, Manzanilla-Puppo J, García-Pérez JE, Bolanõs F,
Chaves G, Pounds JA, Toral E, Young BE (2005) Catastrophic population
declines and extinctions in Neotropical harlequin frogs (Bufonidae: Atelopus).
Biotropica 37:190–201
LaMarca E, Lötters S (1997) Monitoring of declines in Venezuelan Atelopus
(Amphibia: Anura: Bufonidae). Herpetologia Bonnensis 1997:207–213
LaMarca E, Lips KR, Lötters S, Puschendorf R, Ibáñez R, Rueda‐Almonacid JV,
Schulte R, Marty C, Castro F, Manzanilla-Puppo J, García-Pérez JE, Bolanõs F,
Chaves G, Pounds JA, Toral E, Young BE (2005) Catastrophic population declines
and extinctions in Neotropical harlequin frogs (Bufonidae: Atelopus). Biotropica
37:190–201
Labisko J, Maddock ST, Taylor ML, Chong-Seng L and others (2015) Chytrid fungus
(Batrachochytrium dendrobatidis) undetected in the Two Orders of Seychelles
Amphibians. Herpetological Review 46:41–45
Lambertini C, Rodriguez D, Brito FB, Leite DS, Toledo LF (2013) Diagnóstico do
fungo Quitrídio: Batrachochytrium dendrobatidis. Herpetologia Brasileira 2:12–17
Lambertini C, Becker CG, Jenkinson TS, Rodriguez D, Leite DS, James TY, Zamudio
KR, Toledo LF (2016) Local phenotypic variation in amphibian-killing fungus
predicts infection dynamics. Fungal Ecology 20:15–21
Lambertini C, Becker CG, Bardier C, Leite DS, Toledo LF (2017) Spatial distribution
of Batrachochytrium dendrobatidis in South American caecilians. Diseases of
Aquatic Organisms 124:109–116
Lampo M, Barrio-Amorós C, Han B (2006) Batrachochytrium dendrobatidis infection
in the recently rediscovered Atelopus mucubajiensis (Anura, Bufonidae), a
critically endangered frog from the Venezuelan Andes. EcoHealth 3:299–302
Laurance WF, McDonald KR, Speare R (1996) Epidemic disease and the catastrophic
decline of Australian rain forest frogs. Conservation Biology 10:406–413
89
Levin SA (1992) The Problem of Pattern and Scale in Ecology. Ecology 73:1943–1967
Lips KR, Burrowes PA, Mendelson III JR, Parra‐Olea G (2005) Amphibian Population
Declines in Latin America: A Synthesis 1. Biotropica: The Journal of Biology and
Conservation 37:222–226
Lips KR, Brem F, Brenes R, Reeve JD, Alford RA, Voyles J, Carey C, Livo L, Pessier
AP, Collins JP (2006) Emerging infectious disease and the loss of biodiversity in a
Neotropical amphibian community. Proceedings of The National Academy of
Sciences 103:3165–3170
Lips KR, Diffendorfer J, Mendelson III JR, Sears MW (2008) Riding the wave:
reconciling the roles of disease and climate change in amphibian declines. PLoS
Biology 6:e72
Lisboa BS, Neves JMM, Nascimento FAC, Tavares-Bastos L, Mott T (2013) New
records of Batrachochytrium dendrobatidis in the Atlantic forest of Northeastern
Brazil. North-Western Journal of Zoology 9:210–213
Liu X, Rohr JR, Li Y (2013) Climate, vegetation, introduced hosts and trade shape a
global wildlife pandemic. Proceedings of the Royal Society B 280:20122506
Longcore JE, Pessier AP, Nichols DK (1999) Batrachochytrium dendrobatidis gen. et
sp. nov., a chytrid pathogenic to amphibians. Mycologia 91:219–227
Longo AV, Burrowes PA, Joglar RL (2010) Seasonality of Batrachochytrium
dendrobatidis infection in direct-developing frogs suggests a mechanism for
persistence. Diseases of Aquatic Organisms 92:253–260
Lorch JM, Knowles S, Lankton JS, Michell K, Edwards JL, Kapfer JM, Staffen RA,
Wild ER, Schmidt KZ, Ballman AE, Blodgett D, Farrel TM, Glorioso BM, Last
LA, Price SJ, Schuler KL, Smith CE, Wellehan JFX, Blehert DS (2016). Snake
fungal disease: an emerging threat to wild snakes. Philosophical Transactions of
the Royal Society B 371:20150457
Lötters S, Wagner N, Kerres A, Vences M, Steinfartz S, Sabino-Pinto J, Seufer L,
Preissler K, Schulz V, Veith M (2018) First report of host co-infection of parasitic
amphibian chytrid fungi. Salamandra 54:287–290
Luis AD, Kuenzi AJ, Mills JN (2018) Species diversity concurrently dilutes and
amplifies transmission in a zoonotic host–pathogen system through competing
mechanisms. Proceedings of the National Academy of Sciences 115:7979–7984
90
Mann RM, Hyne RV, Choung CB, Wilson SP (2009) Amphibians and agricultural
chemicals: review of the risks in a complex environment. Environmental
Pollution 157: 2903–2927
Martel A, Spitzen-van der Sluijs A, Blooi M, Bert W, Ducatelle R, Fisher MC, Woeltjes
A, Bosman W, Chiers K, Bossuyt F, Pasmans F (2013) Batrachochytrium
salamandrivorans sp. nov. causes lethal chytridiomycosis in
amphibians. Proceedings of the National Academy of Sciences 38:15325–15329
Martel A, Blooi M, Adriaensen C, Van Rooij P, Beukema W, Fisher MC, Farrer RA,
Schmidt BR, Tobler U, Goka K, Lips K, Muletz C, Zamudio KR, Bosh J, Lötters
S, Wombwell E, Garner TWJ, Cunningham AA, Spitzen-van der Sluijs A,
Salvidio S, Ducatelle R, Nishikawa K, Nguyen TT, Kolby JE, Bocxlaer IV,
Bossuyt F, Pasmans F (2014) Recent introduction of a chytrid fungus endangers
Western Palearctic salamanders. Science 346:630–631
McCoy CM, Lind CM, Farrell TM (2017) Environmental and physiological correlates
of the severity of clinical signs of snake fungal disease in a population of pigmy
rattlesnakes, Sistrurus miliarius. Conservation physiology 5:1–10
McMenamin SK, Hadly EA, Wright CK (2008) Climate change and wetland dissecation
cause amphibian decline in Yellowstone National Park. Proceedings of the
National Academy of Sciences 105:16988–16993
McMichael AJ (2004) Environmental and social influences on emerging infectious
diseases: past, present and future. Philosophical Transactions of the Royal Society
of London B 359:1049–1058
McNew GL (1960) The nature, origin, and evolution of parasitism. In Plant Pathology:
An Advanced Treatise (eds Horsfall JG, Dimond AE) 19–69 (Academic Press,
New York)
Mesquita AF, Lambertini C, Lyra M, Malagoli LR, James TY, Toledo LF, Haddad
CFB, Becker CG (2017) Low resistance to chytridiomycosis in direct-developing
amphibians. Scientific Reports 7:1–7
Morse SS (1995) Factors in the emergence of infectious diseases. Emerging Infectious
Diseases 1:7–15
Muletz‐Wolz CR, Barnett SE, DiRenzo GV, Zamudio KR, Toledo LF, James TY, Lips
KR (2019) Diverse genotypes of the amphibian killing fungus produce distinct
phenotypes through plastic responses to temperature. Journal of Evolutionary
Biology 32:287–298
91
Murray KA, Retallick RW, Puschendorf R, Skerratt LF, Rosauer D, McCallum HI,
Berger L, Speare R, VanDerWal J (2011) Assessing spatial patterns of disease risk
to biodiversity: implications for the management of the amphibian pathogen,
Batrachochytrium dendrobatidis. Journal of Applied Ecology, 48(1), 163-173.
O’Hanlon SJ, Rieux A, Farrer RA, Rosa GM, Waldman B, Bataille A, Kosch TA,
Murray KA, Brankovics B, Fumagalli M, Martin MD, Wales N, Alvarado-Rybak
M, Bates KA, Berger L, Böll S, Brookes L, Clare F, Courtois EA, Cunningham
AA, Doherty-Bone TM, Ghosh P, Gower DJ, Hintz WE, Höglund J, Jenkinson
TS, Lin CF, Laurilla A, Loyal A, Martel A, Meurling S, Miaud C, Minting P,
Pasmans F, Schmeller DS, Schmidt BR, Shelton JMG, Skerrat LF, Smith F, Soto-
Azat C, Spagnoletti M, Tessa G, Toledo LF, Valenzuela-Sanches A, Verster R,
Vörös J, Webb RJ, Wierzbicki C, Wombwell E, Zamudio KR, Aanensen, James
TY, Gilbert MTP, Weldon C, Bosch J, Balloux F, Garner TWJ, Fisher MC (2018)
Recent Asian origin of chytrid fungi causing global amphibian declines. Science
360:621–627
Ochoa AV, Carmona PB, Salcedo LDP Correa MF (2013) Detección y cuantificación
de Batrachochytrium dendrobatidis en anfibios de las regiones andina central,
oriental, Orinoquia y Amazonia de Colombia. Herpetotropicos 8:13–21
Olson DH, Ronnenberg KL (2014) Global Bd Mapping Project: 2014 update. FrogLog
22:17–21
Ouellet M, Mikaelian I, Pauli BD, Rodrigue J, Green DM (2005) Historical evidence of
widespread chytrid infection in North American amphibian populations.
Conservation Biology 19:1431–1440
Padgett-Flohr GE, Longcore JE (2007) Taricha torosa (California newt), fungal
infection. Herpetological Review 78:176–177
Parto P, Vaissi S, Farasat H, Sharifi M (2013) First report of chytridiomycosis
(Batrachochytrium dendrobatidis) in endangered Neurergus microspilotus
(Caudata: Salamandridae) in western Iran. Global Veterinary 11:547–551
Penner J, Adum GB, McElroy MT, Doherty-Bone T, Hirschfeld M, Sandberger L,
Weldon C, Cunningham AA, Ohst T, Wombwell E, Portik DM, Reid D, Hillers
A, Ofori-Boateng C, Oduro W, Plötner J, Ohler A, Leaché AD, Rödel MO (2013).
West Africa-A safe haven for frogs? A sub-continental assessment of the chytrid
fungus (Batrachochytrium dendrobatidis). PLoS One 8:e56236
92
Peterson JD, Steffen JE, Reinert LK, Cobine PA, Appel A, Rollins-Smith L, Mendonça
MT (2013) Host stress response is important for the pathogenesis of the deadly
amphibian disease, chytridiomycosis, in Litoria caerulea. PLoS One 8:e62146
Piotrowski JS, Annis SL, Longcore JE (2004) Physiology of Batrachochytrium
dendrobatidis, a chytrid pathogen of amphibians. Mycologia 96:9–15
Piovia-Scott J, Pope KL, Lawler SP, Cole EM, Foley JE (2011) Factors related to the
distribution and prevalence of the fungal pathogen Batrachochytrium dendrobatidis
in Rana cascadae and other amphibians in the Klamath Mountains. Biological
Conservation 144:2913–2921
Pontes MR, Augusto-Alves G, Lambertini C, Toledo LF (2018) A lizard acting as
carrier of the amphibian-killing chytrid Batrachochytrium dendrobatidis in
southern Brazil. Acta Herpetologica 13:201–205
Pounds JA, Bustamante M R, Coloma LA, Consuegra JA and others (2006) Widespread
amphibian extinctions from epidemic disease driven by global warming. Nature
439:161–167
Preuss JF, Lambertini C, Leite DS, Toledo, LF, Lucas EM (2015) Batrachochytrium
dendrobatidis in near threatened and endangered amphibians in the southern
Brazilian Atlantic Forest. North-Western Journal of Zoology 11:360–362
Puschendorf R, Carnaval AC, VanDerWal J, Zumbado‐Ulate H, Chaves G, Bolaños F,
Alford RA (2009) Distribution models for the amphibian chytrid Batrachochytrium
dendrobatidis in Costa Rica: proposing climatic refuges as a conservation tool.
Diversity and Distributions 15:401–408
Rachowicz LJ, Hero JM, Alford RA, Taylor JW, Morgan JA, Vredenburg VT, Briggs
CJ (2005) The novel and endemic pathogen hypotheses: competing explanations
for the origin of emerging infectious diseases of wildlife. Conservation
Biology 19:1441–1448
Raffel TR, Michel PJ, Sites EW, Rohr JR (2010) What drives chytrid infections in newt
populations? Associations with substrate, temperature, and shade. EcoHealth
7:526–536
Raffel TR, Romansic JM, Halstead NT, McMahon TA and others (2013) Disease and
thermal acclimation in a more variable and unpredictable climate. Nature Climate
Change 3:146–151
93
Raffel TR, Halstead NT, McMahon TA, Davis AK, Rohr JR (2015) Temperature
variability and moisture synergistically interact to exacerbate an epizootic disease.
Proceedings of the Royal Society B 282:20142039
Rangel TF, Diniz‐Filho JAF, Bini LM (2010) SAM: a comprehensive application for
spatial analysis in macroecology. Ecography 33:46–50
Raphael BL, Pramuk J (2007) Treatment of chytrid infection in Typhlonectes spp. using
elevated water temperatures. Proceedings of Amphibian Declines and
Chytridiomycosis, Tempe, AZ, abstracts
Rendle M, Tapley B, Perkins M, Bittencourt-Silva G and others (2015) Itraconazole
treatment of Batrachochytrium dendrobatidis (Bd) infection in captive caecilians
(Amphibia: Gymnophiona) and the first case of Bd infection in a wild neotropical
caecilian. Journal of Zoo and Aquarium Research 3:137–140
Rendle M, Tapley B, Perkins M, Bittencourt-Silva G, Gower DJ, Wilkinson M (2015)
Itraconazole treatment of Batrachochytrium dendrobatidis (Bd) infection in
captive caecilians (Amphibia: Gymnophiona) and the first case of Bd infection in
a wild neotropical caecilian. Journal of Zoo and Aquarium Research 3:137−140
Rödder D, Kielgast J, Bielby J, Schmidtlein S and others (2009) Global amphibian
extinction risk assessment for the panzootic chytrid fungus. Diversity 1:52–66
Rödder D, Kielgast J, Lötters S (2010) Future potential distribution of the emerging
amphibian chytrid fungus under anthropogenic climate change. Diseases of Aquatic
Organisms 92:201–207
Rodriguez D, Becker CG, Pupin NC, Haddad CFB, Zamudio KR (2014) Long‐term
endemism of two highly divergent lineages of the amphibian‐killing fungus in the
Atlantic Forest of Brazil. Molecular Ecology 23:774–787
Rodríguez-Contreras A, Señaris JC, Lampo M, Rivero R (2008) Rediscovery of
Atelopus cruciger (Anura: Bufonidae): current status in the Cordillera de la Costa,
Venezuela. Oryx 42:301–304
Ron SR, Duellman WE, Coloma LA, Bustamante MR (2003) Population decline of the
Jambato toad Atelopus ignescens (Anura: Bufonidae) in the Andes of Ecuador.
Journal of Herpetology 37:116–126
Ron SR (2005) Predicting the distribution of the amphibian pathogen Batrachochytrium
dendrobatidis in the New World 1. Biotropica 37:209–221
Rosenblum EB, James TY, Zamudio KR, Poorten TJ, Ilut D, Rodriguez D, Eastman
JM, Richards-Hrdlicka KR, Joneson S, Jenkinson TS, Longcore JE, Parra Olea G,
94
Toledo LF, Arellano ML, Medina EM, Restrepo S, Flechas SV, Berger L, Briggs
CJ, Stajich JE (2013) Complex history of the amphibian-killing chytrid fungus
revealed with genome resequencing data. Proceedings of the National Academy of
Sciences 110:9385–9390
Ruggeri J, Longo AV, Gaiarsa MP, Alencar LR, Lambertini C, Leite DS, Carvalho-e-
Silva SP, Zamudio KR, Toledo LF, Martins M (2015) Seasonal variation in
population abundance and chytrid infection in stream-dwelling frogs of the Brazilian
Atlantic forest. PloS ONE 10:e0130554
Ruggeri J, Carvalho-e-Silva SP, James TY, Toledo LF (2018) Amphibian chytrid
infection is influenced by rainfall seasonality and water availability. Diseases of
Aquatic Organisms 127:107–115
SAS. 2010. JMP, Version 10. SAS Institute Inc. (Cary, NC)
Savage AE, Grismer LL, Anuar S, Onn CK, Grismer JL, Quah E, Muin MA, Ahmad N,
Lenker M, Zamudio KR (2011) First record of Batrachochytrium dendrobatidis
infecting four frog families from Peninsular Malaysia. EcoHealth 8:121–128
Scheele BC, Pasmans F, Berger L, Skerratt LF, Martel A, Beukema W, Acevedo AA,
Burrowes PA, Carvalho T, Catenazzi A, de la Riva I, Fisher MC, Flechas SV,
Foster CN, Frías-Álvarez P, Garner TWJ, Gratwicke B, Guayasamin JM,
Hirschfeld M, Kolby JE, Kosch TA, La Marca E, Lindenmayer DB, Lips KR,
Longo AV, Maneyro R, McDonald CA, Mendelson III J, Palacios-Rodriguez P,
Parra-Olea G, Richards-Zawacki CL, Rödel MO, Rovito SM, Soto-Azat C,
Toledo LF, Voyles J, Weldon C, Whitfield SM, Wilkinson M, Zamudio KR,
Canessa S (2019) Amphibian fungal panzootic causes catastrophic and ongoing
loss of biodiversity. Science in press
Schloegel LM, Toledo LF, Longcore JE, Greenspan SE, Vieira CA, Lee M. Zhao S,
Wangen C, Ferreira CM, Hipolito M, Daves AJ, Cuomo CA, Daszak P, James TY.
(2012) Novel, panzootic and hybrid genotypes of amphibian chytridiomycosis
associated with the bullfrog trade. Molecular Ecology 21:5162–5177
Scholthof KBG (2007) The disease triangle: pathogens, the environment and society.
Nature Reviews Microbiology 5:152
Searle CL, Biga LM, Spatafora JW, Blaustein AR (2011) A dilution effect in the
emerging amphibian pathogen Batrachochytrium dendrobatidis. Proceedings of
the National Academy of Sciences 108:16322–16326
95
Seimon TA, Seimon A, Daszak P, Halloy SR and others (2007) Upward range
extension of Andean anurans and chytridiomycosis to extreme elevations in
response to tropical deglaciation. Global Change Biology 13:288–299
Semlitsch, R. D. Amphibian Conservation (2003) Smithsonian Instituition. Unites
States of America.
Skerratt LF, Berger L, Speare R, Cashins S, McDonald KR, Phillott AD, Hines HB,
Kenyon N (2007) Spread of chytridiomycosis has caused the rapid global decline and
extinction of frogs. EcoHealth 4:125–134
Snieszko SF (1974) The effects of environmental stress on outbreaks of infectious
diseases of fishes. Journal of Fish Biology 6:197–208
Soto‐Azat C, Clarke BT, Poynton JC, Cunningham AA (2010) Widespread historical
presence of Batrachochytrium dendrobatidis in African pipid frogs. Diversity and
Distributions 16:126–131
Stegen G, Pasmans F, Schmidt BR, Rouffaer LO, Van Praet S, Schaub M, Canessa S,
Laudelout A, Kinet T, Adriaensen C, Haesebrouck F, Bert W, Bossuit F, Martel A
(2017) Drivers of salamander extirpation mediated by Batrachochytrium
salamandrivorans. Nature 544:353–363
Stevenson LA, Alford RA, Bell SC, Roznik EA, Berger L, Pike DA (2013) Variation in
thermal performance of a widespread pathogen, the amphibian chytrid fungus
Batrachochytrium dendrobatidis. PloS One 8:e73830
Stuart S N, Chanson JS, Cox NA, Young BE, Rodrigues AS, Fischman DL, Waller RW.
(2004) Status and trends of amphibian declines and extinctions worldwide. Science
306:1783–1786
Swei A, Rowley JJ, Rödder D, Diesmos ML, Diesmos AC, Briggs CJ, Brown R, Cao
TT, Cheng TL, Choing RA, Han B, Hero JM, Hoang HD, Kusrini MD, Le DTT,
McGuire JÁ, Meegaskumbura M, Min MS, Mulcahy DG, Neang T, Phimmachak S,
Rao DQ, Reeder NM, Schoville SD, Sivongxay N, Srei N, Stöck M, Stuart BL,
Torres LS, Tran DTA, Tusntall TS, Vieites D, Vredenburg VT (2011) Is
chytridiomycosis an emerging infectious disease in Asia? PloS One, 6:e23179
Talley BL, Muletz CR, Vredenburg,VT, Fleischer RC, Lips KR (2015) A century of
Batrachochytrium dendrobatidis in Illinois amphibians (1888–1989). Biological
Conservation 182:254–261
Taylor EH (1968) The caecilians of the world: a taxonomic review. Lawrence:
University of Kansas Press
96
Therneau T (2012) A Package for Survival Analysis in S. R package version 2.37–2,
URL: http://CRAN.R-project.org/package = survival.
Toledo LF, Haddad CFB, Carnaval ACOQ, Britto FB (2006a) A Brazilian anuran
(Hylodes magalhaesi: Leptodactylidae) infected by Batrachochytrium
dendrobatidis: a conservation concern. Amphibian and Reptile Conservation
4:17–21
Toledo LF, Britto FB, Araújo OGS, Giasson LMO, Haddad CFB (2006b) The
occurrence of Batrachochytrium dendrobatidis in Brazil and inclusion of 17 new
cases. South American Journal of Herpetology 1:185–191
Toledo, L. F. 2017. Comment on “amphibians on the brink”. Sci. Signal. (E-Letter, 22
August 2017)
Tompkins DM, Dunn AM, Smith MJ, Telfer S (2011). Wildlife diseases: from
individuals to ecosystems. Journal of Animal Ecology 80:19–38
Tompkins DM, Carver S, Jones ME, Krkošek M, Skerratt LF (2015) Emerging
infectious diseases of wildlife: a critical perspective. Trends in
Parasitology 31:149–159
Tuite AR, Greer AL, Fisman DN (2013) Effect of latitude on the rate of change in
incidence of Lyme disease in the United States. CMAJ open 1:E43
Valencia-Aguilar A, Ruano-Fajardo G, Lambertini C, Leite DS, Toledo LF, Mott T
(2015) Chytrid fungus acts as a generalist pathogen infecting species-rich
amphibian families in Brazilian rainforests. Diseases of Aquatic Organisms
114:61–67
Van Rooij P, Martel A, Nerz J, Voitel S, Van Immerseel F, Haesebrouck F, Pasmans F
(2011) Detection of Batrachochytrium dendrobatidis in Mexican bolitoglossine
salamanders using an optimal sampling protocol. EcoHealth 8:237–243
Vasquez-Ochoa AV, Carmona PB, Salcedo LDP, Correa MF (2013) Detección y
cuantificación de Batrachochytrium dendrobatidis en anfibios de las regiones
andina central, oriental, Orinoquia y Amazonia de Colombia. Herpetotropicos
8:13–21
Venesky MD, Kerby JL, Storfer A, Parris MJ (2011). Can differences in host behavior
drive patterns of disease prevalence in tadpoles? PLoS One 6:e24991
Verant ML, Boyles JG, Waldrep Jr W, Wibbelt G, Blehert DS (2012) Temperature-
dependent growth of Geomyces destructans, the fungus that causes bat white-nose
syndrome. PloS One 7:e46280
97
Vieira CA, Almeida CHLN, Lambertini C, Leite DS, Toledo LF (2012) First record of
chytridiomycosis (Batrachochytrium dendrobatidis; Rhizophydiales;
Chytridiaceae) in the state of Paraná, southern Brazil. Herpetological Review
43:93–94
Vieira CA, Toledo, LF, Longcore JE, Longcore JR (2013) Body length of Hylodes cf.
ornatus and Lithobates catesbeianus tadpoles, depigmentation of mouthparts, and
presence of Batrachochytrium dendrobatidis are related. Brazilian Journal of
Biology 731:195–199
Vitt LJ, Caldwell JP (2014) Herpetology. An introductory biology of amphibians and
reptiles (4th
ed.). Oklahoma: Academic Press
Voyles J, Young S, Berger L, Campbell C, Voyles WF, Dinudom A, Speare R (2009)
Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian
declines. Science 326:582–585
Voyles J, Johnson LR, Briggs CJ, Cashins SD, Alford RA, Berger L, Skerratt LF,
Speare R, Rosenblum EB (2012) Temperature alters reproductive life history
patterns in Batrachochytrium dendrobatidis, a lethal pathogen associated with the
global loss of amphibians. Ecology and Evolution 2:2241–2249
Voyles J, Johnson LR, Rohr J, Kelly R, Barron C, Miller D, Minster J, Rosenblum EB
(2017) Diversity in growth patterns among strains of the lethal fungal pathogen
Batrachochytrium dendrobatidis across extended thermal optima. Oecologia
184:363–373
Vredenburg V T, Knapp R A, Tunstall T S, Briggs CJ (2010) Dynamics of an emerging
disease drive large-scale amphibian population extinctions. Proceedings of the
National Academy of Science 107:9689–9694
Walker SF, Bosch J, Gomez V, Garner TW, Cunningham AA, Schmeller DS, Nynierola
M, Henk DA, Ginestet C, Arthur CP, Fisher, MC (2010) Factors driving
pathogenicity vs. prevalence of amphibian panzootic chytridiomycosis in Iberia.
Ecology Letters 13:372–382
Warnecke L, Turner JM, Bollinger TK, Lorch JM, Misra V, Cryan PM, Wibbelt G,
Blehert D, Willis CK (2012) Inoculation of bats with European Geomyces
destructans supports the novel pathogen hypothesis for the origin of white-nose
syndrome. Proceedings of the National Academy of Sciences 109:6999–7003
Weldon C, Du Preez LH, Hyatt AD, Muller R, Speare R (2004) Origin of amphibian
chytrid fungus. Emerging Infectious Diseases 10:2100–2105
98
Wells KD (2007) The ecology and behavior of amphibians. Chicago, IL: The University
of Chicago Press
Wilkinson M, San Mauro D, Sherratt E, Gower DJ (2011) A nine-family classification
of caecilians (Amphibia: Gymnophiona). Zootaxa 2874:41–64
Williams ES, Yuill T, Artois M, Fischer J, Haigh SA (2002) Emerging infectious
diseases in wildlife. Revue scientifique et technique-Office international des
Epizooties 21:139–158
99
ANEXOS
1. Licença de coleta de anfíbios para a região amazônica
100
101
102
2. Licença de coleta de anfíbios para a região da Mata Atlântica
103
104
105
106
3. Comitê de ética para amostragem e realização de experimentação em
laboratório
107
4. Cadastro no Conselho de Gestão do Patrimônio Genético – SISGen
108
109
110
111
112
113
114
5. Declaração
