Morphological Description of the Immature Stages of Nyssomyia umbratilis (Ward & Frahia) (Diptera: Psychodidae: Phlebotominae), the Main Vector of Leishmania guyanensis Floch (Kinetoplastida: Trypanosomatidae) in the Brazilian Amazon Region.

Nyssomyia umbratilis (Ward & Frahia) is the main vector of Leishmania guyanensis in the Brazilian Amazon region, where it is widely distributed. Studies have hypothesized that this phlebotomine is part of a cryptic species complex, spatially delimited by the Amazonian river systems, and with different transmission potential of L. guyanensis. In the present study, the immature stages of N. umbratilis are described from laboratory-reared specimens originating from the states of Amazonas and Pará, Brazil. Based on scanning electron and optical microscopy examinations, fine morphological structures (eggshell, setae, and mouthparts of larvae and pupae) are presented. A new form of sandfly pupae sexing (non-invasive) is also provided. The correct identification of insect is extremely important for the epidemiology of certain diseases. However, only the morphological characters of adult can be insufficient to separate accurately the closely related species. It is expected that the present description may contribute to solve the taxonomic problem involving N. umbratilis.

New microsatellite markers for the neotropical malaria vector Anopheles nuneztovari sensu lato.

Anopheles nuneztovari sensu lato consists of cryptic species and genetic lineages, one of which is an important human malaria vector in the northern part of South America. Population structure and evolutionary genetics studies may help in the definition and delimitation of the species and lineages within this species complex, which is relevant information for organizations involved in malaria control efforts. In this study, 10 new microsatellite markers were isolated from 2 repeat-enriched genomic libraries of A. nuneztovari s.l. and were characterized in 37-48 mosquitoes of this species. All loci were highly polymorphic and encompassed 5-25 alleles per locus. The observed (HO) and expected (HE) heterozygosities ranged from 0.354 to 0.866 and from 0.613 to 0.932, respectively. Six of the 10 new loci showed significant deviations from the Hardy-Weinberg equilibrium, and no linkage disequilibrium was detected. The loci described in this study were more polymorphic than the 18 previously characterized loci and appear to be promising markers for use in investigating the fine-scale population genetic structure and the boundaries of the cryptic species and lineages within the A. nuneztovari complex.

Reduced levels of genetic variation in Aedes albopictus (Diptera: Culicidae) from Manaus, Amazonas State, Brazil, based on analysis of the mitochondrial DNA ND5 gene.

Aedes albopictus, a mosquito originally from Southeast Asia, is considered to be one of the main vectors of dengue fever, yellow fever and other arboviruses. We examined the genetic variability and population structure of 68 individuals of Ae. albopictus collected from five neighborhoods of the city of Manaus, based on the mitochondrial gene coding for NADH dehydrogenase subunit 5 (ND5). Two haplotypes were found, separated by a single mutational event (T <--> C), with extremely low levels of genetic variability (h = 0.187 +/- 0.059; pi = 0.00044 +/- 0.00014). Based on AMOVA, we concluded that most of the variation (99.08%) occurred within populations, though the levels of variation were not significant. Neutrality tests (Tajima's D and Fu's Fs) were non-significant, indicating that these populations are in genetic equilibrium. The most frequent haplotype (H1) is restricted to Brazilian populations of Ae. albopictus, while the rarer haplotype (H2) is shared with populations from the United States and Asia. We suggest that the reduced variability and low genetic structure identified in our study is a consequence of the recent introduction of this species in Manaus, possibly through a founder effect, followed by expansion throughout the city neighborhoods. Genetic similarity would therefore be due to insufficient time to have accumulated genetic differences between the populations of Ae. albopictus and not to extensive gene flow among them.

Molecular differentiation in natural populations of Anopheles oswaldoi sensu lato (Diptera: Culicidae) from the Brazilian Amazon, using sequences of the COI gene from mitochondrial DNA.

Anopheles (Nyssorhynchus) oswaldoi (Peryassú, 1922) s. l., which has been incriminated as a potential human malaria vector in Western Brazilian Amazon, may constitute a cryptic species complex. However, the most recent study with isozymes indicated high similarity among samples from the States of Acre, Amazonas and Rondônia in the Brazilian Amazon. In the present study, 45 individuals were sequenced from Sena Madureira (State of Acre), Coari (State of Amazonas), São Miguel (State of Rondônia), and Moju (State of Pará), using the cytochrome oxidase I gene from mitochondrial DNA. Twenty-five haplotypes were identified in the four localities, and no haplotype was shared among them. The lowest haplotype number was detected in the Coari sample. The dendrogram based on maximum parsimony analysis yielded four groups: I) haplotypes 1, 2, 3, 4, and 5 from Sena Madureira and haplotypes 17 and 18 from São Miguel; II) haplotypes 13 to 16 and 19 to 22 from São Miguel; III) haplotypes 23 to 25 from Moju, and IV) haplotypes 6 to 9 from Sena Madureira and haplotypes 10 to 12 from Coari. The genetic distance (uncorrected p) obtained among the four groups ranged from 0.08 to 5.3%, whereas the highest values (4.97 to 5.3%) were found between groups I (Sena Madureira) and III (Moju). Based on male genitalia identification, it was suggested that group I may be A. oswaldoi s. s. whereas group IV may be A. konderi. Groups II and III could constitute other lineages or species within A. oswaldoi s. l., whose taxonomic status remains to be clarified. These results suggest that additional studies are necessary using samples of A. oswaldoi s. l. from a larger geographic area.

Molecular differentiation in natural populations of Anopheles oswaldoi sensu lato (Diptera: Culicidae) from the Brazilian Amazon, using sequences of the COI gene from mitochondrial DNA

Anopheles (Nyssorhynchus) oswaldoi (Peryassú, 1922) s. l., which has been incriminated as a potential human malaria vector in Western Brazilian Amazon, may constitute a cryptic species complex. However, the most recent study with isozymes indicated high similarity among samples from the States of Acre, Amazonas and Rondônia in the Brazilian Amazon. In the present study, 45 individuals were sequenced from Sena Madureira (State of Acre), Coari (State of Amazonas), São Miguel (State of Rondônia), and Moju (State of Pará), using the cytochrome oxidase I gene from mitochondrial DNA. Twenty-five haplotypes were identified in the four localities, and no haplotype was shared among them. The lowest haplotype number was detected in the Coari sample. The dendrogram based on maximum parsimony analysis yielded four groups: I) haplotypes 1, 2, 3, 4, and 5 from Sena Madureira and haplotypes 17 and 18 from São Miguel; II) haplotypes 13 to 16 and 19 to 22 from São Miguel; III) haplotypes 23 to 25 from Moju, and IV) haplotypes 6 to 9 from Sena Madureira and haplotypes 10 to 12 from Coari. The genetic distance (uncorrected p) obtained among the four groups ranged from 0.08 to 5.3%, whereas the highest values (4.97 to 5.3%) were found between groups I (Sena Madureira) and III (Moju). Based on male genitalia identification, it was suggested that group I may be A. oswaldoi s. s. whereas group IV may be A. konderi. Groups II and III could constitute other lineages or species within A. oswaldoi s. l., whose taxonomic status remains to be clarified. These results suggest that additional studies are necessary using samples of A. oswaldoi s. l. from a larger geographic area.

Enzymatic analysis in Anopheles nuneztovari Gabaldón (Diptera, Culicidae)

Enzymatic analysis in Anopheles nuneztovari was made using four populations from the Brazilian Amazon and two from Colombia. The enzymes ME and XDH presented a monomorphic locus in all of the studied populations. EST and LAP presented a higher number of loci. In EST, genetic variation was observed in the five loci; LAP presented four loci, with allec variation in two loci. In IDH, three activity regions were stained, with genetic variation for locus Idh-1 in the Brazilian Amazon populations. A locus for MDH was observed, with genetic variation in the six populations. A region was verified for ACON, with four alleles in Sitronela and three in the other populations. PGM constituted one locus, with a high variability in the Brazilian Amazon populations. A locus was observed for 6-PGD with allelic variation in all of the populations with the exception of Tibú. Enzyme PGI presented two loci, both with genetic variability in the Tucuruí population. The enzyme alpha-GPD showed an activity region with polymorphism in the Tucuruí, Tibú and Sitronela populations. The phenotypic variations detected for these enzymes suggest that four (EST, LAP, ACON and PGM) possess monomeric structures and five (IDH, MDH, 6-PGD, PGI and alpha-GPD) dimeric structures in their proteins. These enzymes constitute in important markers to estimate variability and genetic divergence in natural populations of A. nuneztovari

Enzymatic analysis in Anopheles nuneztovari Gabaldón (Diptera, Culicidae).

Enzymatic analysis in Anopheles nuneztovari was made using four populations from the Brazilian Amazon and two from Colombia. The enzymes ME and XDH presented a monomorphic locus in all of the studied populations. EST and LAP presented a higher number of loci. In EST, genetic variation was observed in the five loci; LAP presented four loci, with allec variation in two loci. In IDH, three activity regions were stained, with genetic variation for locus Idh-1 in the Brazilian Amazon populations. A locus for MDH was observed, with genetic variation in the six populations. A region was verified for ACON, with four alleles in Sitronela and three in the other populations. PGM constituted one locus, with a high variability in the Brazilian Amazon populations. A locus was observed for 6-PGD with allelic variation in all of the populations with the exception of Tibú. Enzyme PGI presented two loci, both with genetic variability in the Tucuruí population. The enzyme alpha-GPD showed an activity region with polymorphism in the Tucuruí, Tibú and Sitronela populations. The phenotypic variations detected for these enzymes suggest that four (EST, LAP, ACON and PGM) possess monomeric structures and five (IDH, MDH, 6-PGD, PGI and alpha-GPD) dimeric structures in their proteins. These enzymes constitute in important markers to estimate variability and genetic divergence in natural populations of A. nuneztovari.

Population structure and genetic divergence in Anopheles nuneztovari (Diptera: Culicidae) from Brazil and Colombia.

Anopheles nuneztovari is considered an important vector of human malaria in several localities in Venezuela and Colombia. Its status as a vector of human malaria is still unresolved in areas of the Brazilian Amazon, in spite of have been found infected with Plasmodium sp.. For a better understanding of the genetic differentiation of populations of A. nuneztovari, electrophoretic analysis using 11 enzymes was performed on four populations from Brazil and two from Colombia. The results showed a strong differentiation for two loci: alpha-glycerophosphate dehydrogenase (alpha-Gpd) and malate dehydrogenase (Mdh) from 16 loci analyzed. Diagnostic loci were not detected. The populations of A. nuneztovari from the Brazilian Amazon showed little genetic structure and low geographic differentiation, based on the F(IS) (0.029), F(ST) (0.070), and genetic distance (0.001-0.032) values. The results of the isozyme analysis do not coincide with the indication of two lineages in the Amazon Basin by analysis of mitochondrial DNA, suggesting that this evolutionary event is recent. The mean F(ST) value (0.324) suggests that there is considerable genetic divergence among populations from the Brazilian Amazon and Colombia. The genetic distance among populations from the Brazilian Amazon and Colombia is ranges from 0.047 to 0.148, with the highest values between the Brazilian Amazon and Sitronela (SIT) (0.125-0.148). These results are consistent with those observed among members of anopheline species complexes. It is suggested that geographic isolation has reduced the gene flow, resulting in the genetic divergence of the SIT population. Dendrogram analysis showed three large groups: one Amazonian and two Colombia, indicating some genetic structuring. The present study is important because it attempted to clarify the taxonomic status of A. nuneztovari and provide a better understanding of the role of this mosquito in transmission of human malaria in northern South America.

Ecologic observations on anopheline vectors of malaria in the Brazilian Amazon.

Human intervention in the Brazilian Amazon region promotes contacts between humans and vectors that may favor the propagation of anopheline mosquitoes and the spread of malaria in the absence of planning and infrastructure to control this disease. Vector ecology studies were carried out to determine the risk areas. These data should help in designing appropriate malaria control measures. Data from 14 different regions are reported. Vectors are able to adapt to different environments, which made it necessary to study each area. The parameters studied were Anopheles breeding sites, species distribution, incidence, feeding preferences, hours of maximum activity of adult mosquitoes, seasonality, resting places, and the presence of Plasmodium. Species complexes were also studied. Anopheles darlingi may be responsible for maintaining malaria in human populations in this region. A reduction in the population density of A. darlingi in a particular geographic area can sometimes cause the disappearance of malaria. This species feeds at night but has a peak of activity at the beginning of the evening and another at dawn. Other species are mainly crepuscular and all anophelines demonstrated pronounced exophilia. The timing of feeding activities was found to vary in areas altered by human intervention and also depended on the time of the year and climatic conditions. The larvae were more abundant in the rivers with a less acidic pH and rural areas showed the highest larval index.