Kaio Cesar Chaboli Alevi Citotaxonomia e evolução cromossômica na subfamília Triatominae São José do Rio Preto 2017 Campus de São José do Rio Preto Kaio Cesar Chaboli Alevi Citotaxonomia e evolução cromossômica na subfamília Triatominae Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Biociências, área de concentração Genética, junto ao Programa de Pós- Graduação em Biociências, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto. Financiadora: FAPESP – Proc. 2013/19764- 0 e CAPES Orientador: Profª. Drª. Maria Tercília Vilela de Azeredo Oliveira São José do Rio Preto 2017 Alevi, Kaio Cesar Chaboli. Citotaxonomia e evolução cromossômica na subfamília Triatominae / Kaio Cesar Chaboli Alevi -- São José do Rio Preto, 2017 386 f.: il., tabs. Orientador: Maria Tercília Vilela de Azeredo Oliveira Tese (doutorado) - Universidade Estadual Paulista “Júlio de Mesquita Filho”, Instituto de Biociências, Letras e Ciências Exatas 1. Biologia. 2. Biologia – Classificação. 3. Citogenética. 4. Barbeiro (Inseto). I. Universidade Estadual Paulista “Júlio de Mesquita Filho”, Instituto de Biociências, Letras e Ciências Exatas. II. Título. CDU – 576.3:575 Ficha catalográfica elaborada pela Biblioteca do IBILCE UNESP - Câmpus de São José do Rio Preto Kaio Cesar Chaboli Alevi Citotaxonomia e evolução cromossômica na subfamília Triatominae Tese apresentada como parte dos requisitos para obtenção do título de Doutor em Biociências, área de concentração Genética, junto ao Programa de Pós- Graduação em Biociências, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto. Financiadora: FAPESP – Proc. 2013/19764- 0 e CAPES Comissão Examinadora Profa. Dra. Maria Tercília Vilela de Azeredo Oliveira UNESP – São José do Rio Preto Orientador Profa. Dra. Claudia Regina Bonini Domingos UNESP – São José do Rio Preto Prof. Dr. Carlos Eduardo Almeida UNICAMP – Campinas Profa. Dra. Aline Rimoldi Ribeiro UNICAMP – Campinas Prof. Dr. Luis Lênin Vicente Pereira UNILAGO – São José do Rio Preto São José do Rio Preto 23 de outubro de 2017 Este trabalho foi realizado no Laboratório de Biologia Celular, do Departamento de Biologia, do Instituto de Biociências, Letras e Ciências Exatas de São José do Rio Preto – IBILCE/UNESP. Apoio financeiro na forma de bolsa de estudos financiada pela FAPESP (Processo nº 2013/19764-0) e CAPES. Dedicatória Dedico este trabalho aos meus pais, Maria de Lourdes Chaboli Alevi e Cid Nelson Alevi, exemplos de conduta, amor, fé e perseverança, que deixaram de realizar os seus sonhos para que eu pudesse realizar os meus. Agradecimentos Agradecimentos Em honra àqueles que melhoram minha essência, deixando um pouco de si, meu eterno agradecimento: Aos meus pais, Maria de Lourdes Chaboli Alevi e Cid Nelson Alevi, que, de forma singular, mostraram os verdadeiros valores do ser humano. Vocês são os meus bens mais preciosos. À minha orientadora, Profa. Dra. Maria Tercília Vilela de Azeredo Oliveira, pela amizade, confiança e companheirismo. Sou imensamente grato pela disponibilidade em me orientar no TCC da graduação, pois essa oportunidade foi fundamental para que eu conhecesse e me apaixonasse pelo estudo citogenético dos triatomíneos. Obrigado, ainda, pelos valiosos ensinamentos e pelo exemplo de conduta pessoal e profissional humanizado. À Profa. Dra. Claudia Regina Bonini Domingos e ao Prof. Dr. João Aristeu da Rosa, pela amizade e exemplo de profissionalismo. A confiança e a credibilidade que vocês depositam no meu trabalho são estimuladoras e revitalizadoras. Ao Prof. Dr. Luís Lênin Vicente Pereira, por quem tenho tremendo respeito, carinho e admiração, pelos diversos momentos prazerosos e produtivos. Obrigado pela disponibilidade de ter participado das minhas defesas de TCC e, agora, da minha defesa de tese. Obrigado, ainda, por toda a valorização profissional e pelo incentivo na carreira de docente e na pesquisa. Ao Prof. Dr. Carlos Eduardo Almeida e a Profa. Dra. Aline Rimoldi Ribeiro pela disponibilidade em participar da defesa da tese. Vocês são importantes referências no estudo dos triatomíneos que servem de inspiração para mim. À Elizabete Hiromi Habaro e Magda de Souza Alcantara Pires, pela grande amizade. Obrigado pelas horas de conversas e, principalmente, pelo grande incentivo a acreditar mais no meu potencial. A todos os meus ex-professores (pré-escola, ensino fundamental, ensino médio, ensino superior e pós-graduação) que atuam na mais bela das profissões e são os grandes responsáveis pela sólida formação que proporcionou o meu doutoramento. Ao Kauan Chaboli Alevi e ao Raul Eduardo Menezes Cardozo que me proporcionam dias mais felizes. À minha avó, Nair Bevilaqua Chaboli (in memorian), pessoa amável e sábia que, infelizmente, nos deixou há um mês, mas se mantém presente em nossos corações. Obrigado pelos ensinamentos sobre a importância e o poder do amor. As minhas tias Antônia, Lucimara, Uzimar e Dulcilei, que me proporcionam felicidades rotineiras e tornam a vida mais leve e fácil de ser vivida. Aos meus primos Elisângela, Junior, Carol, Beatriz, Elaine, Gislaine, Gianluka, Ana Carolina, Lisnaldo, Diego, Nayana, Juliana e Gabriela que, só de existirem, já contribuem para a minha felicidade. A todos os amigos que fiz durante a infância, o ensino fundamental, o ensino médio, a graduação e a pós-graduação. Felizmente são muitos nomes, mas cada um sabe do amor que tenho por vocês. A todos os alunos que passaram pelo Laboratório de Biologia Celular e que tive o prazer de coorientar. Cada um de vocês contribuiu imensamente para a minha formação pessoal e profissional. À Fundação de Amparo á Pesquisa do Estado de São Paulo (FAPESP) (Processo nº 2013/19764-0) e a CAPES pelas bolsas de doutorado. “Descobrir consiste em olhar para o que todo mundo está vendo e pensar uma coisa diferente” Roger Von Oech Resumo RESUMO A taxonomia e a sistemática dos triatomíneos baseiam-se, principalmente, em caracteres morfológicos. No entanto, existem espécies muito semelhantes ou até mesmo idênticas (espécies crípticas) do ponto de vista morfológico. Dessa forma, novas abordagens são necessárias para caracterizar um táxon e análises citogenéticas têm se mostrado como importantes ferramentas para a caracterização taxonômica e o conhecimento evolução desses vetores. Assim, o presente trabalho teve como objetivos principais: caracterizar os subcomplexos Brasiliensis, Rubrovaria, Matogrossensis, Maculata e Rubrofasciata; auxiliar na revalidação de Triatoma bahiensis; avaliar o status específico de Rhodnius montenegrensis, Rhodnius sp. (afim de R. neglectus), T. pintodiasi, T. lenti e T. sherlocki; analisar a relação evolutiva da tribo Rhodniini; caracterizar o fenômeno de persistência nucleolar na subfamília Triatominae e analisar a variabilidade genética intraespecífica de T. sordida proveniente do Brasil. Os resultados obtidos permitiram caracterizar citogeneticamente as espécies do subcomplexo Brasiliensis; revalidar T. bahiensis e corroborar que essa espécie, bem como T. lenti e T. petrocchiae são membros do subcomplexo Brasiliensis; corroborar o status específico de T. lenti, T. sherlocki e T. bahiensis pela presença de barreira de isolamento reprodutivo pós- zigótica (desmoronamento do híbrido); sugerir que durante a muda imaginal, os triatomíneos cessam a meiose e estimulam a espermiogênese para diminuir os gastos energéticos e garantir a chance de paternidade; caracterizar citogeneticamente os subcomplexos Matogrossensis e Rubrovaria e distingui-los dos outros subcomplexos da América do Sul; reagrupar as espécies dos subcomplexos Matogrossensis, Sordida, Maculata e Rubrovaria, com base na disposição do DNAr 45S; propor a criação de um novo subcomplexo (subcomplexo T. vitticeps), com base em dados fenotípicos e genotípicos; caracterizar citogeneticamente o subcomplexo Maculata e diferenciar T. wygodzinskyi e T. arthurneivai; diferenciar T. rubrofasciata de todas as outras espécies de triatomíneos pela citotaxonomia e cariossistemática; corroborar, por meio da análise cariotípica e das espermátides, que a tribo Rhodniini é um grupo monofilético; caracterizar a evolução cromossômica no grupo pallescens e relacionar a perda de heterocromatina com a ocupação de diferentes ambientes; corroborar o status específico de R. montenegrensis (com base na posição do DNAr 45S) e de T. pintodiasi (com base no comportamento meiótico e na distância genética para o gene mitocondrial 16S); demonstrar que R. neglectus não apresenta variação cromossômica intraespecífica e descrever uma nova espécies do gênero Rhodnius, a saber, R. taquarussuensis sp. n.; discorrer sobre a evolução cariotípica nas tribos Rhodniini, Triatomini e Cavernicolini; confirmar o fenômeno de persistência nucleolar como uma sinapomorfia de Triatominae e, por fim, demonstrar que T. sordida do Brasil não apresenta variação cromossômica intraespecífica e corroborar o status desses vetores como T. sordida sensu strito. Esses resultados mostraram-se de extrema importância para elucidar várias questões sobre a biologia, taxonomia e evolução dos triatomíneos. Palavras-chave: Citogenética. Taxonomia. Evolução, Triatomíneos. Doença de Chagas. Abstract ABSTRACT The taxonomy and systematics of triatomines are mainly based on morphological characters. However, there are very similar or even identical species (cryptic species) from the morphological point of view. Thus, new approaches are necessary to characterize a taxon and cytogenetic analyzes have been shown as important tools for the taxonomic characterization and the evolutionary knowledge of these vectors. Thus, the main objectives of this study were: characterize the Brasiliensis, Rubrovaria, Matogrossensis, Maculata and Rubrofasciata subcomplexes; assist in the revalidation of Triatoma bahiensis; evaluate the specific status of Rhodnius montenegrensis, Rhodnius sp., T. pintodiasi, T. lenti and T. sherlocki; analyze the evolutionary relationship of the Rhodniini tribe; characterize the nucleolar persistence phenomenon in the Triatominae subfamily and analyze the intraspecific genetic variability of T. sordida from Brazil. The results obtained allowed characterize cytogenetically the species of the Brasiliensis subcomplex; revalidate T. bahiensis and corroborate that this species, as well as T. lenti and T. petrocchiae are members of the Brasiliensis subcomplex; corroborate the specific status of T. lenti, T. sherlocki and T. bahiensis by the presence of a post-zygotic reproductive isolation barrier (collapse of the hybrid); suggest that during the imaginal molt, cell division is disrupted (reduce energy costs) and the differentiation into sperm is stimulated (ensure the paternity of the adult male); characterize cytogenetically the Matogrossensis and Rubrovaria subcomplexes and distinguish them from other subcomplexes of South American; regroup the species of the Matogrossensis, Sordida, Maculata and Rubrovaria subcomplexes, based on the arrangement of the 45S rDNA; propose the creation of a new subcomplex (T. vitticeps subcomplex), based on phenotypic and genotypic data; characterized cytogenetically the Maculata subcomplex and differentiate the T. arthurneivai and T. wygodzinskyi; differentiate T. rubrofasciata from all other triatomine species by cytotaxonomy and karyosystematics; corroborate, by means of karyotypic and spermatids analysis, the monophyly of the Rhodniini tribe; characterize the chromosomal evolution in the pallescens group and relate the loss of heterochromatin with the occupation of different environments; corroborate the specific status of R. montenegrensis (based on the position of the 45 rDNA) and T. pintodiasi (based on meiotic behavior and genetic distance for the 16S mitochondrial gene); demonstrate that R. neglectus does not present intraspecific chromosomal variation and describe a new species of the genus Rhodnius, namely, R. taquarussuensis sp. n.; discuss karyotype evolution in the Rhodniini, Triatomini and Cavernicolini tribes; confirming the nucleolar persistence phenomenon as a Triatominae synapomorphy, and, finally, demonstrate that T. sordida from Brazil does not present intraspecific chromosome variation and corroborate the status of these vectors as T. sordida sensu strito. These results were extremely important to elucidate several questions about the biology, taxonomy and evolution of triatomines. Keywords: Citogenetics. Taxonomy. Evolution. Triatomines. Chagas Disease. Sumário SUMÁRIO 1. INTRODUÇÃO................................................................................................................... 1 2. OBJETIVOS....................................................................................................................... 10 3. MATERIAL E MÉTODOS............................................................................................... 12 4. RESULTADOS (CAPÍTULOS)........................................................................................ 15 4.1 CAPÍTULO 1 - Spermatogenesis in Triatoma melanica Neiva and Lent, 1941 (Hemiptera, Triatominae)......................................................................................................... 16 4.2 CAPÍTULO 2 - Cytogenetic and morphologic approaches of hybrids from experimental crosses between Triatoma lenti Sherlock & Serafim, 1967 and T. sherlocki Papa et al., 2002 …………....................................................................................................... 23 4.3 CAPÍTULO 3 - Cytotaxonomy of the Brasiliensis subcomplex and the Triatoma brasiliensis complex (Hemiptera, Triatominae)…………………………...………………… 45 4.4 CAPÍTULO 4 - Revalidation of Triatoma bahiensis Sherlock & Serafim, 1967 (Hemiptera: Reduviidae) and phylogeny of the T. brasiliensis species complex.................................................................................................................................... 57 4.5 CAPÍTULO 5 - Hybrid collapse confirms the specific status of Triatoma bahiensis Sherlock & Serafim, 1967 (Hemiptera, Triatominae), an endemic species in Brazil......................................................................................................................................... 87 4.6 CAPÍTULO 6 - Reproductive biology of Triatoma brasiliensis (Hemiptera, Triatominae) during the imaginal molt……………….……………..……………………….. 93 4.7 CAPÍTULO 7 - Tracking the chromosomal diversification in Triatoma brasiliensis complex (Hemiptera, Triatominae)…….……………………………………………………. 98 4.8 CAPÍTULO 8 - Diploid chromosome set of kissing bug Triatoma baratai (Hemiptera, Triatominae)……………………………………………………………………………….… 104 4.9 CAPÍTULO 9 - Description of the diploid chromosome set of Triatoma pintodiasi (Hemiptera, Triatominae)………………………….………………………………………… 111 4.10 CAPÍTULO 10 - Mitochondrial gene confirms the specific status of Triatoma pintodiasi Jurberg, Cunha & Rocha, 2013 (Hemiptera, Triatominae), an endemic species in Brazil……………………………………………………………………..…………………. 132 4.11 CAPÍTULO 11 - Chromosomal characteristics and distribution of constitutive heterochromatin in the Matogrossensis and Rubrovaria subcomplexes……………………... 137 4.12 CAPÍTULO 12 - New arrangements on several species subcomplexes of Triatoma genus based on the chromosomal position of ribosomal genes (Hemiptera - Triatominae)… 148 4.13 CAPÍTULO 13 - Differentiation between Triatoma arthurneivai (Lent & Martins, 1940) and T. wygodzinskyi (Lent, 1951) (Hemiptera: Reduviidae: Triatominae) using cytotaxonomy………………………………………………………………………………... 175 4.14 CAPÍTULO 14 - Cytotaxonomy of the Maculata subcomplex (Hemiptera, Triatominae)…………………………………………………………………………………. 184 4.15 CAPÍTULO 15 - Triatoma vitticeps subcomplex (Hemiptera, Reduviidae, Triatominae): a new grouping of Chagas disease vectors from South America…………….. 190 4.16 CAPÍTULO 16 - Karyosystematic of Triatoma rubrofasciata (De Geer, 1773) (Hemiptera, Reduviidae, Triatominae)..................................................................................... 200 4.17 CAPÍTULO 17 - Cytogenetics characterization of Triatoma rubrofasciata (De Geer) (Hemiptera, Triatominae) spermatocytes and its cytotaxonomic application……………….. 210 4.18 CAPÍTULO 18 - Karyotype of Rhodnius montenegrensis (Hemiptera, Triatominae)... 218 4.19 CAPÍTULO 19 - Spermiotaxonomy of the tribe Rhodniini (Hemiptera, Triatominae).. 226 4.20 CAPÍTULO 20 - Chromosomal evolution in the pallescens group (Hemiptera, Triatominae)…………………………………………………………………………………. 231 4.21 CAPÍTULO 21 - Chromosomal divergence between Rhodnius montenegrensis Rosa et al. (2012) and R. robustus Larrousse, 1927 (Hemiptera, Triatominae)…………………… 240 4.22 CAPÍTULO 22 - Entoepidemiology of Chagas disease in the Western region of the State of São Paulo from 2004 to 2008, and cytogenetic analysis in Rhodnius neglectus (Hemiptera, Triatominae)……………………………………………………………………. 246 4.23 CAPÍTULO 23 - A new species of Rhodnius from Brazil (Hemiptera, Reduviidae, Triatominae)…………………………………………………………………………………. 260 4.24 CAPÍTULO 24 - New evidence on the genetic structure of Brazilian populations of Triatoma sordida (Stål, 1859) (Hemiptera, Triatominae), by means of chromosomal markers………………………………………………………………………………………. 293 4.25 CAPÍTULO 25 - Karyotype evolution of Chagas disease vectors (Hemiptera, Triatominae)…………………………………………………………………………………. 304 4.26 CAPÍTULO 26 - Nucleolar persistence in spermatogenesis of the genus Rhodnius (Hemiptera, Triatominae)….………………………………………………………………. 311 4.27 CAPÍTULO 27 - Nucleolar persistence: peculiar characteristic of spermatogenesis of the vectors of Chagas disease (Hemiptera, Triatomine)……………………………………... 319 4.28 CAPÍTULO 28 - Coloration of the testicular peritoneal sheath as a synapomorphy of triatomines (Hemiptera, Reduviidae)………………………………...………………………. 325 4.29 CAPÍTULO 29 - Study of the salivary glands in Triatominae (Hemiptera, Reduviidae, Triatominae): their color and application to the Chagas disease vector evolution……………………………………………………………………………………... 333 5. DISCUSSÃO…………...............………………………………………………………… 340 6. CONCLUSÕES………………………………………………………................……….. 350 7. REFERÊNCIAS………………………………………………………………...………... 353 1 Introdução 2 1. INTRODUÇÃO A doença de Chagas é um problema de saúde pública na América Latina e está se espalhando, cada vez mais, para novas regiões geográficas, como a Europa, a América do Norte, o Japão e a Austrália, associada, principalmente, à migração de pessoas infectadas pelo protozoário Trypanosoma cruzi (Chagas 1909) (Kinetoplastida, Trypanosomatidae), agente etiológico da doença de Chagas (GASCON; BERN; PINAZO, 2010; JACKSON; PINTO; PETT, 2014; WHO, 2017). Embora estima-se que oito milhões de pessoas estejam infectadas por T. cruzi e dez mil pacientes chagásicos morram anualmente, aproximadamente, 70 milhões de pessoas vivem em risco de contrair a doença de Chagas, tornando-a a principal causa de morte por doença parasitária na América Latina, bem como a principal causa da miocardiopatia infecciosa no mundo (MARTINS-MELO et al., 2012; CUCUNUBÁ et al., 2016; DNDi, 2017; WHO, 2017). A principal forma de transmissão do T. cruzi é vetorial, por meio das fezes de triatomíneos contaminados com o parasito, pois esses insetos são hematófagos e possuem o hábito de defecar durante o repasto sanguíneo (CHAGAS, 1909; WHO, 2017). Os triatomíneos pertencem à ordem Hemiptera, subordem Heteroptera, família Reduviidae e subfamília Triatominae (LENT; WYGODZINSKY, 1979). Atualmente, são admitidas 152 espécies na subfamília Triatominae, agrupadas em 18 gêneros e cinco tribos (Tabela 1) (GALVÃO, 2014; ALEVI et al., 2015a; MENDONÇA et al., 2016; SOUZA et al., 2016; ROSA et al., 2017). O Brasil apresenta grande diversidade de espécies (GALVÃO, 2014) que, em consequência de ações antrópicas (como o desmatamento e as queimadas), estão migrando para o ambiente domiciliar, processo conhecido como domiciliação (DIAS; SCHOFIELD, 1998; ALMEIDA et al., 2009). Embora existam espécies com maior ou menor grau de importância na transmissão da doença de Chagas [com destaque para Triatoma infestans, Rhodnius prolixus, T. dimidiata, Panstrongylus megistus e T. brasiliensis, que possuem importância mundial na transmissão da doença, assim como para T. infestans, T. brasiliensis, T. pseudomaculata, T. sordida e P. megistus, que apresentam maiores competências vetoriais no Brasil (GALVÃO, 2014)], todos os triatomíneos, de ambos os sexos e em qualquer fase do desenvolvimento, são considerados como potenciais vetores dessa doença. 3 Tabela 1 – Revisão do número de espécies da subfamília Triatominae. TRIBO GÊNERO NÚMERO DE ESPÉCIES Alberproseniini Alberprosenia 2 Bolboderini Belminus 8 Bolbodera 1 Microtriatoma 2 Parabelminus 2 Cavernicolini Cavernicola 2 Rhodniini Psammolestes 3 Rhodnius 21 Triatomini Dipetalogaster 1 Eratyrus 2 Hermanlentia 1 Linshcosteus 6 Meccus 6 Mepraia 3 Nesotriatoma 3 Panstrongylus 15 Paratriatoma 1 Triatoma 73 Total 152 Além da grande importância epidemiológica, os triatomíneos destacam-se, também, por constituírem um excepcional modelo para estudos celulares, pois apresentam características cromossômicas peculiares, como cromossomos holocêntricos (com cinetócoro difuso) (UESHIMA, 1966), meiose invertida para os cromossomos sexuais (em que a primeira fase da divisão meiótica é equacional, ou seja, separa as cromátides irmãs) (UESHIMA, 1966) e persistência nucleolar durante a meiose (TARTAROTTI; AZEREDO- OLIVEIRA, 1999). O fenômeno de persistência nucleolar é caracterizado pela presença do nucléolo ou de corpúsculos nucleolares durante todas as fases da meiose (TARTAROTTI; AZEREDO- OLIVEIRA, 1999). Esse comportamento nucleolar é incomum quando comparado aos outros eucariontes, uma vez que o nucléolo geralmente se fragmenta no final da prófase e só é reorganizado no final da anáfase/começo da telófase (GONZÁEZ-GARCIA et al., 1995). Até o momento, a nucleologênese de 22 espécies de triatomíneos (distribuídas nos gêneros Triatoma, Rhodnius e Panstrongylus) foi descrita (TARTAROTTI; AZEREDO-OLIVEIRA, 4 1999; MORIELLE; AZEREDO-OLIVEIRA, 2004; SEVERI-AGUIAR; AZEREDO- OLIVEIRA, 2005a; SEVERI-AGUIAR et al., 2006; MORIELLE; AZEREDO-OLIVEIRA, 2007; COSTA; AZEREDO-OLIVEIRA; TARTAROTTI, 2008; ALEVI et al., 2013a, 2014a; PEREIRA et al., 2015). Alevi et al. (2014a) sugerem que novas espécies e, principalmente, novos gêneros de triatomíneos sejam analisados para avaliar se esse fenômeno é uma sinapomorfia da subfamília Triatominae. A taxonomia e a sistemática dos triatomíneos baseiam-se, principalmente, em caracteres morfológicos (LENT; WYGODZINSKY, 1979; GALVÃO et al., 2003, 2014; ROSA et al., 2010, 2014). No entanto, existem espécies muito semelhantes ou até mesmo idênticas (espécies crípticas) do ponto de vista morfológico (MONTEIRO et al., 2000, 2003; PANZERA et al., 2006, 2015). Dessa forma, a utilização de novas abordagens é necessária para caracterizar um táxon e as análises citogenéticas têm se mostrado como importantes ferramentas para diferenciar esses insetos hematófagos (PÉREZ et al., 1992; PANZERA et al., 1995, 1007; ALEVI et al., 2012a, 2013b, 2015a, b; ALEVI; ROSA; AZEREDO- OLIVEIRA, 2014b). Os estudos citogenéticos na subfamília Triatominae foram iniciados com a descrição do cariótipo de Triatoma sanguisuga (Leconte, 1855) (PAYNE, 1909). Todavia, apenas em 1950 os estudos citogenéticos foram retomados e novos cariótipos foram descritos (SCHREIBER; PELLEGRINO, 1950). Ueshima, em 1966, descreveu o conjunto cromossômico diplóide e o comportamento meiótico de vinte novas espécies, propôs que 22 cromossomos (20 autossomos + XY) é o número tipo para a subfamília Triatominae e ressaltou que os estudos citogenéticas são importantes ferramentas taxonômicas para esses vetores, iniciando, assim, a citotaxonomia dos triatomíneos. Atualmente, 88 espécies apresentam o cariótipo descrito que varia de 21 a 25 cromossomos (Tabela 2) (ALEVI et al., 2015a). Schofield; Galvão (2009) propuseram o agrupamento dos triatomíneos em complexos e subcomplexos específicos (Tabela 3). Esses autores compilaram diferentes informações de várias espécies, como, por exemplo, caracteres morfológicos, distribuição geográfica e dados genéticos. No entanto, foram utilizados os dados disponíveis na literatura até 2009 e, com a publicação de novos resultados, alguns agrupamentos foram questionados (ALEVI et al., 2012a; GARDIM et al., 2014; JUSTI et al., 2014). 5 Tabela 2 – Espécies da subfamília Triatominae com o cariótipo descrito. Cariótipo (2n) Gênero: espécie (s) 21 = 18A + X1X2Y Panstrongylus: megistus Triatoma: nitida 22 = 20A + XY Psammolestes: coreodes, tertius Rhodnius: brethesi, colombiensis, domesticus, ecuadoriensis, milesi, montenegrensis, nasutus, neglectus, neivai, pallescens, pictipes, prolixus, robustus, stali Dipetalogaster: maximus Paratriatoma: hirsuta Triatoma: arthurneivai, baratai, brasiliensis (b. brasiliensis, b. macromelanosoma), carcavalloi, circummaculata, costalimai, delpontei, garciabesi, guasayana, guazu, infestans, juazeirensis, jurbergi, klugi, lectularia, lenti, maculata, matogrossensis, melanica, patagonica, petrocchiae, pintodiasi, platensis, pseudomaculata, rubrovaria, sherlocki, sordida, vandae, williami, wygodzinskyi 23 = 20A + X1X2Y Belminus: herreri, corredori Eratyrus: cuspidatus, mucronatus Meccus: bassolsae, longipennis, mazzottii, pallidipennis, phyllosoma, picturatus Mepraia: gajardoi, parapatrica, spinolai Nesotriatoma: bruneri, flavida Panstrongylus: chinai, geniculatus, howardi, lignarius, rufotuberculatus, tupynambai Triatoma: barberi, dimidiata (d. capita, d. dimidiata, d. maculipennis), gerstaeckeri, hegneri, mexicana, peninsularis, protracta, rubida, ryckmani, sanguisuga, sinaloensis, tibiamaculata 24 = 20A + X1X2X3Y Panstrongylus: lutzi Triatoma: eratyrusiformis, melanocephala, vitticeps 25 = 22A + X1X2Y Triatoma: rubrofasciata 6 Tabela 3 – Complexos e subcomplexos de triatomíneos. COMPLEXOS SUBCOMPLEXOS Phyllosoma Dimidiata Phyllosoma (=Meccus) Flavida (=Nesotriatoma) Rubrofasciata Protracta Lecticularia Dispar Infestans Brasiliensis Infestans Maculata Matogrossensis Rubrovaria Sordida Spinolai (=Mepraia) O subcomplexo Brasiliensis foi, inicialmente, proposto por Schofield; Galvão (2009) com nove espécies, a saber, T. brasiliensis Neiva, 1911, T. juazeirensis Costa & Félix, 2007, T. melanica Neiva & Lent, 1941, T melanocephala Neiva & Pinto, 1923, T. petrocchiae Pinto & Barreto, 1925, T. lenti Sherlock & Serafim, 1967, T. sherlocki Papa et al., 2002, T. tibiamaculata (Pinto, 1926) e T. vitticeps (Stål, 1859). No entanto, Alevi et al. (2012a), por meio de dados cariossistemáticos, propuseram a exclusão de T. tibiamaculata, T. melanocephala e T. vitticeps do subcomplexo Brasiliensis, por apresentarem cariótipos diferentes dos observados para os outros membros do subcomplexo (derivado da fragmentação do cromossomo sexual X). Esses resultados foram corroborados por análises citogenéticas (ALEVI; ROSA; AZEREDO-OLIVEIRA, 2014b; ALEVI et al., 2014c), morfométricas (OLIVEIRA et al., 2017) e filogenéticas (GARDIM et al., 2014; OLIVEIRA et al., 2017). Costa e colaboradores, com base em análises isoenzimáticas (COSTA et al., 1997a), morfológicas (COSTA et al., 1997b; COSTA et al., 2013), da superfície exocorial do ovo (COSTA et al., 1997b), biológicas (COSTA; SILVA, 1998a), ecológicas (COSTA et al., 1998b), genéticas (MONTEIRO; COSTA; BEARD, 1999), citogenéticas (PANZERA et al., 2000), biogeográficas (COSTA; PETERSON; BEARD, 2002), de cruzamentos experimentais (COSTA et al., 2003), moleculares (MONTEIRO et al., 2004) e da morfometria dos testículos (FREITAS et al., 2008) propuseram que o complexo T. brasiliensis é um grupo monofilético 7 formado pelas espécies T. brasiliensis, T. melanica e T. juazeirensis e pela subespécie T. b. macromelanosoma Galvão, 1965. Mendonça et al. (2009), por meio de reconstrução filogenética com dois genes mitocrondriais (CytB e 16S), sugeriram que T. sherlocki deveria fazer parte do complexo T. brasiliensis. A inclusão foi confirmada, posteriormente, por análises citogenéticas (ALEVI et al., 2013b) e cruzamento híbrido experimental (CORREIA et al., 2013). Além disso, foi sugerido que T. lenti venha a ser o sexto membro do complexo (ALEVI et al., 2013b) e, recentemente, com base em dados moleculares (genes mitocondriais 12S, 16S, COI e Cytb) e dados morfométricos (morfométrica geométrica de asas e cabeças), T. petrocchiae foi agrupado ao complexo T. brasiliensis (OLIVEIRA et al., 2017). Espécies do subcomplexos Matogrossensis (T. baratai Carcavallo & Jurberg, 2000, T. costalimai Verano & Galvão, 1958, T. deaneorum Galvão, Souza & Lima, 1967, T. guazu Lent & Wygodzinsky, 1979, T. jurbergi Carcavallo, Galvão & Lent, 1998, T. matogrossensis Leite & Barbosa, 1953, T. vandae Carcavallo et al., 2002 e T. williami Galvão, Souza & Lima, 1965) e Rubrovaria [T. carcavalloi Jurberg, Rocha & Lent, 1998, T. circummaculata (Stål, 1859) , T. klugi Carcavallo et al., 2001, T. limai Del Ponte, 1929, T. oliverai (Neiva, Pinto & Lent, 1939) e T. rubrovaria (Blanchard, 1843)] (SCHOFIELD; GALVÃO, 2009) foram, inicialmente, agrupadas no complexo oliverai (NOIREAU et al., 2002). Dados moleculares demonstram que apenas o subcomplexo Rubrovaria forma um grupo monofilético (JUSTI et al., 2014), ressaltando a necessidade de novos estudos para as espécies do subcomplexo Matogrossensis. Além disso, duas espécies foram descritas e agrupadas nesses complexos, a saber, T. jatai Gonçalves et al., 2013 (subcomplexo Matogrossensis) (GONÇALVES et al., 2013) e T. pintodiasi Jurberg et al., 2013 (subcomplexo Rubrovaria) (JURBERG et al., 2013). O subcomplexo Maculata é composto pelas espécies T. maculata (Erichson, 1848), T. pseudomaculata Corrêa & Espínola, 1964, T. arthurneivai Lent & Martins, 1940 e T. wygodzinskyi Lent 1951 (SCHOFIELD; GALVÃO, 2009). Por meio de morfometria da cabeça e do tórax, Santos et al. (2007) alertaram para um possível problema taxonômico envolvendo T. arthurneivai e T. wygodzinskyi. Recentes estudos de morfometria geométrica demonstraram que os insetos capturados fora da região da Serra do Cipó são espécimes de T. wygodzinskyi (CARBAJAL-DE-LA-FUENTE et al., 2010). Os autores relatam que as populações de T. arthurneivai de São Paulo, estudadas por mais de quarenta anos por muitos autores (CORRÊA; ALVES; PASCALE, 1962; CORRÊA; PINTO ALVES; NODA, 1965; PINTO ALVES; NODA, 1964; FORATTINI; JUAREZ; RABELLO, 1968; FORATTINI; 8 RABELLO; PATTOLI, 1972; JUAREZ et al., 1970; BARRETTO; RIBEIRO, 1981; HYPSA et al., 2002; PAULA; DIOTAIUTI; SCHOFIELD, 2005; ROSA et al., 2005; SANTOS et al., 2007; BARGUES et al., 2008), correspondem a T. wygodzinskyi. A citotaxonomia dos triatomíneos é uma importante ferramenta para resolver problemas sistemáticos e taxonômicos: Jurberg et al. (1998) e Frías-Lasserre (2010), por exemplo, utilizaram dados citogenéticos para revalidar a espécie T. garciabesi Carcavallo et al., 1967 e descrever Mepraia parapatrica Frías-Lasserre, 2010; Panzera et al. (1995, 1997), Santos et al. (2007), Alevi; Rosa; Azeredo-Oliveira (2014b) e Alevi et al. (2015b), por meio da análise citogenética de prófases iniciais, caracterizaram as espécies dos subcomplexos Infestans, Sordida, Maculata, Matogrossensis, Rubrovaria e Brasiliensis, respectivamente. Além disso, a citotaxonomia também pode auxiliar na diferenciação de espécies morfologicamente relacionadas (ALEVI et al., 2013c, d, 2015b) e é importante ferramenta para estudar variação intraespecífica nos triatomíneos (Tabela 4). Tabela 4 – Variação cromossômica intraespecífica na subfamília Triatominae. ESPÉCIES VARIAÇÃO CROMOSSÔMICA INTRAESPECÍFICA REFERÊNCIAS R. ecudoriensis Presente Pita et al. (2013) R. pallescens Presente Gómez-Palacio et al. (2008) P. geniculatus Presente Crossa et al. (2002) T. dimidiata Presente Panzera et al. (2006) T. infestans Presente Panzera et al. (2004, 2012) T. sordida Presente Panzera et al. (1997, 2015) R. neglectus Ausente Alevi et al. (2015c) R. prolixus Ausente Ravazi et al. (2017) R. nasutus Ausente Ravazi et al. (2017) P. megistus Ausente Alevi et al. (2015d) T. brasiliensis Ausente Panzera et al. (2000) T. pseudomaculata Ausente Imperador et al. (2016) A tribo Rhodniini é composta por 23 espécies, sendo 20 do gênero Rhodnius e três do gênero Psammolestes (ALEVI et al., 2015a; SOUZA et al., 2016). Esses insetos apresentam homogeneidade cariotípica (PITA et al., 2013, ALEVI et al., 2015a) e apenas quatro espécies 9 apresentam heterocromatina constitutiva nos autossomos (PITA et al., 2013). Além disso, Rhodnius spp. são muito semelhantes do ponto de vista morfológico (MONTEIRO et al., 2000) e, com base em análises moleculares, foram agrupados em grupos ou complexos monofiléticos (MONTEIRO et al., 2003; ROSA et al., 2012; JUSTI; GALVÃO, 2017). O grupo pallescens, por exemplo, é formado pelas espécies R. colombiensis Mejia, Galvão & Jurberg, 1999, R. pallescens Barber, 1932 e R. ecuadoriensis Lent & León, 1958 (SCHOFIELD; DUJARDIN, 1999) e Abad-Franch; Monteiro (2007) sugerem que análises citogenéticas podem auxiliar na compreensão da taxonomia e evolução desse importante grupo de vetores. Análises citogenéticas moleculares também são importantes ferramentas que auxiliam no entendimento evolutivo dos triatomíneos. Panzera et al. (2012), por meio da técnica de hibridização in situ (FISH) com sonda de DNA ribossômico (DNAr) 45S, apresentaram o padrão evolutivo das Regiões Organizadoras Nucleolares (RONs) para 38 espécies de triatomíneos. Até então, poucos estudos com FISH tinham sido realizados na subfamília Triatominae, todos comandados por Azeredo-Oliveira (SEVERI-AGUIAR; AZEREDO- OLIVEIRA, 2005b; SEVERI-AGUIAR et al., 2006; MORIELLE-SOUZA; AZEREDO- OLIVEIRA, 2007; BARDELLA et al., 2010). Pita et al. (2013) analisaram a disposição das RONs em 10 espécies da tribo Rhodniini e observaram que em Rhodnius e Psammolestes as RONs ficam restritas apenas aos cromossomos sexuais (X, Y ou X e Y), diferente, por exemplo, das espécies do gênero Triatoma (tribo Triatomini) em que as RONs podem ser observadas nos cromossomos sexuais e/ou nos autossomos (PANZERA et al., 2012). Apesar dos conhecimentos relatados sobre as características citogenéticas desses insetos hematófagos de importância medico-sanitária, ainda é necessário ampliar os estudos citotaxonômicos na subfamília Triatominae. Dessa forma, os dados apresentados mostram-se importantes, em diferentes aspectos, para auxiliar na taxonomia e sistemática, assim como no entendimento da história evolutiva desses vetores, pois a doença de Chagas, embora descrita há mais de 100 anos, é a doença parasitária com o maior índice de mortalidade nas Américas e o controle vetorial é a principal forma de minimizar a incidência dessa enfermidade. Assim, a caracterização citotaxonômica desses vetores pode gerar subsídios para auxiliar na atividade dos agentes de saúde, uma vez que a correta classificação dos triatomíneos permite que as principais espécies vetoras sejam foco específico dos programas de controle de vetores. 10 Objetivos 11 2. OBJETIVOS a) Caracterizar a espermatogênese das espécies dos subcomplexos Brasiliensis, Rubrovaria, Matogrossensis, Maculata e Rubrofasciata, por meio de técnicas citogenéticas clássicas e moleculares, com ênfase citotaxonômica. b) Descrever citogeneticamente uma população de T. lenti com diferença no padrão cromático, a fim de auxiliar na possível revalidação de T. bahiensis. c) Descrever citogeneticamente a espécie R. montenegrensis, por meio de técnicas citogenéticas clássicas e moleculares, a fim de contribuir com a taxonomia da tribo Rhodniini. d) Avaliar a relação evolutiva das espécies do grupo pallescens (R. pallescens, R. colombiensis e R. ecuadoriensis), por meio de análises citogenéticas. e) Descrever a espermiogênese das espécies da tribo Rhodniini, com foco citotaxonômico. f) Descrever citogeneticamente Rhodnius sp. e comparar com diferentes populações de R. neglectus (espécie afim), com o intuito de auxiliar na descrição da nova espécie. g) Descrever o comportamento nucleolar de triatomíneos, com ênfase no fenômeno de persistência nucleolar. h) Estudar a variabilidade cromossômica intraespecífica em populações de T. sordida do Brasil, a fim de avaliar o fenômeno de especiação críptica. 12 Material e Métodos 13 3. MATERIAL E MÉTODOS 3.1. Obtenção e procedência das espécies As espécies de triatomíneos utilizadas são provenientes do “Insetário de Triatominae”, instalado na Faculdade de Ciências Farmacêuticas (FCFAR/UNESP), Câmpus de Araraquara, São Paulo, sob coordenação do Prof. Dr. João Aristeu Rosa; do Insetário do Laboratório Nacional e Internacional de Referência em Taxonomia de Triatomíneos, instalado na FIOCRUZ, Rio de Janeiro, coordenado pelo Dr. José Jurberg; e do Insetário do Laboratório de Triatomíneos e Epidemiologia da Doença de Chagas, instalado no CPqRR/FIOCRUZ, Minas Gerais, coordenado pela Dra. Liléia Diotaiuti. 3.2 Órgão analisado Foram analisados os túbulos seminíferos de, pelo menos, três machos adultos de cada espécie de triatomíneo, pois a espermatogênese em Hemiptera é contínua na fase adulta, permitindo que os diferentes estágios da espermatogênese (espermatocitogênenese, meiose e espermiogênese) sejam estudados. 3.3 Fixação dos túbulos seminíferos Após a dissecção, os testículos foram transportados para uma solução fisiológica (Demerec), onde foi realizada a limpeza e individualização dos túbulos seminíferos. Posteriormente, cada túbulo foi colocado em um recipiente de vidro contendo três partes de metanol para uma de ácido acético (3:1), e conservado no freezer à -20°C. 3.4 Preparação das lâminas Os túbulos seminíferos foram retirados da solução fixadora e colocados em uma lâmina, onde foram realizados dois banhos de água destilada, por cinco minutos cada. Posteriormente, foi acrescentada uma gota de ácido acético 45%, durante 10 minutos. Após esse período, o túbulo seminífero foi dilacerado e sobre esse material foi colocada uma lamínula para a realização do esmagamento celular. A remoção da lamínula ocorreu em nitrogênio líquido. 3.5 Técnicas citogenéticas convencionais 3.5.1 Orceína Lacto-Acética (DE VAIO et al., 1985, com modificações de acordo com ALEVI et al., 2012a): Estudo do cariótipo, da espermatogênese e da espermiogênese. 14 3.5.2 Bandamento C (SUMNER, 1972): Estudo do padrão de heterocromatina constitutiva. 3.5.3 Impregnação por íons prata (HOWELL e BLACK, 1980): Estudo do comportamento nucleolar. 3.6 Técnica citogenética molecular 3.6.1 Hibridização in situ (FISH) (PANZERA et al., 2012): Estudo da disposição das RONs. 3.6.2 CMA3/DAPI (SCHIMID, 1980, com modificações de acordo com SEVERI-AGUIAR et al., 2006): Estudo da riqueza de AT e CG no material genético. 3.7 Forma de análise dos resultados O material submetido às técnicas citogenéticas convencionais foi analisado ao microscópio de luz Jenaval (Zeiss), acoplado à câmera digital e ao sistema analisador de imagens Axio Vision LE 4.8 (Copyright ©2006-2009 Carl Zeiss Imaging Solutions Gmb H). O material submetido à técnica citogenética molecular foi analisado em microscopia de fluorescência Olympus BX-FLA. 15 Resultados 16 4. RESULTADOS (CAPÍTULOS) 4.1 Capítulo 1 (Artigo científico publicado na revista Journal of Vector Ecology FI: 1,47) ALEVI, K. C. C.; ROSA, J. A.; AZEREDO-OLIVEIRA, M. T. V. Spermatogenesis in Triatoma melanica Neiva and Lent, 1941 (Hemiptera, Triatominae). Journal of Vector Ecology, v. 39, p. 231-233, 2014. Spermatogenesis in Triatoma melanica Neiva and Lent, 1941 (Hemiptera, Triatominae) SCIENTIFIC NOTE Costa and collaborators proposed the Triatoma brasiliensis complex using many approaches, including egg morphology (Costa et al. 1997a), morphometry of the testis (Freitas et al. 2008), hybrid cross (Costa et al. 2003), isoenzymes (Costa et al. 1997b), molecular data (Monteiro et al. 2004), morphological data (Costa et al. 1997a), biological data, and ecological data (Costa et al. 1998). This complex is comprised of the subspecies T. b. brasiliensis and T. b. macromelanosoma, and also the species T. juazeirensis and T. melanica. By means of phylogenetic reconstruction, Mendonça et al. (2009) proposed the inclusion of T. sherlocki to this complex. This inclusion was recently confirmed through the use of both cytogenetic analysis (Alevi et al. 2013a) and cross-mating experiments (Correia et al. 2013). Although the T. brasiliensis complex is a monophyletic group, T. melanica is considered an independent evolutionary unit and is thought to be the most differentiated form of the complex, with a genetic composition that is incompatible and hybrids that are inviable with other members of the T. brasiliensis complex (Costa et al. 2003). In 1941, this species was described by Neiva and Lent as a subspecies of T. brasiliensis (T. b. melanica). Using different studies such as morphology, biology, ecology, crossing experiments, allozymes, and mtDNA sequences, Costa et al. (2006) increased the specific status of T. b. melanica to T. melanica (Costa et al. 2006). Because T. melanica has only been collected in natural ecotopes and was considered to be important in the maintenance of the wild cycle of Trypanosoma cruzi (an etiologic agent of Chagas disease) (Costa 1999), all approaches used to study biology and reproduction of this 17 vector are important. Thus, the present study aims to describe the spermatogenesis of T. melanica. Seminiferous tubules of two adult males of T. melanica from the Triatominae Insectarium within the Department of Biological Sciences, in the College of Pharmaceutical Sciences, at Sao Paulo State University’s Araraquara campus, Brazil (FCFAR/UNESP) were first shredded, smashed, and set on a slide in liquid nitrogen. They were then stained using the cytogenetic technique of lacto-acetic orcein (De Vaio et al. 1985, with modifications according to Alevi et al. 2012). Spermatogenesis consists of three different phases: spermatocytogenesis, which is a phase of proliferation; meiosis, which is the multiplication phase; and spermiogenesis, which is the differentiation phase (Johnson et al. 1997). Spermatocytogenesis was represented only by mitotic metaphase (Figure 1). Note all 20 autosomes and two sex chromosomes (arrows). All stages of meiosis were observed (Figure 2), including the diffuse stage (prophase) (Figure 2A), metaphase I in polar (Figure 2B) and lateral view (Figure 2C), anaphase I (Figure 2D) and II (Figure 2E) and telophase (Figure 2F). In addition, the elongation of haploid cells was observed during spermiogenesis (Figure 3). Analyses of mitotic and meiotic metaphases made it possible to confirm the karyotype described for T. melanica (2n = 20A + XY) (Panzera et al. 2000). However, T. melanica presented a peculiar behavior during mitotic metaphase: the chromatids of sex chromosomes were visible. All species of the T. brasiliensis complex have the same chromosomal characteristics: namely, 22 chromosomes (2n = 20A + XY) with heterochromatic blocks at one or both chromosomal ends of all autosomal pairs and a large heterochromatic chromocenter formed by the association of both sex chromosomes plus two autosomal pairs and many heterochromatic blocks dispersed inside the nucleus (Panzera et al. 2000; Alevi et al. 2013a). The analysis of prophase revealed the same results described when the C-banding technique was used by Panzera et al. (2000). Thus, our analysis confirms the association of T. melanica with the species of the complex. Through molecular data (16S and Cytb), T. melanica was considered to be a sister to T. sherlocki (Mendonça et al. 2009). Alevi et al. (2013b, c) propose the analysis of spermatids as a cytotaxonomic tool that can be used to compare related species. During spermiogenesis of T. melanica, two heteropycnotic filaments were noted in each of the haploid cells. These characteristics are quite different from those described for T. sherlocki, which presents early 18 spermatids and which possesses a heteropyknotic corpuscle that becomes a the periphery filament during cell elongation (Alevi et al. 2013b). Thus, this paper describes the spermatogenesis of T. melanica, confirms the relationship this species shares with members of the T. brasiliensis complex, and differentiates T. melanica from T. sherlocki (sister species). Although morphometric analyses were not performed, we noted that cells of T. melanica are relatively larger than those of other members of complex. In addition to genetic load, this phenomenon may be a factor that is related to reproductive incompatibility in experimental hybrid crosses, thus representing an important pre-zygotic barrier between these hematophagous insects. REFERENCES CITED Alevi, K.C.C., P.P. 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Inv. Rep. Dev. 58: 9-12. Correia, N., C.E. Almeida, V. Lima-Neiva, M. Gumiel, L.L. Dornak, M.M. Lima, L.M. Medeiros, V.J. Mendonça, J.A. Rosa and J. Costa. 2013. Cross-mating experiments detect reproductive compatibility between Triatoma sherlocki and other members of the Triatoma brasiliensis species complex. Acta Trop. 128:162-167. Costa, J. 1999. The synanthropic process of Chagas disease vectors in Brazil, with special attention to Triatoma brasiliensis Neiva, 1911 (Hemiptera, Reduviidae, Triatominae) population, genetical, ecological, and epidemiological aspects. Mem. Inst. Oswaldo Cruz. 94: 239–241. 19 Costa, J., A. Argolo and M. Felix. 2006. Redescription of Triatoma melanica Neiva & Lent, 1941, new status (Hemiptera: Reduviidae: Triatominae). Zootaxa. 1385: 47-58. Costa, J., C.E. Almeida, J.P. Dujardin and C.B. Beard. 2003. Crossing Experiments Detect Genetic Incompatibility among Populations of Triatoma brasiliensis Neiva, 1911 (Heteroptera, Reduviidae, Triatominae). 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Freitas, S.P.C., J.E. Santos-Mallet, J. Costa, A.L.B. Souza, J.E. Serrão and T.C.M. Gonçalves. 2008. A comparative study of testis follicles in species of Triatoma (Hemiptera, Triatominae). An. Biol. 58: 227-233. Johnson, L., T.L. Blanchard, D.D. Varner and W.L. Scrutchfield. 1997. Factors affecting spermatogenesis in the stallion, Theriogenology. 48: 1199-1216. Mendonça, V.J., M.T.A. Silva, R.F. Araujo, J.M. Junior, M.B. Junior, C.E. Almeida, J. Costa, M.A.S. Graminha, R.M.B. Cicarelli and J.A. Rosa. 2009. Phylogeny of Triatoma sherlocki (Hemiptera: Reduviidae: Triatominae) inferred from two mitochondrial genes suggests its location within the Triatoma brasiliensis complex, Am. J. Trop. Med. Hyg. 81: 858-864. Monteiro, F.A., M.J. Donnelly, C.B. Beard and J. Costa. 2004. Nested clade and phylogeographic analyses of the Chagas disease vector Triatoma brasiliensis in Northeast Brazil. Mol. Phylogenet. Evol. 32: 46-56. Panzera, F, R. Pérez, P. Nicolini, P. Hornos, J. Costa, E. Borges, L. Diotaiuti and J. Scholfield. 2000. Chromosome homogeneity in populations of Triatoma brasiliensis Neiva 1911 (Hemiptera – Reduviidae – Triatominae). Cad Saúde Pública. 16: 83-88. 20 Figure 1. Mitotic metaphase of Triatoma melanica. (A) Note 20 autosomes and the two sex chromosomes (arrows). Bar: 10 µm. 21 Figure 2. Stages of meiosis of Triatoma melanica. (A) Diffuse stage (prophase). Note the large heterochromatic chromocenter (arrow) and many heterochromatic blocks dispersed within the nucleus. (B, C) Metaphase I. Note the karyotype 2n = 20A + XY. (D) Anaphase I. Note the separation of homologous chromosomes in autosomes and the sister chromatids in sexual chromosomes (arrow). (E) Anaphase II. (F). Telophase. Bar: 10 µm. 22 Figure 3. Spermiogenesis of Triatoma melanica. (A-D) Note the presence of two heteropycnotic filaments during the elongation of haploid cells. Bar: 10 µm. 23 4.2 Capítulo 2 (Artigo científico publicado na revista Infection, Genetics and Evolution FI 2,88) MENDONÇA, V. J.; ALEVI, K. C. C.; MEDEIROS, L. M. O.; NASCIMENTO, J. D.; AZEREDO-OLIVEIRA, M. T. V.; ROSA, J. A. Cytogenetic and morphologic approaches of hybrids from experimental crosses between Triatoma lenti Sherlock & Serafim, 1967 and T. sherlocki Papa et al., 2002 (Hemiptera: Reduviidae). Infection, Genetics and Evolution, v. 26, p. 123-131, 2014. Cytogenetic and morphologic approaches of hybrids from experimental crosses between Triatoma lenti Sherlock & Serafim, 1967 and T. sherlocki Papa et al., 2002 (Hemiptera: Reduviidae) Abstract The reproductive capacity between Triatoma lenti and T. sherlocki was observed with the aim to verify the fertility and viability of the offspring, and by cytogenetic, morphologic and morphometric approaches to analyze the differences inherited. Experimental crosses were performed in both directions. The fertility rate of the eggs in crosses involving T. sherlocki females was 65% and 90% in F1 and F2 offspring, respectively, while in reciprocal crosses was 7% and 25% in F1 and F2 offspring, respectively. The cytogenetic analyses of the male meiotic process of the hybrids were performed using the lacto-acetic orcein, C-banding and Feulgen techniques. The male F1 offspring presented a normal chromosome behavior, similar as reported in parental species. However, cytogenetic analysis of F2 offspring showed errors in chromosome pairing. This post-zygotic isolation that prevents hybrid in nature may be collapse of the hybrid. This phenomenon is due to a genic deregulation occurred in the chromosomes of F1. The results were similar in the hybrids the both crosses. Morphological approaches, as color and size of connexive and presence of red rings on the legs were similar to T. sherlocki, while the wings size was similar to T. lenti in F1 offspring. The eggshells showed characteristics similar to species of origin, whereas the median process of the pygophore showed intermediate characteristics in the F1, and segregating pattern in F2 offspring. Geometric morphometric techniques of the wings showed that both F1 and F2 offspring were similar to T. lenti. Our studies about the reproductive capacity in experimental hybrids between T. lenti and T. sherlocki confirm that both species are evolutionarily closed 24 hence included in the brasiliensis subcomplex. The extremely reduced fertility observed in the F2 hybrids confirming the status specific of species analyzed. 1. Introduction Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, which is transmitted to mammalian hosts primarily through feces during the blood meal of insect of the Triatominae subfamily (Chagas, 1909; Lent and Wygodzinsky, 1979). It has been estimated that there are 7 to 8 million people infected by T. cruzi in Latin America and 25 million are exposed to infection (Moncayo and Silveira, 2009; Coura and Viñas, 2010; WHO, 2013). Triatoma sherlocki was first studied by Cerqueira (1982) that performed experimental crosses between T. sherlocki with T. brasiliensis, T. infestans and T. lenti. Experimental crosses with T. lenti and T. infestans has not obtaining hybrids, whereas obtaining fertile hybrids with T. brasiliensis and T. sherlocki, classifying the latter primarily as a subspecies of T. brasiliensis, designated T. b. santinacensis (Cerqueira, 1982). Morphological analysis of genital structures, pronotum and scutelum, suggest T. sherlocki related to T. lenti however peculiar characteristics as the reduced hemelytra, rings of color orange redish in the femur, allow the description of T. sherlocki as a valid species (Papa et al., 2002). According to Schofield and Galvão (2009) the brasiliensis subcomplex comprises nine species distributed in South America: T. brasiliensis, T. juazeirensis, T. lenti, T. melanica, T. melanocephala, T. petrocchiae, T. sherlocki, T. tibiamaculata and T. vitticeps. The parameters used to group these species were principally morphological and geographical distribution. By means of cytogenetic data, Alevi et al. (2012a) proposes the exclusion of T. melanocephala, T. tibiamaculata and T. vitticeps of brasiliensis subcomplex and Alevi et al. (2012b, 2013a) confirm the inclusion of T. lenti. Mendonça et al. (2009), by analyses with mitochondrial genes and Alevi et al. (2013a), by cytogenetic analysis support the inclusion of the T. sherlocki in the brasiliensis subcomplex. Experimental crosses between subcomplex brasiliensis members (as defined by Costa et al., 2013) showed hybridization potential and reproductive compatibility under laboratory conditions (Costa et al., 2003; Almeida et al., 2012; Correia et al., 2013). Human modifications to ecological landscapes both may increase epidemiologic risks and facilitate endemic disease emergence and create new suitable environments for 25 integration and mating between species, potentially resulting in natural hybrids (e.g., T. infestans with T. rubrovaria) (Salvatella et al., 1990). Considering that triatomine hybridization (a) allows formulation of hypotheses concerning origin and divergence of species, (b) may help understand the systematics of the group (Pérez et al., 2005), and (c) experimental hybridizations have enabled quantitative analyses of taxonomic relationships correlated with degrees of morphological similarities between species (Usinger, 1966), the present investigation was aimed to determine the reproductive compatibility between T. sherlocki and T. lenti and to analyze by cytogenetic and morphologic approaches the characteristics of the hybrids from crosses experimental of these species. 2. Methods 2.1 Insects Specimens used in these crosses were obtained from colonies established for at least six generations with Triatominae from non-overlapping areas. T. sherlocki (Fig. 1A) samples used in this study were taken from a colony generated from 26 specimens collected in a wild and rock upland environment, near the village of Santo Inácio in the district of Gentio do Ouro, state of Bahia, Brazil, in 2003. T. lenti (Fig. 1B) samples are from a colony generated from 23 specimens adults and 5 nymphs collected in peridomestic environment in the rural village of Macaúbas/BA, state of Bahia, Brazil, in 2009. Both colonies are maintained at the Triatominae Insectarium of the São Paulo State University (UNESP, Araraquara/SP, Brazil). 2.2 Experimental crosses T. sherlocki and T. lenti were crossed in both directions: T. lenti females × T. sherlocki males and T. sherlocki females × T. lenti males. The insects were sexed at the 5th instar nymphs and males and females were kept separately until they reach the adult stage, in order to get adults virgins (Martínez-Ibarra et al., 2011). For those crosses, three couples from each set were placed in plastic jars (5 cm diameter x 10 cm height) and maintained at room temperature. The fertility rate and oviposition were calculated from each cross. The eggs were collected daily throughout the oviposition period of females. Viable F1 offspring were maintained to the adult stage, according to Belisário et al. (2007). 26 To determine whether the F1 offspring were fertile, crosses between F1 x F1, from the same couple, were performed to obtain F2 offspring. Likewise of experimental crosses of parental, the fertility rate and oviposition were calculated throughout the oviposition period of females, and to ascertain the fertility of the F2 offspring. 2.3 Morphologic and Morphometric Analysis The phenotype of the offspring of each couple was described according to the major characteristics that species presented, as reduced hemelytra, rings of color orange redish in the femura and connexive (Papa et al., 2002). Scanning Electron Microscopy (SEM) was used for the morphologic analysis of ten eggs and three median process of the pygophore of male genitalia for comparison among the parental and offspring. These images were examined by under a Topcon SM-300 microscope at the Instituto de Química (UNESP, Araraquara/SP, Brazil). The samples eggs and median process of the pygophore were washed, dehydrated in an alcohol series and oven-dried at 50°C, afterwards, sputtering metallization was performed for 2 minutes at 10 mA (Rosa et al., 2012). Geometric morphometric techniques were applied to wings to evaluate whether the morphotype exhibited by offspring presented any differences in shape compared T. lenti and T. sherlocki (Almeida et al., 2012, Campos et al., 2011). Thirteen anatomical landmarks (Schachter-Brooide et al., 2004) were collected at intersections between venations and processed by the same researcher using modules available at the CLIC (Collection of Landmarks for Identification and Characterization, http://www.mpl.ird.fr/morphometrics/clic/index.html (Dujardin et al., 2010), and the COOWin software (Dujardin, 2004), as described by Dujardin (2008). We identified a total of 11 types I landmarks (venation intersections) and two type II landmarks (Bookstein, 1990). Analyses were computed as nonuniform (partial warps) and uniform components, which describe regional and global deformations of the wing architecture (Bookstein, 1991). Prior to the generalized procrustes analysis, an isometric estimator of size variation (centroidsize) was calculated as the square root of the sum of the squared distances between the center of the configuration of landmarks and each individual landmark (Bookstein, 1991). A factorial map was built to illustrate the variation, which resulted from the first and second principal components of the analysis, representing 95% of the shape. 27 2.4 Cytogenetic approaches Seminiferous tubules of ten F1 and F2 offspring adult males, from the crosses between T. sherlocki females × T. lenti males, after being shredded, crushed and fixed in liquid nitrogen on glass slide, using lacto-acetic orcein (De Vaio et al., 1985), C-banding (Sumner, 1972) and Feulgen reaction (Mello and Vidal, 1978). For the analysis of the affinity genomic hybrid methodology was used to Techio et al. (2005), with modifications, since besides diakinesis also analyze cells in meiosis I and meiosis II. 3. Results 3.1 Experimental crosses The fertility rate of the eggs from couple involving T. sherlocki females × T. lenti males was 65%, while the reciprocal crosses revealed 7% of fertile eggs (Table 1). The mortality rate, before reaching adulthood, was 80% in the F1 offspring in both crosses. The fertility rate of the eggs from F1 offspring was greater in couple involving T. sherlocki females × T. lenti males than in couple involving T. lenti females × T. sherlocki males, 90% and 25%, respectively. In the crosses involving T. lenti females × T. sherlocki males, only one couple obtained viable eggs. The fertility of the F2 offspring was only observed in couple involving T. sherlocki females × T. lenti males and the percentage of eggs viable was 2%, showing 100% of mortality rate before reaching adulthood. 3.2 Morphologic and Morphometric Analysis Phenotypes of F1 offspring as size and color of connexive, presence or absence of rings color orange redish in the femura and size of hemelytra were identical in both directions of the couple (Fig. 2). The F1 offspring revealed morphological characteristics as color and size of the connexive and the presence of red rings on the legs similar to T. sherlocki, however, the size of the wings was intermediate (Fig. 2A, B). In the F2 offspring, these same characteristics showed patterns segregating in adults (Fig. 2C-G). Under Scanning Electron Microscopy (1000X) the exochorion showed difference between T. lenti and T. sherlocki (Fig. 3A, C). In the F1 offspring, the exochorion showed similar characteristics that presented from the female of the original crossing (Fig. 3B, D). 28 The male genitalia, analyzed by median process of the pygophore revealed that the main difference between the two species is slender point in T. sherlocki and a wider base (Fig. 4A), whereas in T. lenti the rounded point and narrower base (Fig. 4B). Intermediate characteristics was observed in the F1 offspring that differentiated the parental species (Fig. 4C). In the F2 offspring, the median process of the pygophore was similar as T. sherlokci revealed a characteristics segregating (Fig. 4D). For this analysis were used hybrids from couple T. sherlocki female x T. lenti male. For the wings geometric morphometrics the factorial map built with 40, 34, 43 and 15 specimens of T. sherlocki, T. lenti, F1 and F2, respectively, distinguished both species and hybrids in well-defined groups (Fig. 5). Considering the shape variation components, the contribution of the first principal (PC1) component accounted for 34% of the total variation, whereas the second principal component (PC2) accounted for 14%. 3.3 Cytogenetic approaches We analyze the spermatogenesis of F1 (Fig. 6) through lacto-acetic orcein technique and F2 (Fig. 9) through Feulgen reaction, which show striking differences in their meiotic chromosome behavior. In the F1 offspring of both crosses, i.e., T. sherlocki females × T. lenti males and T. lenti females and T. sherlocki males, we observed the same cytogenetic characteristics (Figures 6-8). During diffuse stage, a heteropycnotic corpuscle is observed formed by the association between both sex chromosomes and some autosomes (Fig. 6A, arrow). In diplotene, some of the ten bivalents showed chiasmata (Fig. 6B, asterisk). In metaphase I, the 10 autosomal bivalents and two sex chromosomes are clearly seen (Fig. 6C), while that the anaphase I are normal (Fig. 6D). The spermatids present a heteropycnotic filament on their periphery (Fig. 6E, F) and the spermatozoan it seen normal (Fig. 6G). With C-banding, we observed that the hybrids showed the same diploid chromosome number and arrangement of constitutive heterochromatin than the parental species. During early meiotic prophase we observed a large chromocenter constituted by the association of both sex chromosomes plus two autosomal pairs (arrow), and multiple C-dots spread in the nucleus (Fig. 7A). In metaphase I, the ten autosomal bivalents showed C-blocks at one or both chromosomal ends (Fig. 7B). By means of the Feulgen reaction analyzed 100 cells in diplotene (Fig. 8A) metaphase I (Fig. 8B) and metaphase II (Fig. 8C), in order to assess the compatibility between the 29 genomic parental species. It was observed that all cells showed 100% pairing. However, analysis of 50 F2 offspring cells diakinesis/metaphase I showed that in 90% of the cells showed some errors in pairing of autosomes, resulting in monovalent chromosomes (Fig. 9A- D). Not been possible view Metaphase II in F2 offspring. The spermatids present a heteropycnotic filament on their periphery (Fig. 10A-C, arrows). 4. Discussion Morphological approaches has been an important tool in the characterization, identification and description of the species of the brasiliensis subcomplex (Costa et al, 2006, Costa and Felix, 2007) as well as the characterization of hybrid forms (Almeida et al., 2012, Costa et al., 2003, 2013). Based on morphological observations, Costa et al. (2009) propose that T. b. macromelasoma is a hybridization product between T. b. brasiliensis and T. juazeirensis. Hybridization between closely related triatomine species is a well-known phenomenon detected in nature and in experimental laboratory cross-mating (Costa et al., 2003; Mas-Coma and Bargues, 2009; Perez et al., 2005; Usinger et al., 1966). The subspecies T. b. brasiliensis and T. b. macromelasoma and species T. juazeirensis presented genetic compatibility and generate fertile hybrids in the F1 and F2. However, the ability of hybridization may not be the only factor taken into account, as the species T. melanica presents genetic incompatibility and hybrid unviable with brasiliensis subcomplex members (Costa et al., 2003). Experimental crosses were made between T. sherlocki and the species of T. brasiliensis complex to confirm T. sherlocki as a member of the T. brasiliensis complex (Correia et al., 2013). In this study all experimental combinations of T. sherlocki with members of the T. brasiliensis complex species produced viable eggs with variable percentages of survivor’s index (52.3-73.5%), confirming the inclusion in brasiliensis subcomplex. Crossing experiments already had been carried out to check reproductive compatibility in the brasiliensis subcomplex members (according to Schofield and Galvão, 2009) and T. petrocchiae and T. lenti failed to produce F1 offspring viable with T. brasiliensis (Espínola, 1971, Heitzmann-Fontenelle, 1984). Cerqueira (1982) in studies involving interspecific crosses between T. lenti and T. sherlocki, did not get viable hybrids which contradicts the results presented in this work. 30 T. sherlocki and T. lenti are closely related geographically and ecologically. According to ecological niche modeling, T. sherlocki showed distribution for the city of Ipupiara (Almeida et al., 2009), region known for the distribution of T. lenti (Sherlock and Serafim, 1967; 1972). Although the specimens used in the experiment were of different origin (peridomiciliary for T. lenti and wild for T. sherlocki), both species can be found in two types of ecotypes (Sherlock and Serafim, 1967; 1972; Almeida et al., 2009). Since the ecological and geographic boundaries between T. sherlocki and T. lenti are not clear, the possible existence of natural hybrids would be expected. However, due to reproductive isolation pre-and post-zygotic described in this work, the formation of natural hybrids is unfeasible. According to Mas-Coma and Bargues (2009), T. delpontei x T. infestans and T. platensis x T. infestans hybrids have been detected in natural populations, it is still unclear if these represent accidental events or reflect the existence of large and stable hybrid populations. Unlike the reproductive success of T. sherlocki females × T. lenti males, the reciprocal crosses did not show the same pattern. The crosses involving T. lenti females × T. sherlocki males showed high mortality and low fertility of eggs in the F1 and F2 offspring. This difference in reproductive success between pairs of couples was also reported by Sasabe et al. (2007) when analyzing the genetic basis of interspecific differences in genital morphology of closely related carabid beetles. Almeida et al. (2012) showed that laboratory-bred hybrids of T. sherloki with T. juazeirensis possess intermediate morphological traits. Intermediate morphological traits were founded in the male genitalia, median process of the pygophore, in the F1 offspring in the crosses between T. sherloki and T. lenti. Furthermore, intermediate forms in nature have been observed between T. b. brasiliensis, T. b. macromelasoma, and T. juazeirensis in Pernambuco State (Costa et al., 2009). Morphological approaches of the F1 offsprings, in both couples, as color and size of the connexive and the presence of red rings on the legs were similar to T. sherlocki. Phenotypes of F1 offspring of crosses between M. phyllosomus and M. pallidipennis showed that all F1 individuals were morphologically similar to M. pallidipennis (Martínez-Ibarra et al., 2011). Hybrid fertility and fitness are key parameters determining the long-term outcome of the mixture of two species. The egg counts suggest that hybrid females are as fertile, at least as far as egg production, as parental populations, suggesting little or no prezygotic isolation 31 (Jiggins and Mallet, 2000, Barton and Cara, 2009). On the other hand, analysis of genotype frequencies of the different sexes and developmental stages strongly suggest the disappearance of hybrid females as they aged, possibly because of increased mortality. A lack of hybrid fitness may thus lead to a postzygotic barrier allowing for the maintenance of reproductive isolation of parental genotypes (Wiwegweaw et al., 2009). The presence of recombinants between parental genotypes suggests that gene flow does occur between sibling species but that selection processes act to maintain their distinctiveness. The F1 offspring showed a normal meiotic behavior. Pérez et al. (2005) also observed that in hybrids of the species T. infestans and T. platensis meiotic division occurred normally. However, in experimental hybrids between T. infestans and T. rubrovaria, Pérez et al. (2005) assesses sterility of F1 offspring in pairing failures associated with the homeologus, which lead to the production of abnormal spermatids. By analyzing spermiogenesis was observed that the spermatids present the same characteristics described in T. lenti, by Alevi et al. (2013b), i.e., peripherals heteropycnotic filaments. The parental species, T. lenti and T. sherlocki, show the same pattern of constitutive heterochromatin in the autosomes, as well as all other species that are part of brasiliensis subcomplex (Alevi et al., 2013a). Alevi et al. (2013c) by analyzing the pattern heterochromatic confirmed the relationship of T. lenti with the brasiliensis subcomplex. Experimental crosses are considered as a tool to evaluate the proximity found among species and, therefore, propose the group or not. However, we emphasize that to confirm the true position of T. lenti in the complex, many other approaches must be used, and since T. melanica presents genetic incompatibility with the triatomines of the complex and is still considered a member (Costa et al., 2003). Pérez et al. (2005), when analyzed hybrid resulting from a cross between evolutionarily related species, proposed that differences in the pattern of constitutive heterochromatin between homologous chromosomes in parental species are not a barrier that influences synaptic recombination. So, as the parents do not show differences in heterochromatic pattern, F1 hybrids retained the same characteristics as T. lenti and T. sherlocki, out more, a large chromocenter made up of the association of both sex chromosomes plus two autosomal pairs, and multiple C-dots spread in the nucleus. The location of the autosomal C-blocks (at one or both chromosomal ends in all autosomal pairs), the diploid chromosome number consisting of 20 autosomes plus two sex chromosomes (XY 32 in males and XX in females) and the amount of autosomal C-heterochromatin (25-32%) (Panzera et al., 2010; Alevi et al., 2013a). In the F1 offspring, as well as T. lenti (Alevi et al., 2013a) and T. sherlocki (Panzera et al., 2010) showed a diploid chromosome set of 2n = 20A + XY. Furthermore, we observed that homologous chromosomes showed 100% homeology. This result allows us to measure phylogenetic proximity between T. lenti and T. sherlocki since, according to Dewey (1982), the classical analysis of genomics, which involves evaluating the behavior of chromosomes in metaphase I in interspecific hybrids, allows establishing phylogenetic relationships in groups of different species, as well to be employed in defining taxonomic and evolutionary placements. Similar statements are made by Riley (1966) in holding that the two species have distinct genomes when their chromosomes are different in structure and gene content, so that no occurs pairing between one or more pairs of homeologus during meiosis of hybrids. This behavior leads to sterility, and consequently the genetic isolation between species. Thus, if we analyzed only the F1 derived from crossing experiment, we propose an early homoploid speciation. Costa et al. (2009), suggest, through morphological analyses, that T. b. macromelanosoma is a species derived from crosses between the species T. b. brasiliensis and T. juazeirensis. However, when analyzing the F2, we found that possibly other evolutionary barriers to prevent hybridization. Through the analysis in the F2 offspring were observed errors in chromosome pairing. These results possibly are related to one mechanism of post-zygotic isolation proposed by Dobzhansky (1970), out more, collapse of the hybrid. The collapse is a little known phenomenon that the F2 offspring or backcross has reduced viability or fertility, as observed in this work by the amount of F3 offspring. We believe that this phenomenon is due to a genic deregulation occurred in the chromosomes of F1. Probably, this imbalance is the result of crossing over that occurs in the F1 homeologus which results in lack of homology in euchromatic regions and prevents the normal pairing of chromosomes of some in F2. Thus, in the F2 offspring, although some cells analyzed (10%) were normal (present 100% homeology between autosomes), most of the cells were abnormal, with an absence of homeology among autosomes, resulting in monovalent. This phenomenon results in the formation of inviable sperm. However, no significant changes in haploid cells during spermiogenesis, as observed by Schreiber et al. (1975). Questions regarding the genetics and ecology of T. sherlocki and T. lenti are still in the initial studies, since the few published works on these two species. It would therefore be 33 reasonable to initiate quantitative trait locus (QTL) mapping, which provides fundamental information such as the number of loci involved in a certain trait (morphological or behavioral), locations on chromosomes, and magnitude of individual genetic effects. Thus, we believe that T. lenti is specie of T. brasiliensis complex, although new analysis to confirm it should be made to this matter. Furthermore, it can be seen that although T. sherlocki and T. lenti species are evolutionarily and citotaxonomically closed, there is a post-zygotic reproductive barrier that reduces the fertility in the F2 offspring, confirming the status specific of species analyzed. References Alevi, K.C.C., Mendonça, P.P., Pereira, N.P., Fernandes, A.L.V.Z., Rosa, J.A., Azeredo- Oliveira, M.T.V., 2013b. 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Sherlock, I.A., Serafim, E.M., 1972. Fauna Triatominae do Estado da Bahia, Brasil. I – As espécies e distribuição geográfica. Rev. Soc. Bras. Med. Trop. 6, 265-276. Sumner, A.T., 1972. A simple technique for demonstrating centromeric heterochromatin. Experimental Cell Res. 75, 305-306. Techio, V.H., Davide, L.C., Pereira, A.V., 2005. Genomic analysis in Pennisetum purpureum x P. glaucum hybrids. Caryologia. 58, 28-33. Usinger, R.L., Wygodzinsky, P., Ryckman, E.R., 1966. The biosystematics of Triatominae. Annu. Rev. Entomol. 11, 309-329. WHO - World Health Organization., 2013. Chagas disease (American trypanosomiasis) [http://www.who.int/mediacentre/factsheets/fs340/en/#]. Wiwegweaw, A., Seki, K., Utsuno, H., Asami, T., 2009. Fitness consequences of reciprocally asymmetric hybridization between simultaneous hermaphrodites. Zoolog. Sci. 26, 191-196. 38 Table 1. Crosses involving T. sherlocki x T. lenti showing the fertile eggs and rate mortality. T. sherlocki females × T. lenti males T. lenti females × T. sherlocki males fertile eggs mortality fertile eggs mortality F1 65% 80% 7% 80% F2 90% 90% 25% 40% F3 2%* - - - *only three nymphs emerged Figure 1. Distribution of T. sherlocki and T. lenti in the Bahia state, Brazil. T. sherlocki (A) is distributed in the region of Gentio do Ouro and T. lenti (B), in the region of Macaúbas. 39 Figure 2. Phenotypes of F1 and F2 offspring from crosses involving T. sherlocki females × T. lenti males. A-B: female and male F1 offspring, respectively. C-G: F2 offspring adults C: similar to T. lenti; D: connexivum and rings of color orange redish in the femura similar to T. sherlocki and size of hemelytra similar to T. lenti; E-F: connexivum and absence rings of color orange redish in the femura similar to T. lenti and reduced hemelytra similar to T. sherlocki; G: connexivum and absence rings of color orange redish in the femura similar to T. lenti and color of connexivum and reduced hemelytra similar to T. sherlocki. 40 Figure 3. Egg exochorion detail by SEM (1000X). A: Triatoma lenti; B: F1 offspring from couple T. lenti females × T. sherlocki males; C: Triatoma sherlocki; D: F1 offspring from couple T. sherlocki females × T. lenti males. 41 Figure 4. Median process of the pygophore by SEM (100X). A: T. sherlocki; B: T. lenti; C: F1 offspring; D: F2 offspring. (F1 and F2 offspring from couple T. sherlocki females × T. lenti males). Figure 5. Factorial map of the wing shape. The factorial map of the wing shape for the species T. sherlocki, T. lenti and the F1 offspring and F2 offspring. 42 Figure 6. Spermatogenesis of F1 hybrids stained with lacto-acetic orcein. A-B: Prophase I. Note corpuscle heteropyknotic in the initial diffuse (A, arrow) and in diplotene was possible to visualize chiasmus (B, asterisk). C: Metaphase I. Note that it is possible to observe ten pairs of autosomes and the sex chromosomes, being the Y greater (arrow). D: Anaphase. E-F: Spermiogenesis. Note the peripheral heteropycnotic in spermatids (arrows). G: Spermatozoid. Bar: 10 um. 43 Figure 7. Constitutive heterochromatin pattern in F1 hybrids stained with C-banding. A-B: Constitutive heterochromatin pattern in hybrids. Note the chromosome configuration during early meiotic prophase (A), out more, a large chromocenter made up of the association of both sex chromosomes plus two autosomal pairs (arrow), and multiple C-dots spread in the nucleus. In metaphase I (B), note that diploid chromosome number consisting of 20 autosomes plus two sex chromosomes (XY in males and XX in females) and the amount of autosomal C-heterochromatin (25 - 32%). Bar: 10 um. Figure 8. Cells meiotic of F1 hybrids stained with Feulgen reaction. A: Diplotene. B: Metaphase I. C: Metaphase II. Note the sex chromosome Y (arrow). Bar: 10 um. 44 Figure 9. Diakinesis the F2 generation with Feulgen reaction. A-D: Diakinesis. Note autosomes monovalent by not the pairing among homeologous and sex chromosomes (asterisk). Bar: 10 um. Figure 10. Spermiogenesis of F2 generation. A-C: Spermiogenesis. Note the peripheral heteropycnotic in spermatids (arrows). Bar: 10 um. 45 4.3 Capítulo 3 (Artigo científico publicado na revista Zootaxa FI 0,97) ALEVI, K. C. C.; ROSA, J. A.; AZEREDO-OLIVEIRA, M. T. V. Cytotaxonomy of the Brasiliensis subcomplex and the Triatoma brasiliensis complex (Hemiptera: Reduviidae: Triatominae). Zootaxa, v. 3838, p. 583-589, 2014. Cytotaxonomy of the Brasiliensis subcomplex and the Triatoma brasiliensis complex (Hemiptera, Triatominae) Abstract We analyzed the classical cytotaxonomy of the Brasiliensis subcomplex (Triatoma brasiliensis Neiva, T. juazeirensis Costa & Felix, T. melanica Costa, Argolo & Felix, T. melanocephala Neiva & Pinto, T. petrocchiae Pinto & Barreto, T. lenti Sherlock & Serafim, T. sherlocki Papa, Jurberg, Carcavallo, Cerqueira & Barata, T. tibiamaculata Pinto and T. vitticeps Stal) and the T. brasiliensis complex (T. b. brasiliensis, T. b. macromelasoma Neiva & Lent, T. juazeirensis, T. melanica and T. sherlocki). The five members of the T. brasiliensis complex share the same cytogenetic characteristics. Merely T. sherlocki show differences in spermatids, which confirms the status of more differentiated member of the complex. T. lenti also presented the same cytogenetic characteristics described for the species of the T. brasiliensis complex, which supports possible grouping of the species as sixth member of the complex, although further analysis as molecular and experimental crosses are needed to corroborate this hypothesis. T. petrocchiae, T. vitticeps, T. tibiamaculata and T. melanocephala presented one or more characteristics that allow questioning grouping in the proposed Brasiliensis subcomplex. Thus, we suggested that Brasiliensis subcomplex and T. brasiliensis complex should be constituted by the same triatomines (T. b. brasiliensis, T. b. macromelasoma, T. juazeirensis, T. melanica and T. sherlocki). However, we draw attention to T. lenti and suggest that although new analyzes should be performed, possibly this species is the sixth member of the T. brasiliensis complex. Introduction The Triatominae subfamily is composed of 148 species distributed in 18 genera and six tribes (Abad-Franch et al. 2013; Alevi et al. 2013a; Jurberg et al. 2013; Poinar et al. 2013). These insects present five nymphal stages (N1, N2, N3, N4, N5) and one adult. After 46 hatching, the triatomine are hematophagous strict and, once infected with the protozoan Trypanosoma cruzi Chagas (Kinetoplastida: Trypanosomatidae), can transmit the Chagas disease. Transmission occurs by the habit of defecating during the repast (Noireau et al. 2009). The triatomines were grouped in complexes and subcomplexes specific by Schofield & Galvão (2009). The authors grouped in Infestans complex and the Brasiliensis subcomplex the species: Triatoma brasiliensis Neiva, T. juazeirensis Costa & Felix, T. melanica Costa, Argolo & Felix, T. melanocephala Neiva & Pinto, T. petrochiae Pinto & Barreto, T. lenti Sherlock & Serafim, T. sherlocki Papa, Jurberg, Carcavallo, Cerqueira & Barata, T. tibiamaculata Pinto and T. vitticeps Stal. However, to collate the species in the subcomplex, the authors used parameters as morphological characters and geographical disposition. This subcomplex species is endemic to Northeast Brazil and is of great epidemiological importance (Costa 2000; Almeida et al. 2009). At first, it was believed that the species of the Brasiliensis subcomplex were only populations of T. brasiliensis with chromatic polymorphism (Lent & Wygodzinsky, 1979). However, Costa and collaborators, by means of different aspects, redescribed T. melanica (Costa et al. 2006) and T. b. macromelasoma Neiva & Lent (Costa et al. 2013), and described T. juazeirensis (Costa & Felix 2007). Furthermore, based on characteristics monophyletic, as egg morphology (Costa et al. 1997a), morphometry of the testis (Freitas et al. 2008), hybrid cross (Costa et al. 2003), isoenzymes (Costa et al. 1997b), molecular data (Monteiro et al. 2004), morphological data (Costa et al. 1997a), biological data, and ecological data (Costa et al. 1998), proposed that species should be grouped in T. brasiliensis complex, composed of two subspecies (T. b. brasiliensis and T. b. macromelasoma) and two species (T. juazeirensis and T. melanica). Mendonça et al. (2009) proposed the inclusion of T. sherlocki to this complex. This inclusion was recently confirmed through the use of both cytogenetic analysis (Alevi et al. 2013b) and cross-mating experiments (Correia