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Coccídios (Protozoa: Apicomplexa) Em Peixes Da Planície De Inundação Do Rio Curiaú, Estado Do Amapá: Prevalência E Caracterização Molecular

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Coccídios (Protozoa: Apicomplexa) Em Peixes Da Planície De Inundação Do Rio Curiaú, Estado Do Amapá: Prevalência E Caracterização Molecular Universidade Federal do Amapá Pró-Reitoria de Pesquisa e Pós-Graduação Programa de Pós-Graduação em Biodiversidade Tropical Mestrado e Doutorado UNIFAP / EMBRAPA-AP / IEPA / CI-Brasil COCCÍDIOS (PROTOZOA: APICOMPLEXA) EM PEIXES DA PLANÍCIE DE INUNDAÇÃO DO RIO CURIAÚ, ESTADO DO AMAPÁ: PREVALÊNCIA E CARACTERIZAÇÃO MOLECULAR MACAPÁ, AP 2018 MÁRCIO CHARLES DA SILVA NEGRÃO COCCÍDIOS (PROTOZOA: APICOMPLEXA) EM PEIXES DA PLANÍCIE DE INUNDAÇÃO DO RIO CURIAÚ, ESTADO DO AMAPÁ: PREVALÊNCIA E CARACTERIZAÇÃO MOLECULAR Dissertação apresentada ao Programa de Pós-Graduação em Biodiversidade Tropical (PPGBIO) da Universidade Federal do Amapá, como requisito parcial à obtenção do título de Mestre em Biodiversidade Tropical. Orientador: Dr. Lúcio André Viana Dias MACAPÁ, AP 2018 MÁRCIO CHARLES DA SILVA NEGRÃO COCCÍDIOS (PROTOZOA: APICOMPLEXA) EM PEIXES DA PLANÍCIE DE INUNDAÇÃO DO RIO CURIAÚ, ESTADO DO AMAPÁ: PREVALÊNCIA E CARACTERIZAÇÃO MOLECULAR _________________________________________ Dr. Lúcio André Viana Dias Universidade Federal do Amapá (UNIFAP) ____________________________________________ Dr. Marcos Tavares Dias Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) ____________________________________________ Dra. Marcela Nunes Videira Universidade Estadual do Amapá (UEAP) Aprovada em 11 de abril de 2018, Macapá, AP, Brasil. À Deus pela vida; Aos meus pais; Aos meus irmãos; Aos meus tios e primos; A todos os meus amigos. AGRADECIMENTOS Agradeço a Deus, pela vida e oportunidades. Aos meus pais Benedito Vilhena Negrão e Maria Esmeralda da Silva Negrão, pelo amor dedicado, pela educação transmitida e exemplos de caráter, personalidade e luta; Ao meu irmão Emerson, in memorian, que me enchia de elogios ao se referir a minha capacidade de desenvolvimento intelectual e postura profissional. Hoje me enche de saudade; Às minhas irmãs Eliane, Elem e Lana, grandes incentivadoras que renovam as minhas forças para continuar sempre em frente; Aos meus tios, tias, primos, primas, por me amarem de forma incondicional, por confiarem em minhas escolhas, apoiando e incentivando sempre; Ao meu orientador Dr. Lúcio André Viana Dias, pelos ensinamentos, por sua dedicação ao trabalho sem descuidar de sua família, por acreditar que eu conseguiria, pelo exemplo de competência e ousadia que inspiraram a elaboração desta dissertação, pela amizade, cuidado e, sobretudo, pela paciência em todos os momentos; A todos os professores e colaboradores do Programa de Mestrado em Biodiversidade Tropical da Universidade Federal do Amapá (UNIFAP), pelo empenho e dedicação ao desenvolvimento da pesquisa científica na Amazônia; À Universidade Estadual do Amapá (UEAP), pela estrutura do Laboratório de Morfofisiologia e Sanidade Animal (LABMORSA) para a realização do trabalho; À Dra. Marcela Nunes Videira, Chefe do Laboratório de Morfofisiologia e Sanidade Animal da UEAP, que permitiu a realização das coletas e análises em seu laboratório, disponibilizando seu espaço, material, arcabouço teórico e know-how. Aos colegas do grupo de pesquisa de Sanidade de Organismos Aquáticos na Amazõnia (SOOA) Abthyllane, Josafá, Priscila e Roger, pela ajuda e pelo companheirismo nas coletas e análises; À Dra. Cecile de Souza Gama, do Laboratório de Ictiologia do Instituto de Pesquisas Científicas e Tecnológicas do Estado do Amapá (IEPA), pelo auxílio na identificação taxonômica das espécies de peixes. Aos colegas da turma de Mestrado, pela força e companheirismo nos momentos difíceis do curso. RESUMO Negrão, Márcio Charles da Silva . Coccídios (Protozoa: Apicomplexa) em peixes da planície de inundação do Rio Curiaú, Estado do Amapá: Prevalência e Caracterização Molecular. Macapá, 2018. Dissertação (Mestre em Biodiversidade Tropical) – Programa de Pós-graduação em Biodiversidade Tropical – Pró-Reitoria de Pesquisa e Pós-Graduação - Universidade Federal do Amapá. Os peixes são vulneráveis aos coccídios, sendo registrados diferentes estágios desses parasitos em todas as classes de vertebrados. O objetivo deste estudo foi investigar coccídios do gênero Calyptospora em espécies de peixes da planície de inundação do Rio Curiaú, estado do Amapá, Brasil. Foram coletados 130 peixes, das espécies Astyanax bimaculatus, Metynnis lippincottianu, Hoplias malabaricus, Hoplerythrinus unitaeniatus; Astronotus ocellatus, Cichla ocellaris, Crenicichla lepidota, Geophagus proximus, Heros efasciatus, Mesonauta festivus, Pterophyllum scalare e Satanoperca jurupari. Os peixes foram eutanasiados, pesados (g), medidos (cm), e dissecados para coleta de fragmentos de fígado, coração e vesícula e examinados pelo método de exame a fresco entre lâmina e lamínula, em microscópio de luz (ML), para verificar a presença de oocistos de Calyptospora spp. Os fragmentos de fígado de G. proximus e H. malabaricus foram armazenados em álcool etílico 80%. Posteriormente, o DNA total de cada amostra foi extraído e submetido a reação em cadeia da polimerase, mediante o uso de primers designados para detecção de um seguimento do gene SSU rDNA de Calyptospora spp. Os produtos das amplificações foram sequenciados e as seqüências obtidas foram comparadas com outras disponíveis no Genbank quanto à similaridade. Foi registrado a presença de oocistos de Calyptospora em 55% dos 130 animais examinados, ou seja, 10 das 12 espécies estudadas. Não foram observadas infecções em A. bimaculatus e M. lippincottianus, peixes de hábito herbívoro. As taxas de infecção variaram entre os órgãos parasitados, onde em todos os hospedeiros positivos foram encontrados oocistos no fígado, seguido por 30% na vesícula biliar e 9% no coração. As sequencias obtidas na biologia molecular apresentaram 99% de similaridade com Calyptospora serralsami descrita de piranha Serrasalmus rhombeus no Brazil. No presente estudo foi registrado pela primeira vez a ocorrência de Calyptospora sp. em A. Ocellatus, G. proximus, H. efasciatus, H. unitaeniatus, H. malabaricus, M. festivus P. Scalare e S. jurupari. Foi determinado a ocorrência de C. serrasalmi na planície e em dois novos hospedeiros. Por último, novos primers foram criados neste estudo e testados com sucesso para diagnóstico de Calyptospora. Palavras-chaves: Parasito; Calyptospora; Ictiofauna; Ocorrência; Amazônia. ABSTRACT Negrão, Márcio Charles da Silva . Coccidia (Protozoa: Apicomplexa) in fish from the floodplain of the Curiaú River, State of Amapá: Prevalence and Molecular Characterization. Macapá, 2018. Dissertação (Mestre em Biodiversidade Tropical) – Programa de Pós-graduação em Biodiversidade Tropical – Pró-Reitoria de Pesquisa e Pós- Graduação - Universidade Federal do Amapá. Fish are vulnerable to coccidia, with different stages of these parasites being recorded in all vertebrate classes. The objective of this study was to investigate coccidia of the genus Calyptospora in fish species from the Curiaú River floodplain, Amapá state, Brazil. A total of 130 fish belonging to species Astyanax bimaculatus, Metynnis lippincottianu, Hoplias malabaricus, Hoplerythrinus unitaeniatus; Astronotus ocellatus, Cichla ocellaris, Crenicichla lepidota, Geophagus proximus, Heros efasciatus, Mesonauta festivus, Pterophyllum scalare e Satanoperca jurupari. The fish were euthanized, weighed (g), measured (cm), and dissected for collection of fragments of liver, heart and vesicle and examined by fresh method between lamina and laminula under light microscopy (ML) to verify the presence of oocysts of Calyptospora spp. The liver fragments of G. proximus and H. malabaricus were stored in ethyl alcohol 80%. Subsequently, total DNA from each sample was extracted and subjected to polymerase chain reaction using primers designated to detect a follow-up of the SSU rDNA gene from Calyptospora spp. The products of the amplifications were sequenced and the sequences obtained were compared with others available from Genbank for similarity The presence of Calyptospora oocysts was recorded in 55% of the 130 animals examined, that is, 10 of the 12 species studied. No infections were observed in A. bimaculatus and M. lippincottianus, herbivorous habit fish. Infection rates varied among the parasitized organs, where in all positive hosts oocysts were found in the liver, followed by 30% in the gallbladder and 9% in the heart. The sequences obtained in molercular biology presented 99% similarity with Calyptospora serralsami described of piranha Serrasalmus rhombeus in Brazil. In the present study, the occurrence of Calyptospora sp. in A. Ocellatus, G. proximus, H. efasciatus, H. unitaeniatus, H. malabaricus, M. festivus P. Scalare and S. jurupari. The occurrence of C. serrasalmi in the plain and two new hosts was determined. Finally, new primers were created in this study and tested successfully for Calyptospora diagnosis. Key words: Parasite; Calyptospora; Icthyofauna; Occurrance; Amazon. SUMÁRIO 1. INTRODUÇÃO GERAL.......................................................................................................... 10 1.1. Distribuição taxonômica de espécies de peixes dulcícolas no Brasil......................................11 1.2. Caracterização da ictiofauna da Amazônia brasileira..............................................................12 1.3. A bacia e a ictiofauna do Rio Curiaú............................................................................................ 13 1.3.1. Aspectos biológicos de Astyanax bimaculatus (Linnaeus, 1758)............................16 1.3.2. Aspectos biológicos de Astronotus ocellatus (Agassiz 1831)....................................17 1.3.3. Aspectos biológicos de Cichla ocelaris (Cuvier 1819).................................................19
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