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Instituto De Biologia UNIVERSIDADE ESTADUAL DE CAMPINAS Instituto de Biologia JULIANA DAMIELI NASCIMENTO ESTUDO DOS ASPECTOS MORFOLÓGICOS, FILOGENÉTICOS E PROTEÔMICO DE RHODNIUS NEGLECTUS (HEMIPTERA, REDUVIIDAE) STUDY OF ASPECTS MORPHOLOGICAL, PHYLOGENETIC AND PROTEOMIC OF RHODNIUS NEGLECTUS (HEMIPTERA, REDUVIIDAE) Campinas 2020 JULIANA DAMIELI NASCIMENTO ESTUDO DOS ASPECTOS MORFOLÓGICOS, FILOGENÉTICOS E PROTEÔMICO DE RHODNIUS NEGLECTUS (HEMIPTERA, REDUVIIDAE) STUDY OF ASPECTS MORPHOLOGICAL, PHYLOGENETIC AND PROTEOMIC OF RHODNIUS NEGLECTUS (HEMIPTERA, REDUVIIDAE) 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 Relações Antrópicas, Meio Ambiente e Parasitologia Thesis presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Doctorate in Animal Biology in the field of Anthropic Relations, Environment and Parasitology. Orientador: Prof. Dr. João Aristeu da Rosa ESTE TRABALHO CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA JULIANA DAMIELI NASCIMENTO, E ORIENTADO PELO PROF. DR. JOÃO ARISTEU DA ROSA Campinas 2020 Ficha catalográfica Universidade Estadual de Campinas Biblioteca do Instituto de Biologia Mara Janaina de Oliveira - CRB 8/6972 Nascimento, Juliana Damieli, 1990- N17e Estudo dos aspectos morfológicos, filogenéticos e proteômico de Rhodnius neglectus (Hemiptera, Reduviidae) / Juliana Damieli Nascimento. – Campinas, SP : [s.n.], 2020. Orientador: João Aristeu da Rosa. Tese (doutorado) – Universidade Estadual de Campinas, Instituto de Biologia. 1. Rhodnius. 2. Filogenia. 3. Proteômica. I. Rosa, João Aristeu. II. Universidade Estadual de Campinas. Instituto de Biologia. III. Título. Informações para Biblioteca Digital Título em outro idioma: Study of aspects morphological, phylogenetic and proteomic of Rhodnius neglectus (Hemiptera, Reduviidae) Palavras-chave em inglês: Rhodnius Phylogeny Proteomics Área de concentração: Relações Antrópicas, Meio Ambiente e Parasitologia Titulação: Doutora em Biologia Animal Banca examinadora: João Aristeu da Rosa [Orientador] Carlos Eduardo Almeida Mara Cristina Pinto Vagner José Mendonça Cléber Galvão Ferreira Data de defesa: 09-04-2020 Programa de Pós-Graduação: Biologia Animal Identificação e informações acadêmicas do(a) aluno(a) - ORCID do autor: https://orcid.org/0000-0002-6892-2453 - Currículo Lattes do autor: http://lattes.cnpq.br/2335413580447528 Campinas, 09/04/2020. COMISSÃO EXAMINADORA Prof. Dr. João Aristeu da Rosa (Presidente) Prof. Dr. Carlos Eduardo de Almeida Profa. Dra. Mara Cristina Pinto Prof. Dr. Vagner José Mendonça Prof. Dr. Cleber Galvão Ferreira Os membros da Comissão Examinadora acima assinaram a Ata de Defesa, que se encontra no processo de vida acadêmica do aluno. A Ata da defesa com as respectivas assinaturas dos membros encontra-se no SIGA/Sistema de Fluxo de Dissertação/Tese e na Secretaria do Programa Pós-Graduação em Biologia Animal da Unidade do Instituto de Biologia. DEDICATÓRIA A minha família, razão de tudo. AGRADECIMENTOS Agradeço a Deus, por minha saúde, paciência e discernimento durante esses quatro anos de estudo e trabalho. A minha família, em especial à minha mãe Sirlei, meu pai Nelson, meus irmãos Andréia e José Mário, meus sobrinhos Raul e Lívia, meus cunhados Luciana e Luís Fernando, e ao meu namorado, Rafael, que são minha base. Agradeço por compartilharem ao meu lado esta etapa, por todo o apoio moral, amoroso e financeiro durante este período e em todas as minhas decisões. Obrigada por serem meus exemplos, acreditarem em mim, pela compreensão nos momentos de ausência, pelos conselhos e por todas as palavras motivadoras que me consolaram e deram força nos momentos mais difíceis. Ao Prof. Dr. João Aristeu da Rosa, pela orientação, oportunidade de crescimento profissional e pessoal, principalmente, confiança, incentivo e amizade. Obrigada professor pelo apoio na execução desse projeto, pela convivência e oportunidade de aprendizado. Ao Prof. Dr. Juan David Ramírez pela orientação durante meu estágio na Universidad del Rosário, Faculdade de Ciências Naturais e Matemáticas, Colômbia. Muito obrigada professor pelo aprendizado e amizade, sobretudo pela confiança depositada, dedicação e carinho. Aos alunos do grupo de pesquisas em Microbiologia da Universidad del Rosario, Giovani Herrera, Lissa Cruz Saavedra, Marina Muñoz, Adriana Higuera Gelvez e Luz Patiño Blanco por todo companheirismo, paciência e ensinamentos. A Profa. Dra. Márcia Aparecida da Silva Graminha pela contribuição significativa para o desenvolvimento deste trabalho na Faculdade de Ciências Farmacêuticas de Araraquara, laboratório de Parasitologia clínica. Muito obrigada professora, por toda ajuda e companheirismo nos momentos mais difíceis da pesquisa. Assim como a sua aluna de Pós-Doutorado Ângela Arenas que foi essencial para o desenvolvimento inicial do projeto. A Profa Dra. Ana Marisa Fusco Almeida por disponibilizar o núcleo de proteômica da Faculdade de Ciências Farmacêuticas de Araraquara para realização do trabalho, assim como ao Dr. Paulo César Gomes, técnico responsável da espectrometria de massas e todos os alunos orientados pela professora. Aos Drs. Mara Cristina Pinto, Marlene T. Ueta, Regina Maura Bueno Franco, Danilo Ciccone Miguel, pela disponibilidade em compor a banca do exame de qualificação. Aos demais professores e técnicos do departamento de Parasitologia da Universidade Estadual de Campinas, por toda contribuição para meu aprendizado durante o mestrado e doutorado. As minhas amigas, Aline Rimoldi Ribeiro, Larissa Aguiar Almeida, Fabrícia Regiane Borges e Luana Elis Sabino, sempre presentes, que conviveram com minha ausência em vários momentos e nunca se afastaram ou deixaram de me apoiar. Obrigada pelos momentos de descontração e imensurável ajuda. Aos colegas do Laboratório de Parasitologia da Faculdade de Ciências Farmacêuticas de Araraquara em especial Rossana Falcone, Gabriela Kunii, Dennys Sammilian, Flávia Benini e Cintia Oliveira pelo ambiente de trabalho, pelas discussões científicas, momentos de descontração e imensurável ajuda, sem os quais parte do trabalho não seria possível. A todos os colegas, técnicos, alunos e professores do departamento de Ciências Biológicas da Faculdade de Ciências Farmacêuticas de Araraquara que auxiliaram de algum modo para meu aprendizado acadêmico-profissional e realização das obrigações necessárias para a complementação dessa etapa acadêmica. A Universidade Estadual de Campinas por prover condições para minha formação acadêmico-profissional e pelos subsídios fornecidos a esta pesquisa. A Universidade Estadual Paulista Júlio de Mesquita Filho, Faculdade de Ciências Farmacêuticas de Araraquara por prover condições para a realização dos experimentos científicos, pela formação acadêmico-profissional e pelos subsídios fornecidos. O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001. RESUMO A doença de Chagas tem por agente etiológico o protozoário Trypanosoma cruzi que é transmitido pelas fezes dos triatomíneos. A subfamília Triatominae contém 19 gêneros e 154 espécies, todas potenciais vetoras de T. cruzi, exceto três que são fósseis. A diferenciação das espécies de Triatominae é realizada principalmente por caracteres morfológicos e moleculares, diante dessa premissa este trabalho teve como intuito identificar a espécie de Rhodnius sp. coletada no município de Taquarussu/MS em 2010. Para tanto foram utilizados caracteres morfológicos e citogenéticos. O desenvolvimento desse estudo resultou na descrição de R. taquarussuensis sp. n. Posteriormente por meio de análises das divergências genéticas e delimitações das espécies pelo estudo da variação de dois genes mitocondriais e quatro nucleares, assim como cruzamentos interespecíficos foi demonstrado que R. taquarussuensis sp. n. trata-se de uma linhagem fenotípica de R. neglectus. Após a publicação de R. taquarussuensis sp. n como sinônimo júnior de R. neglectus, foi realizado o estudo de espermatecas de fêmeas fecundadas. Para este estudo, foram dissecadas e retiradas 40 espermatecas fecundadas, logo após foram realizadas a extração das proteínas, análise do perfil proteico, digestão tríptica, espectrometria de massas e identificação das proteínas por meio do banco de dados de R. neglectus em comparação com os bancos SwissProt e NCBI. Foram identificadas 34 proteínas pertencentes a quatro classes proteicas distintas, dessas classes destacam-se as musculares, vacuolar H+-ATPase (V-ATPases) e Chaperonas com funções correlacionadas na forma como as espermatecas mantém os espermatozoides viáveis e nutridos até a fecundação dos ovócitos, processo necessário para a perpetuação das espécies. ABSTRACT Chagas disease is caused by the protozoan Trypanosoma cruzi, which is transmitted through the feces of triatomines. The subfamily Triatominae contains 19 genera and 154 species, all potential vectors of T. cruzi, except three fossils. The differentiation of the species of Triatominae is carried out mainly by morphological and molecular characters, in view of this premise this study aimed to identify the species of Rhodnius sp collected in the municipality of Taquarussu / MS in 2010. For this purpose, morphological and cytogenetic characters were used. The development of this study resulted in the description of R. taquarussuensis sp. n. Subsequently, by analyzing genetic divergences and species
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