Linhagens genéticas em Anopheles cruzii Dyar & Knab, 1908 (Diptera: Culicidae) : aspectos moleculares, ecológicos e morfométricos

Orientador : Prof. Dr. Mario Antônio Navarro da SilvaTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências Biológicas (Entomologia). Defesa: Curitiba, 24/08/2017Inclui referências ao final de cada capítuloResumo: A família Culicidae (Diptera) compreende um diverso grupo de mosquitos distribuídos entre as regiões temperadas e tropicais no mundo, na qual estão incluídas 3.555 espécies, sendo muitas destas capazes de transmitir diversos patógenos para os humanos. Anopheles cruzii é considerado o principal vetor do agente etiológico malária nas regiões Sul e Sudeste do Brasil. Devido à sua importância epidemiológica, alguns estudos sobre a caracterização morfológica e molecular da espécie foram realizados. Estes estudos sugerem que An. cruzii pode representar um complexo de espécies, porém a distribuição, quantidade e o status destas diferentes linhagens ainda é incerto. Diante deste cenário, o objetivo da tese foi caracterizar as linhagens genéticas de An. cruzii quanto a variações morfológicas, moleculares e ecológicas. Com isso, a tese apresenta-se dividida em dois capítulos, o primeiro intitulado "Análise da estrutura populacional de Anopheles (Kerteszia) cruzii Dyar & Knab, 1908 (Diptera: Culicidae) com base em marcadores mitocondriais e morfometria geométrica" e o segundo "Sobreposição de nicho ecológico entre linhagens genéticas de Anopheles (Kerteszia) cruzii Dyar & Knab, 1908 (Diptera: Culicidae)". Como resultados do primeiro capítulo quatro linhagens genéticas foram descritas para An. cruzii, sendo estas localizadas em Camacan-BA, Itaparica-BA, Morretes-PR + Tapiraí-SP e Florianópolis-SC + Itatiaia-RJ. Todas estas linhagens apresentaram altos índices Fst, ausência de migrantes e representação em clados exclusivos nas análises filogenéticas de máxima verossimilhança. A variação fenotípica interpopulacional também foi constatada, sendo as asas das populações de Camacan-BA e da Ilha do Mel-PR distintas morfologicamente das demais populações analisadas. No segundo capítulo foi avaliada a existência de conservação de nicho ecológico entre as diferentes linhagens genéticas de An. cruzii. Os resultados revelaram a existência de sobreposição de hábitat disponível para a ocorrência das duas linhagens nos Estados de São Paulo e Rio de Janeiro. Os resultados apresentados sugerem que as características do nicho climático dos grupos A e B talvez não tenham sido as mais importantes para dirigir um possível processo de especiação em andamento, já que observou-se a sobreposição de nichos entre as linhagens analisadas. Palavras-chave: diversidade genética, variações fenotípicas, nicho ecológico.Abstract: The Culicidae (Diptera) family comprises a diverse group of mosquitoes spread through temperate and tropical regions around the world. There are 3,555 species included in this family, many of which are vectors for a number of pathogens to humans. Anopheles cruzii is considered the main vector of the etiologic agent of malaria in the south and southeast regions of Brazil. Given its epidemiologic relevance, a number of studies have been carried on morphologic and molecular characteristics of An. cruzii. Those studies suggest that An. cruzii may represent a complex of species, however the distribution, quantity and status of the different lineages is still uncertain. In light of this, the objective of this thesis is to characterize the genetic lineages of An. cruzii in regards with morphological, molecular and ecological variances. The thesis is divided in two chapters, the first one named "Analysis of population structure of Anopheles (Kerteszia) cruzii Dyar & Knab, 1908 (Diptera: Culicidae) through mitochondrial markers and wing geometric morphometry" and the second one "Ecological niche overlap among genetic lineages of Anopheles (Kerteszia) cruzii Dyar & Knab, 1908 (Diptera: Culicidae)". As results of the first chapter four genetic lineages were described for An. cruzii, being these located in Camacan-BA, Itaparica-BA, Morretes-PR + Tapiraí-SP and Florianópolis-SC + Itatiaia-RJ. All of these lineages presented high levels of Fst, absence of migrants and exclusive clades in phylogenetic analyzes of maximum likelihood. The interpopulational phenotypic variation was also observed, being the wings of the populations of Camacan-BA and Ilha do Mel-PR morphologically distinct from the other populations analyzed. The second chapter evaluated the existence of ecological niche conservatism among the different genetic lineages of An. cruzii. The results revealed the existence of available habitat overlap for the occurrence of the two lineages in the States of São Paulo and Rio de Janeiro. The results suggest that the climatic niche characteristics of the groups A and B may not have been the most important to guide a possible speciation process in progress, since the overlap of niches among the analyzed lineages was observed. Keywords: genetic diversity, phenotypic variability, ecological niche

Espectroscopia de infravermelho próximo e microestruturas de escamas de Sabethes (Sabethes) albiprivus Theobald, 1903 (Diptera : Culicidae)

Orientador : Prof. Dr. Mário Antônio Navarro da SilvaDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências Biológicas (Entomologia). Defesa: Curitiba, 21/08/2013Inclui referênciasÁrea de concentração : EntomologiaResumo: O gênero Sabethes Robineau-Desvoidy é constituído por 39 espécies, distribuídas em cinco subgêneros, Davismyia, Sabethoides, Sabethinus, Peytonulus e Sabethes, sendo este último o que apresenta a maior riqueza com 18 espécies descritas. Sabethes (Sabethes) albiprivus Theobald, 1903 é uma espécie com distribuição restrita à América do Sul, já foi encontrada naturalmente infectada com o vírus da febre amarela e apresenta consideráveis variações morfológicas quanto ao seu tamanho e coloração do reflexo das escamas que recobrem o tórax, abdome, lobos antepronotais e occipício. Diante destas considerações, o objetivo deste trabalho foi caracterizar e descrever as variações morfológicas dos exemplares, em conjunto com um estudo das microestruturas presentes nas escamas que revestem o corpo deste culicídeos. Foram analisados 210 exemplares adultos, machos e fêmeas, montados em alfinetes entomológicos com o auxílio de estéreomicroscópio, caracterizando-se os padrões de coloração encontrados. Para a obtenção dos espectros de absorbância (NIRS), os exemplares foram posicionados entre a sonda de fibra óptica e o acessório de reflectância sendo o programa Unscrambler® utilizado para a análise estatística de componentes principais. Os exemplares analisados através da MEV foram posicionados sobre suportes de alumínio, metalizados com ouro e observados no microscópio JEOL JSM-6360LV. A partir da observação de fêmeas adultas foi possível descrever sete morfotipos, diagnosticáveis pelo reflexo das escamas que revestem os lobos antepronotais, occipício, abdome e mesonoto, sendo dois morfotipos pertencentes ao grupo de exemplares com escamas claras e cinco de escamas escuras. Estes mesmos dois grupos foram identificados utilizando-se a técnica de NIRS, demonstrando que estes exemplares possuem variações em sua composição química que permitem diagnosticar estes dois grupos. Porém, as análises de componentes principais não detectaram diferenças significativas no padrão de coloração em relação à variação ambiental e estacional. A análise das nanoestruturas presentes nas escamas que revestem o mesonoto, occipício e lobos antepronotais através da técnica de microscopia eletrônica de varredura, levou a divisão de Sa. (Sab.) albiprivus nos mesmos dois grupos encontrados através da técnica de NIRS. Nas três metodologias utilizadas, NIRS, MEV e coloração de reflexos de escamas, definiram-se dois agrupamentos para Sa. (Sab.) albiprivus, sendo que estes não apresentam padrões de variação ambiental intraespecífica. Com isso, acredita-se que a espécie represente um complexo de espécies. Palavras-chave: Composição química, escamas, exoesqueleto, morfotipos.Abstract: The genus Sabethes Robineau-Desvoidy is composed of 39 species, distributed in five subgenera, Davismyia, Sabethoides, Sabethinus, Peytonulus and Sabethes, being this last one the richest subgenera within the genera, with a total of 18 described species. Sabethes (Sabethes) albiprivus, Theobald, 1903 is restricted to South America, has been found naturally infected with the yellow fever virus and has morphological variations regarding its size and color of the reflection of the scales that cover its thorax, abdomen, antepronotal lobes and occiput. In sight of such considerations, the objective of this study was characterizing and describing the morphological variations of the samples, as well as studying the existing microstructures on the scales that cover the body of those Culicidae. 210 adult specimens were analyzed, male and female, placed on entomological pins, with a stereo microscope, characterizing the identified patterns of color. In order to obtain the absorbance spectra (NIRS), the samples were positioned between the optic-fiber probe and the reflectance diffuse enhancement through Unscrambler® for the statistical analysis of the principal components. The samples analyzed through SEM were positioned on aluminum stubs, metalized with gold and observed with the microscope JEOL JSM-6360LV. From the observation of adult female it was possible to describe seven morphotypes, diagnosable through the reflection of the scales that cover the antepronotal lobes, occiput, abdomen and mesonotum, being two morphotypes part of the group of samples with clear scales and five from the group with dark scales. These same two groups were identified through the NIRS technique, demonstrating that the samples have variations on their chemical composition that allow the diagnostic of these two groups. But the PCA analysis did not detect significant differences on the color pattern regarding environmental and seasonal variations. The analysis of the nanostructures existing on the scales that cover the mesonotum, occiput and antepronotal lobes through the scanning electron microscopy technique resulted on the division of Sa. (Sab.) albiprivus in the same two groups. On the three adopted methodologies, NIRS, SEM and color of reflection of scales, two groupings were identified for Sa. (Sab.) albiprivus, which do not present intraspecific environmental variation. Given this, it is believed that the species represents a species complex. Keywords: Chemical composition, scales, exoskeleton, morfphotypes

Near-infrared spectroscopy and microstructure of the scales of Sabethes ( Sabethes ) albiprivus (Diptera: Culicidae)

ABSTRACT Near-infrared spectroscopy and microstructure of the scales of Sabethes (Sabethes) albiprivus (Diptera: Culicidae). Sabethes (Sabethes) albiprivus Theobald individuals vary considerably in size and color of the reflections of the scales on their thorax, abdomen, antepronotal lobes and occiput. The goal of this study was to investigate and to characterize the differences in the color of the scales among preserved specimens and to analyze the differences in the microstructures of the scales that cover their bodies using near-infrared spectroscopy, and to evaluate whether the latter is efficient in distinguishing the populations. A total of 201 adult females were analyzed for the characterization of color patterns. In addition, absorbance spectra and scanning electron microscope images were obtained from them. As a result of color analysis, two variations were identified, one represented by specimens with yellow or green scales and the other with blue or purple scales. The same two variations were corroborated using NIRS. Analysis of the microstructure of the scales lining the mesonotum, occiput and antepronotal lobes resulted in the same variations. The three methodologies, near-infrared spectroscopy, scanning electron microscopy and coloration of the reflections of the scales revealed two variations within Sa. albiprivus

Near-infrared spectroscopy and microstructure of the scales of Sabethes ( Sabethes ) albiprivus (Diptera: Culicidae)

ABSTRACT Near-infrared spectroscopy and microstructure of the scales of Sabethes (Sabethes) albiprivus (Diptera: Culicidae). Sabethes (Sabethes) albiprivus Theobald individuals vary considerably in size and color of the reflections of the scales on their thorax, abdomen, antepronotal lobes and occiput. The goal of this study was to investigate and to characterize the differences in the color of the scales among preserved specimens and to analyze the differences in the microstructures of the scales that cover their bodies using near-infrared spectroscopy, and to evaluate whether the latter is efficient in distinguishing the populations. A total of 201 adult females were analyzed for the characterization of color patterns. In addition, absorbance spectra and scanning electron microscope images were obtained from them. As a result of color analysis, two variations were identified, one represented by specimens with yellow or green scales and the other with blue or purple scales. The same two variations were corroborated using NIRS. Analysis of the microstructure of the scales lining the mesonotum, occiput and antepronotal lobes resulted in the same variations. The three methodologies, near-infrared spectroscopy, scanning electron microscopy and coloration of the reflections of the scales revealed two variations within Sa. albiprivus

First record of Culex (Melanoconion) spissipes (Theobald, 1903) (Diptera: Culicidae) in the Atlantic Forest of Bacia Platina, southern Brazil

In this article the occurrence of Culex (Melanoconion) spissipes is recorded at the city of Terra Roxa, state of Paraná, South of Brazil. This record covers the species distribution in the Atlantic Forest of the Bacia Platina

First records of the occurrence of twelve species of Sabethini (Diptera, Culicidae) in the state of Paraná, southern Brazil

We report on the first records of Sabethini mosquitoes in the state of Paraná, based on specimens collected in forested environments between 2004 and 2005. In total, 12 species were identified, including 7 species of Wyeomyia Theobald, 1901 and 5 species of Sabethes Robineau-Desvoidy, 1827. Among them, 6 are new records for the southern region of the country, and 6 are new records for the state of Paraná. These results indicate the occurrence of relict species of mosquitoes in Paraná, as they were collected from portions of forests that preserve original characteristics

Novel molecular evidence of population structure in Anopheles (Kerteszia) bellator from Brazilian Atlantic Forest

Anopheles bellator is a primary malaria vector in the Atlantic Forest. Partial sequences of timeless and Clock genes were used to assess the genetic differentiation of five Brazilian populations, which showed strong population structure (e.g. high F ST values and fixed differences) in all pairwise comparisons between Bahia sample and the others from Paraná, São Paulo and Rio de Janeiro states. Also, the resulting phylogenetic trees clearly grouped the sequences from Bahia in a different cluster with high bootstrap values. Among southern and southeastern populations low levels of genetic differentiation were found suggesting a general stability of the genetic structure

Commented list of diurnal mosquitoes (Diptera: Culicidae) occurring in forested areas of the state of Paraná, southern Brazil

This study was conducted to determine the composition and distribution of anthropophilic diurnal mosquitos in forest environments in the state of Paraná, southern Brazil. Mosquitoes were collected between April 2004 and June 2006, in the remnant forests of rural and urban areas of 37 municipalities. A total of 15 genera and 121 species, or groups of species. The largest number of species, or groups of species, belonged to Wyeomyia (32 species or morphospecies), followed by Sabethes (20), Aedes (15) and Anopheles (13) genera. The results are discussed and compared with other entomological surveys conducted in Paraná. Several species collected are potential vectors of infectious agents to humans