Morphological analysis of three populations of Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae) from Colombia

Based on the results of comparative analyses of 1,039 specimens of several progenies of Anopheles nuneztovarifrom three localities in Colombia, eight costal wing spot patterns were observed. Patterns I and III were the most frequent: 77.96% and 11.36%, respectively. Using the diagnostic characters ratio of the length of the basal dark area of hind tarsomere II/length of hind tarsomere II, ratio of the length of the humeral pale spot/length of the pre-humeral dark spot, and the ratio of the length of the subcostal pale spot/length of the distal sector dark spot (DS-III2/Ta-III2, HP/PHD, SCP/DSD) approximately 5% of the adult females were misidentified as a species of Nyssorhynchus, different from An. nuneztovari. Approximately 5% of the specimens showed DS-III2/Ta-III2 ratio less than 0.25 (range 0.21 - 0.24), and among them 3.34% shared a HP/PHD ratio less than 1.50. Consequently, 1.52% of An. nuneztovari individuals can be misidentified as Anopheles oswaldoi. In those specimens with the DS-III2/Ta-III2 ratios higher than 0.25, 34.45% displayed SCP/DSD values greater than 0.50 and of these, 3.65% displayed HP/PHD values greater than 1.8. This combination of characters could lead one to misidentify samples of An. nuneztovari as Anopheles rangeli. Similarly, 2.43% of the females could be identified erroneously as either Anopheles aquasalis or Anopheles benarrochi. Individuals with a HP/PHD ratio greater than 2.0, could be misidentified as Anopheles trinkae, Anopheles strodei or Anopheles evansae. A distinct combination of diagnostic characters for An. nuneztovari from Colombia is proposed.

Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) rDNA and partial sequences of the cytochrome coxidase subunit I (COI) mtDNA and white gene nDNA were obtained from specimens of Anopheles nuneztovari A collected in Macapá (state of Amapá), Óbidos, Prainha and Almeirim (state of Pará), Itacoatiara and Parintins (state of Amazonas), Brazil, and compared with previously published sequences of A. nuneztovari s.l. Results of the Bayesian phylogenetic analyses performed using either COI or combined ITS2, COI and white gene sequences suggest that An. nuneztovari B/C is distinct from specimens obtained in the Amazonas/Solimões River basin. Anopheles goeldii, currently in synonymy with An. nuneztovari, was described from individuals collected in Belterra (= Fordlândia) in the Tapajós River, state of Pará, Southern Amazonas River. Morphological comparisons of the characteristics of the male genitalia indicated that An. nuneztovari A and An. goeldii are similar but distinct from An. nuneztovariB/C by the apex of the aedeagus. In considering the results of the phylogenetic analyses and morphological comparisons, An. goeldii is resurrected from synonymy with An. nuneztovari. Additionally, Anopheles dunhamiis reported for the first time in Parintins. This species can be distinguished from An. goeldiiby characters of the male genitalia and molecular data

LARVICIDAL ACTIVITY OF Bacillus sphaericus 2362 AGAINST Anopheles nuneztovari, Anopheles darlingi AND Anopheles braziliensis (DIPTERA, CULICIDAE)

In this present study, preliminary data was obtained regarding the mortality rate of the Amazonian anophelines, Anopheles nuneztovari, Anopheles darlingi and Anopheles braziliensis when subjected to treatment with Bacillus sphaericus strain 2362, the WHO standard strain. Initially, experiments were conducted to test the mortality rate of the three species of anopheline larvae. The third larval instar of An. nuneztovari and the second and third larval instars of An. darlingi proved to be the least susceptible. In other experiments, the same three mosquito species were tested with the standard strain 2362, An. nuneztovari was the least susceptible to this insect pathogen, while An. braziliensis was the most susceptible. This latter species showed a difference in the level of LC50 concentration, when compared to the former, of 2.4, 2.5 and 1.8 in readings taken 24, 48 and 72 hours after exposure to the bacillus.

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae) from Western and Northeastern Colombia

Random amplified polymorphic DNA (RAPD) markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8) but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8). According to molecular variance analysis, the genetic distance between populations was significant (phiST 0.1131, P < 0.001). The variation among individuals within populations (phiST 0.8869, P < 0.001)was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.

Location of ribosomal genes in the chromosomes of Anopheles darlingi and Anopheles nuneztovari (Diptera, Culicidae) from the Brazilian Amazon

Fluorescence in situ hybridization of Anopheles darlingi and A. nuneztovari demonstrated nucleolar organizer region activity at the end of the fourth larval instar, when the nucleolar organizer regions underwent gradual condensation. The heteromorphic sex chromosomes showed intraindividual size variation in the rDNA blocks located in the pericentromeric region and this coincided with the location of constitutive heterochromatin (C-banding).

Morphological analysis of three populations of Anopheles (Nyssorhynchus) nuneztovari Gabaldón (Diptera: Culicidae) from Colombia

Based on the results of comparative analyses of 1,039 specimens of several progenies of Anopheles nuneztovarifrom three localities in Colombia, eight costal wing spot patterns were observed. Patterns I and III were the most frequent: 77.96% and 11.36%, respectively. Using the diagnostic characters ratio of the length of the basal dark area of hind tarsomere II/length of hind tarsomere II, ratio of the length of the humeral pale spot/length of the pre-humeral dark spot, and the ratio of the length of the subcostal pale spot/length of the distal sector dark spot (DS-III2/Ta-III2, HP/PHD, SCP/DSD) approximately 5% of the adult females were misidentified as a species of Nyssorhynchus, different from An. nuneztovari. Approximately 5% of the specimens showed DS-III2/Ta-III2 ratio less than 0.25 (range 0.21 - 0.24), and among them 3.34% shared a HP/PHD ratio less than 1.50. Consequently, 1.52% of An. nuneztovari individuals can be misidentified as Anopheles oswaldoi. In those specimens with the DS-III2/Ta-III2 ratios higher than 0.25, 34.45% displayed SCP/DSD values greater than 0.50 and of these, 3.65% displayed HP/PHD values greater than 1.8. This combination of characters could lead one to misidentify samples of An. nuneztovari as Anopheles rangeli. Similarly, 2.43% of the females could be identified erroneously as either Anopheles aquasalis or Anopheles benarrochi. Individuals with a HP/PHD ratio greater than 2.0, could be misidentified as Anopheles trinkae, Anopheles strodei or Anopheles evansae. A distinct combination of diagnostic characters for An. nuneztovari from Colombia is proposed.

Resurrection of Anopheles goeldii from synonymy with Anopheles nuneztovari (Diptera, Culicidae) and a new record for Anopheles dunhami in the Brazilian Amazon

Nucleotide sequences of the internal transcribed spacer 2 (ITS2) rDNA and partial sequences of the cytochrome coxidase subunit I (COI) mtDNA and white gene nDNA were obtained from specimens of Anopheles nuneztovari A collected in Macapá (state of Amapá), Óbidos, Prainha and Almeirim (state of Pará), Itacoatiara and Parintins (state of Amazonas), Brazil, and compared with previously published sequences of A. nuneztovari s.l. Results of the Bayesian phylogenetic analyses performed using either COI or combined ITS2, COI and white gene sequences suggest that An. nuneztovari B/C is distinct from specimens obtained in the Amazonas/Solimões River basin. Anopheles goeldii, currently in synonymy with An. nuneztovari, was described from individuals collected in Belterra (= Fordlândia) in the Tapajós River, state of Pará, Southern Amazonas River. Morphological comparisons of the characteristics of the male genitalia indicated that An. nuneztovari A and An. goeldii are similar but distinct from An. nuneztovariB/C by the apex of the aedeagus. In considering the results of the phylogenetic analyses and morphological comparisons, An. goeldii is resurrected from synonymy with An. nuneztovari. Additionally, Anopheles dunhamiis reported for the first time in Parintins. This species can be distinguished from An. goeldiiby characters of the male genitalia and molecular data.

Molecular evidence for a single taxon, Anopheles nuneztovari s.l., from two endemic malaria regions in Colombia

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F ST) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.

Incrimination of Anopheles (Anopheles) intermedius Peryassú, An. (Nyssorhynchus) nuneztovari Gabaldón, An. (Nys.) oswaldoi Peryassú as natural vectors of Plasmodium falciparum in French Guiana

Anopheles darlingi Root is the major vector of human malaria in the Neotropics and has been considered to be the sole malaria vector in French Guiana. The presence of other potential vectors suggests that malaria may be transmitted by other species under certain conditions. From 2006-2011, all anopheline specimens collected from 11 localities were assayed to determine if the Plasmodium circumsporozoite protein was present. In addition to An. darlingi, we found Anopheles oswaldoi, Anopheles intermedius and Anopheles nuneztovari specimens that were infected with Plasmodium sp. Further investigations on the behaviour and ecology of An. oswaldoi, An. intermedius and An. nuneztovari are necessary to determine their role in malaria transmission in French Guiana.

Anopheles goeldii Rozeboom & Gabaldón (Diptera, Culicidae): a species of the Nuneztovari Complex of Anopheles Meigen

Anopheles (Nyssorhynchus) goeldiiRozeboom & Gabaldón, 1941, a species of the Nuneztovari Complex, was described based on morphological characteristics of the male, female, larva, pupa, and eggs. The type locality is Boa Vista (= Fordlândia), a district in the vicinity of Rio Tapajós, in the municipality of Aveiro, in the state of Pará, Brazil. Anopheles goeldii is redescribed based on morphological traits of the fourth instar larva, pupa, egg, and male and female. DNA sequences from the cytochrome oxidase subunit I (COI barcode region) of the mitochondrial genome were utilized for species characterization. Specimens of An. goeldii from the Pará, Amapá, and Amazonas states were employed to redescribe the species and to compare with morphologically similar taxa.

Allozyme differentiation among allopatric populations of Anopheles nuneztovari (Diptera: Culicidae)

Four enzymes in six geographic populations, four Brazilian and two Colombian, of Anopheles nuneztovari were studied. There were differences among the most frequent alleles in the EST5, ACON and MDH loci in the populations from Brazil and Colombia. The α-GPD(*)C allele was encountered very frequently in the Palo/Sit population in Colombia and the α-GPD(*)B allele was found to be fixed in most of the Brazilian populations. An additional α-GPD band was found only in Palo/Sit. The considerable genetic differentiation between populations in western Colombia vs. Brazil suggests that a certain degree of reproductive isolation exists between them.

Susceptibilidade experimental do Anopheles (Nyssorhynchus) nuneztovari Galbadon, 1940 ao Plasmodium vivax Grassi&Feletti, 1890 e Plasmodium falciparum Welch, 1897

A importância do An. nuneztovari como vetor primário de malária já foi comprovado em países da América do Sul como Venezuela, Colômbia e Peru. Na Amazônia brasileira, embora tenha sido encontrado naturalmente infectado com Plasmodium vivax e P. falciparum e em alta densidade, é ainda considerado vetor secundário desta doença. O objetivo deste presente trabalho foi avaliar a susceptibilidade do An. nuneztovari à infecção por plasmódios humanos. Para isso exemplares da geração F1, obtida em laboratório, de An. nuneztovari e An. darlingi (espécie controle) foram alimentados, em alimentador artificial, com sangue de pacientes com diagnóstico inicial de malária causada por P. falciparum, cuja revisão resultou no diagnóstico de infecção mista. Todas as amostras sangüíneas dos pacientes infectaram espécimes das duas espécies, não mostrando diferença significativa entre elas quanto à susceptibilidade. Para detecção de infecção malárica nos mosquitos foi usado o teste ELISA (Enzime – Linked Imunosorbent Assay) cujos resultados foram discordantes do diagnóstico laboratorial, já que o teste detectou infecções pelo P. falciparum, P. vivax VK210 ou P. vivax VK247entre os mosquitos positivos sugerindo que os pacientes apresentavam infecção mista. Também foi observado o curto período de desenvolvimento de oocistos e esporozoítos, de quatro a cinco dias, o que pode ser explicado pela alta temperatura (>30°C) que os mosquitos foram expostos. Assim nossos resultados sugerem possível envolvimento do An nuneztovari na transmissão de malária humana na área estudada e alertam para o papel deste, como possível vetor principal de malária humana na região amazônica brasileira.

Caracterização e relacionamento antigênico de três novos Bunyavirus no grupo Anopheles A (Bunyaviridae) dos arbovirus

São descritos o isolamento e a caracterização de três novos arbovirus isolados na região da Usina Hidro-Elétrica de Tucuruí (UHE-TUC). Os três novos arbovirus pertencem ao grupo Anopheles A(ANA), gênero Bunyavirus (família Bunyaviridae). Os vírus Tucuruí (TUC), Caraipé (CPE) e Arumateua (ART) são relacionados entre si e com o vírus Trombetas (TBT), formando dentro do grupo ANA um complexo chamado Trombetas. Os arbovirus TUC, CPE e ART foram obtidos a partir de lotes de mosquitos Anopheles (Nyssorhynchus) sp capturados em Tucuruí, nas proximidades da usina hidrelétrica de Tucuruí, Estado do Pará, nos meses de fevereiro, agosto e outubro de 1984, respectivamente. Até o final de 1990 os vírus TUC, CPE e ART foram isolados 12, 32 e 28 vezes respectivamente, sempre na região da UHE-TUC, exceção feita ao vírus TUC, do qual se obteve uma amostra procedente de Balbina, onde também foi construída uma hidroelétrica. Até o presente, esses vírus só foram isolados a partir de mosquitos do grupo An. (Nys.) principalmente, a partir das espécies An. (Nys.) nuneztovari e An. (Nys.) triannulatus também consideradas vetores secundários da malária na Amazônia Brasileira. Testes sorológicos executados com soros humanos e de diversas espécies de animais silvestres foram negativos, com exceção de um soro de um carnívoro de espécie Nasua nasua que neutralizou a amostra TUC em títulos de 2.6 índice logaritmico de neutralização (ILN).

Espécies de Anopheles (Culicidae, Anophelinae) em área endêmica de malária, Maranhão, Brasil

OBJETIVO: Estudar a flutuação sazonal, freqüência horária, abundância relativa e riqueza de espécies de Anopheles em ambiente antrópico, para entender a bioecologia do grupo e para a monitorização do programa de controle da malária. MÉTODOS: Os anofelinos foram estudados durante um ano, de outubro de 1996 a setembro de 1997, das 18 às 6 horas, a cada 30 dias, no Município da Raposa, Ilha de São Luís, Maranhão. Utilizou-se o método de captura de fêmeas em iscas humanas no intra e peridomicílio, com tubo de sucção e foco luminoso orientado. RESULTADOS: Foram coletados 1.407 espécimes assim distribuídos: Anopheles aquasalis (82%), Anopheles galvaoi (10,2%) e Anopheles albitarsis (6,4%). As demais espécies, Anopheles evansae, Anopheles nuneztovari e Anopheles triannulatus davisi representaram juntas 1,4%. Os anofelinos ocorreram o ano inteiro, principalmente no período chuvoso, sendo mais freqüentes no intra (75,3%) do que no peridomicílio (24,7%), preferindo sugar sangue no crepúsculo vespertino e nas primeiras horas da noite. CONCLUSÃO: As variações observadas no comportamento do anofelino mostram que as diferentes espécies vêm adaptando-se, em maior ou menor grau, ao convívio com o homem nas suas habitações.