ABSTRACT Aedes (Stegomyia) aegypti is an important vector of dengue, yellow fever, chikungunya and Zika virus. It is well known that resistance monitoring and genetic diversity data help designing the vector control programs. This study aimed to evaluate resistance to pyrethroids (PYs) through the frequency of kdr mutations Val1016IIe and F1534C, and the genetic variation of the mitochondrial gene ND4 in six natural populations of A. aegypti from Paraná - Brazil. Adults were obtained from eggs collected from Alvorada do Sul, Marilena, Maringá, Nova Londrina, Paranavaí and São Carlos do Ivaí. From these adults, 345 were used to identify the 1016 and 1534 sites, and 120 were used to perform the ND4 gene analysis. The studied populations from Paraná showed PYs resistance, low gene flow and genetic diversity. Additionally, a relationship was observed among the haplotypes of populations from the Amazon and Southeastern Brazil, Peru, Mexico, and North America. Stegomyia (Stegomyia dengue fever virus programs (PYs ValIIe Val IIe F1534C FC F C ND A Brazil Sul Marilena Maringá Londrina Ivaí adults 34 101 153 sites 12 analysis Additionally Peru Mexico America 3 10 15 1

ABSTRACT In Colombia Aedes aegypti is present in 80% of the country up to 2,300 m; however, little is known of its genetic relations within a country context and, hence, within a broader context, for example, America. The aforementioned, herein, analyzed the gene flow within a context of the Americas, its directionality and genetic diversity of the mitochondrial lineages in the A. aegypti populations for Colombia. This called for the use of the sequences for A. aegypti available of the mitochondrial ND4 gene in the GenBank for Colombia and the American continent. No presence was found of nuclear mitochondrial pseudogenes (NUMTs) for Colombia. It is estimated that in Colombia the gene flow of the A. aegypti populations is occurring from the southeast and northeast toward the center of the country. In comparison with the mitochondrial sequences for America, the vector’s haplotypes in Colombia suggest connections between the populations of mosquitoes from the south with those from the north of the continent. The gene flow model at continental scale suggests bidirectional connections between the populations from the north of the continent with those from the south, while at South American scale it proposes the gene flow in all the directions with respect to the Colombian. The Colombian A. aegypti vector monitoring and control strategies must pay special attention to the vector’s points of entry into Colombia related with Peru, Venezuela, Brazil, Mexico, and North America to avoid the entry of populations with characteristics like resistance to insecticides or vector competition.

Abstract Histological effects of polybrominated diphenyl ethers (PBDEs) were observed in Chironomus sancticaroli larvae which underwent acute exposure. 2,2′,4-triBDE (BDE-17), 2,2′,4,4′-tetraBDE (BDE-47) and 2,2′,4,4′,5-pentaBDE (BDE-99) were evaluated at 0.5, 2.0 and 20 μg L-1. Cytoplasm vacuolisation of oenocytes was observed in the larvae exposed to BDE-17 and BDE-47. Cuénot cells were disrupted at the brush border as an effect of the three evaluated congeners highlighting BDE-47 at 2.0 μg L-1; 60% of larvae displayed this disruption. The midgut showed changes in the morphology of apex cells located next to the lumen of region I exposed to BDE-17 and BDE-47, while BDE-99 induced a narrowing of the lumen diameter. Significant cytoplasm vacuolisation of the larvae exposed to BDE-47 and BDE-99 was observed in region II of the midgut. Salivary glands showed acidophilic granules in the cytoplasm exposed to BDE-17 and BDE-47. The results showed that the tissues of C. sancticaroli were sensitive to flame retardants; these histopathologies can compromise the health and physiology of this organism, highlighting the concern with the presence of PBDEs in freshwater sediments.

RESUMEN Introducción: Aedes (Stegomyia) albopictus (Skuse, 1894) es un vector para los virus del dengue y chicunguña en la naturaleza, junto con cerca de 24 arbovirus en condiciones de laboratorio. El conocimiento de la diversidad genética de los insectos vectores es fundamental para el control eficaz y la eliminación de enfermedades transmitidas por estos. Objetivo: Aquí se determinó el escenario actual de la diversidad genética en poblaciones naturales de A. albopictus a través de una revisión sistemática. Metodología: Se pudieron establecer los primeros registros y distribución de las poblaciones de A. albopictus en el mundo, así como su diversidad genética, estructura genética poblacional y marcadores moleculares utilizados para determinar su diversidad genética. Resultados: A. albopictus se distribuye en todo el mundo con poblaciones genéticamente estructuradas y baja diversidad; Sin embargo, el 89,5% de la diversidad genética conocida se basa en el uso de RFLP, aloenzimas, isoenzimas y marcadores moleculares mitocondriales que presentan problemas significativos según la literatura. Una vez obtenidos los resultados, se realizó un análisis crítico y se detectaron deficiencias existentes. Conclusión: El conocimiento actual de la diversidad genética de A. albopictus se basa en marcadores genéticos que presentan problemas significativos reportados en la literatura; Por lo tanto, los programas de control de vectores dirigidos a las poblaciones de A. albopictus pueden verse comprometidos.

ABSTRACT Introduction: Aedes (Stegomyia) albopictus (Skuse, 1894) is a vector for dengue and chikungunya viruses in the field, along with around 24 additional arboviruses under laboratory conditions. Knowledge of the genetic diversity of insect vectors is critical for the effective control and elimination of vector-borne diseases. Objective: We determined the current scenario of the genetic diversity in natural populations of A. albopictus through a systematic review. Methodology: It was possible to establish the first reports and distribution of A. albopictus populations in the world, as well as its genetic diversity, population genetic structure and molecular markers used to determine its genetic diversity. Results: A. albopictus is distributed worldwide with genetically structured populations and low diversity; however, 89.5% of the genetic diversity known is based on the use of RFLP, allozymes, isozymes, and mtDNA molecular markers that exhibit significant problems according to the literature. After the results were obtained, a critical analysis was carried out and existing shortcomings were detected. Conclusion: The current knowledge of genetic diversity of A. albopictus is based on genetic markers that exhibit significant problems reported in the literature; therefore, vector control programs targeting A. albopictus populations, may be compromised.

ABSTRACT After a dengue outbreak, the knowledge on the extent, distribution and mechanisms of insecticide resistance is essential for successful insecticide-based dengue control interventions. Therefore, we evaluated the potential changes to insecticide resistance in natural Aedes aegypti populations to Organophosphates (OP) and Pyrethroids (PY) after chemical vector control interventions. After a Dengue outbreak in 2010, A. aegypti mosquitoes from the urban area of Jacarezinho (Paraná, Brazil) were collected in 2011 and 2012. Insecticide resistance to OP Temephos was assessed in 2011 and 2012 by dose–response bioassays adopting WHO-based protocols. Additionally, in both sampling, PY resistance was also investigated by the Val1016Ile mutation genotyping. In 2011, a random collection of mosquitoes was carried out; while in 2012, the urban area was divided into four regions where mosquitoes were sampled randomly. Bioassays conducted with larvae in 2011 (82 ± 10%; RR95 = 3.6) and 2012 (95 ± 3%; RR95 = 2.5) indicated an incipient altered susceptibility to Temephos. On the other hand, the Val1016IIe mutation analysis in 2011, presented frequencies of the 1016Ilekdr allele equal to 80%. Nevertheless, in 2012, when the urban area of Jacarezinho was analyzed as a single unit, the frequency of the mutant allele was 70%. Additionally, the distribution analysis of the Val1016Ile mutation in 2012 showed the mutant allele frequencies ≥60% in all regions. These outcomes indicated the necessity of developing alternative strategies such as insecticide rotations for delaying the evolution of resistance.

ABSTRACTChironomidae immature are used as bioindicators of sediment quality in aquatic ecosystems and ecotoxicological assays. Histological descriptions for this family are outdated and limited and there are no studies with Neotropical species. The aim of this study was to describe the tissue architecture of several organs of the larva of Chironomus sancticaroli. For the description of the histological pattern, the larvae were fixed in Duboscq solution for insects at 56 °C, followed by routine histologic processing, infiltration in paraffin, and the sections were stained with Hematoxylin–Eosin. After examining the slides, the tube digestive, salivary gland, excretory, nervous, endocrine, circulatory, and integumentary systems and fat body were histologically characterized. The histology allows evaluation of cell morphology, and for being not expensive and easily accessible can be routinely used in biomonitoring. In addition, is a useful tool in ecotoxicological assays and allow to evaluate biomarkers at tissue and cell levels.

Phenanthrene, a Polycyclic Aromatic Hydrocarbon, remains adsorbed to sedimentary particles in aquatic environments. It affects mainly benthic organisms, and is considered potentially genotoxic. In ecotoxicology, species of Chironomus Meigen, 1803 are widely known as bioindicators of the effects of chemicals on aquatic organisms. This study investigates the effects of phenanthrene on the size of the head capsule of Chironomus sancticaroli Strixino & Strixino, 1981 larvae after chronic (eight days) exposure, and DNA damage after acute (96 hours) and chronic exposure (eight days), under laboratory conditions. DNA damage, evaluated using the alkaline comet assay, detected effects for both exposure periods, indicating that phenanthrene is toxic for C. sancticaroli. For the acute exposure, we analyzed five concentrations of phenanthrene, between 0.16 mg.l-1 and 1.60 mg.l-1, detecting significant differences (Kruskall-Wallis test with p < 0.05) in the degree of DNA damage in all groups. These effects were not dose-dependent. For the chronic exposure, two concentrations (0.16 mg.l-1, 0.83 mg.l-1) were analyzed, and DNA damage was observed in both. Again, the effects were not dose-dependent. This indicates that phenanthrene is genotoxic to larvae of C. sancticaroli even at low concentrations. The size of the head capsule was evaluated after chronic exposure to concentrations of 0.16 mg.l-1 and 0.83 mg.l-1. Significant differences (ANOVA test with p < 0.05) were detected in the two concentrations, and a reduction in the size of the larval head capsule was observed. This suggests that phenanthrene causes delay in larval development. These results indicate that phenanthrene affects the development of and causes DNA damage in C. sancticaroli larvae. Therefore, we suggest that C. sancticaroli can be used as a biological indicator for environmental contamination with phenanthrene.

Mosquito community composition in dynamic landscapes from the Atlantic Forest biome (Diptera, Culicidae). Considering that some species of Culicidae are vectors of pathogens, both the knowledge of the diversity of the mosquito fauna and how some environment factors influence in it, are important subjects. In order to address the composition of Culicidae species in a forest reserve in southern Atlantic Forest, we compared biotic and abiotic environmental determinants and how they were associated with the occurrence of species between sunset and sunrise. The level of conservation of the area was also considered. The investigation was carried out at Reserva Natural do Morro da Mina, in Antonina, state of Paraná, Brazil. We performed sixteen mosquito collections employing Shannon traps at three-hour intervals, from July 2008 to June 2009. The characterization of the area was determined using ecological indices of diversity, evenness, dominance and similarity. We compared the frequency of specimens with abiotic variables, i.e., temperature, relative humidity and pluviosity. Seven thousand four hundred ten mosquito females were captured. They belong to 48 species of 12 genera. The most abundant genera were Anopheles, Culex, Coquillettidia, Aedes and Runchomyia. Among the species, the most abundant was Anopheles cruzii, the primary vector of Plasmodium spp. in the Atlantic Forest. Results of the analyses showed that the abiotic variables we tested did not influence the occurrence of species, although certain values suggested that there was an optimum range for the occurrence of culicid species. It was possible to detect the presence of species of Culicidae with different epidemiologic profiles and habitat preference.

INTRODUÇÃO:A identificação precisa da variante genética do vírus da dengue é importante para compreender a dispersão, virulência e identificação das cepas responsáveis pelas epidemias. O objetivo da pesquisa foi investigar a variação genética do fragmento da junção do gene capsídeo/pré-membrana dos sorotipos 1 e 2. MÉTODOS: Amostras de onze municípios do Estado Paraná, Brasil, foram cedidas pelo Laboratório Central do Paraná e consistiam em isolados de cultura de células da linhagem C6/36 (Aedes albopictus), positivos para técnica de imunofluorescência indireta. O Ribonucleic acid (RNA) dessas amostras foi extraído, seguido da transcrição reversa, reação em cadeia da polimerase (PCR) e nested PCR. RESULTADOS: Co-infecção por DENV-1 e 2 (virus da dengue 1 e 2) foi observada em quatro pacientes, através da técnica Reverse transcriptase-polymerase chain reaction (RT-PCR). Para o DENV-1 a porcentagem de similaridade variou de 95 a 100% comparando com cepas do Genbank. Para o DENV-2 a porcentagem de similaridade variou de 98 a 100%. De acordo com o cladograma gerado, todas as cepas deste estudo se agruparam no genótipo V para DENV-1. Para o DENV-2 foi encontrada a cepa referente ao genótipo asiático/americano. CONCLUSÕES: O monitoramento das cepas circulantes torna-se uma ferramenta importante na detecção da migração dos subtipos do vírus da dengue envolvidos em epidemias.

INTRODUCTION: The precise identification of the genetic variants of the dengue virus is important to understand its dispersion and virulence patterns and to identify the strains responsible for epidemic outbreaks. This study investigated the genetic variants of the capsid-premembrane junction region fragment in the dengue virus serotypes 1 and 2 (DENV1-2). METHODS: Samples from 11 municipalities in the State of Paraná, Brazil, were provided by the Central Laboratory of Paraná. They were isolated from the cell culture line C6/36 (Aedes albopictus) and were positive for indirect immunofluorescence. Ribonucleic acid (RNA) extracted from these samples was submitted to the reverse transcription polymerase chain reaction (RT-PCR) and nested PCR. RESULTS: RT-PCR revealed that 4 of the samples were co-infected with both serotypes. The isolated DENV-1 sequences were 95-100% similar to the sequences of other serotype 1 strains deposited in GenBank. Similarly, the isolated DENV-2 sequences were 98-100% similar to other serotype 2 sequences in GenBank. According to our neighbor-joining tree, all strains obtained in this study belonged to genotype V of DENV-1. The DENV-2 strains, by contrast, belonged to the American/Asian genotypes. CONCLUSIONS: The monitoring of circulating strains is an important tool to detect the migration of virus subtypes involved in dengue epidemics.

Species of Chironomidae are widely used as bioindicators of water quality, since their larvae undergo morphological deformities when in contact with sediment contaminated with chemicals. In this work we endeavored to study the morphology of head structures (antennae, mandible, mentum, pecten epipharyngis, ventromental plate and premandible) throughout the development of the four larval instars of Chironomus sancticaroli Strixino & Strixino, 1981, which can be used in environmental impact analyses. Our results show that it is possible to differentiate among larval instars by doing a quantitative analysis on the number of striae on the ventromental plates. The six structures analyzed changed during larval ontogeny. These changes are part of the ontogeny of the immature stages not exposed to xenobiotics. We believe that the morphological pattern defined in this work can be used for comparisons with ontogenetic changes observed in field studies conducted in polluted environments.

El dengue es causado por un Flavivirus que presenta una alta diversidad genética, cuatro serotipos y varios genotipos. Esta enfermedad es endémica en la mayoría de los países del continente Americano y sólo las hembras de Aedes (Stegomyia) aegypti (Linnaeus, 1762) son las transmisoras de esta enfermedad de importancia epidemiológica. Un único mosquito infectado permanece así por el resto de su vida pudiendo infectar múltiples hospederos humanos. El detectar el serotipo de virus de dengue presente en los mosquitos permite conocer, ya sea, el serotipo circulante o el ingreso de un nuevo serotipo a una determinada región sin ningún tipo de implicaciones éticas presentando así resultados reproducibles. Ésta revisión aborda las técnicas de detección viral en mosquitos, sus ventajas y limitaciones, así como estudios previos con poblaciones naturales de Aedes aegypti.

Dengue is caused by Flavivirus which exhibits high genetic diversity, with four serotypes and various genotypes. The disease is endemic in most countries in the Americas, and the female Aedes (Stegomyia) aegypti (Linnaeus, 1762) are the only transmitters of epidemiological importance. A single infected mosquito remains so for the rest of his life and can infect multiple human hosts. For dengue virus detection in mosquitoes, the circulating serotype can be revealed or detect an entry of a new serotype in the region, without any ethical implications, and reproducible results. This review covers the techniques for detecting virus in mosquitoes, their advantages and limitations, as well as previous studies with natural populations of Aedes aegypti.

A dengue é causada por um Flavivirus que apresenta elevada diversidade genética, com quatro sorotipos e vários genótipos. A enfermidade é endêmica na maioria dos países das Américas, e as fêmeas de Aedes (Stegomyia) aegypti (Linnaeus, 1762) são as únicas transmissoras, com importância epidemiológica. Um único mosquito infectado permanece assim pelo resto de sua vida, podendo infectar múltiplos hospedeiros humanos. Com a detecção do vírus da dengue em mosquitos, podemos revelar o sorotipo circulante ou a entrada de um novo sorotipo em uma determinada região, sem quaisquer implicações éticas, além de apresentar reprodutibilidade dos resultados. Esta revisão aborda as técnicas para detecção viral em mosquitos, suas vantagens e limitações, bem como as pesquisas já realizadas com populações naturais de Aedes aegypti.

Los marcadores moleculares microsatelitales se caracterizan por su neutralidad, alto polimorfismo y elevada abundancia con amplia distribución en el genoma de eucariotas. Su empleo en estudios relacionados a especies de mosquitos vectores es ideal para el mapeo genético y físico, para la identificación y discriminación de genotipos y estudios de genética de poblaciones. Esta revisión sintetiza y provee información sobre estudios de mosquitos que utilizan marcadores microsatelitales y propone nuevas líneas de investigación para una mejor comprensión de la biología y dinámica de transmisión del virus de la Fiebre Amarilla por especies del género Haemagogus.

Microsatellite molecular markers are characterized by neutrality, high polymorphism and wide distribution with high abundance in the genome of eukaryotes. Its use in studies related to species of mosquito vectors is ideal for genetic and physical mapping, for the identification and discrimination of genotypes and genetic studies of populations. This review summarizes and provides information on studies involving mosquitoes by using microsatellite markers and suggests avenues of research to better understand the biology and dynamics of transmission of Yellow Fever virus by Haemagogus mosquitoes.

Marcadores moleculares microssatélites se caracterizam pela neutralidade, alto polimorfismo e elevada abundância com ampla distribuição pelo genoma de eucariotos. O seu emprego em estudos relacionados a espécies de mosquitos vetoras é ideal para o mapeamento genético e físico, para a identificação e discriminação de genótipos e estudos de genética de populações. Esta revisão sumariza e fornece informações sobre estudos envolvendo culicídeos através da utilização de marcadores microssatélites e propõe sugestões de pesquisas para uma melhor compreensão da biologia e dinâmica de transmissão do vírus da febre amarela por espécies do gênero Haemagogus.

O gênero Parapentaneura foi estabelecido em 2006 e apresenta somente uma espécie, Parapentaneura bentogomensis, proveniente do Estado do Mato Grosso. Esta espécie foi coletada no Estado de São Paulo e então redescrita detalhadamente, apresentando caracteres adicionais. Os espécimes de ambas as localidades foram comparados, possibilitando o aperfeiçoamento da diagnose do gênero. O novo registro amplia consideravelmente a distribuição geográfica de Parapentaneura.

The genus Parapentaneura was established in 2006 and is composed of a single species, Parapentaneura bentogomensis, originally from Mato Grosso State. This species was collected in São Paulo State and is thus redescribed. The specimens from both localities were compared, adding new characters to the description of the species and emending the genus diagnosis. The new record increases considerably the geographical distribution of Parapentaneura.

Realizaram-se bioensaios para detectar a susceptibilidade de Aedes aegypti aos inseticidas químicos, temefós e cipermetrina. Os resultados mostraram que esta espécie é suscetível a temefós e apresenta resistência a cipermetrinae.

Bioassays were performed in order to detect the susceptibility of Aedes aegypti to the chemical insecticides temephos and cypermethrin. The results showed that this species is susceptible to temephos and presents resistance to cypermethrin.

Realizaram-se bioensaios para detectar a susceptibilidade de Aedes aegypti aos inseticidas químicos, temefós e cipermetrina. Os resultados mostraram que esta espécie é suscetível a temefós e apresenta resistência a cipermetrinae.

Bioassays were performed in order to detect the susceptibility of Aedes aegypti to the chemical insecticides temephos and cypermethrin. The results showed that this species is susceptible to temephos and presents resistance to cypermethrin.