Jan E. Conn

Amazonian malaria: asymptomatic human reservoirs, diagnostic challenges, environmentally driven changes in mosquito vector populations, and the mandate for sustainable control strategies.

Across the Americas and the Caribbean, nearly 561,000 slide-confirmed malaria infections were reported officially in 2008. The nine Amazonian countries accounted for 89% of these infections; Brazil and Peru alone contributed 56% and 7% of them, respectively. Local populations of the relatively neglected parasite Plasmodium vivax, which currently accounts for 77% of the regional malaria burden, are extremely diverse genetically and geographically structured. At a time when malaria elimination is placed on the public health agenda of several endemic countries, it remains unclear why malaria proved so difficult to control in areas of relatively low levels of transmission such as the Amazon Basin. We hypothesize that asymptomatic parasite carriage and massive environmental changes that affect vector abundance and behavior are major contributors to malaria transmission in epidemiologically diverse areas across the Amazon Basin. Here we review available data supporting this hypothesis and discuss their implications for current and future malaria intervention policies in the region. Given that locally generated scientific evidence is urgently required to support malaria control interventions in Amazonia, we briefly describe the aims of our current field-oriented malaria research in rural villages and gold-mining enclaves in Peru and a recently opened agricultural settlement in Brazil.

Molecular population genetics of the malaria vector Anopheles darlingi in Central and South America.

To analyze the genetic relatedness and phylogeographic structure of Anopheles darlingi from 19 localities throughout Central and South America, we used a minimum spanning network, diversity measures, differentiation, neutrality tests, and mismatch distribution with mitochondrial cytochrome oxidase subunit I (COI) sequences. All the Central American haplotypes were separated by seven mutational steps from the South American haplotypes and the FST distance-based neighbor-joining tree showed a primary division between Central and South America, evidence for a putative gene pool division. More ancestral and diverse haplotypes were found in Amazonian and southern Brazil populations, suggesting that Central American populations may have originated in South America. The patterns of the mtDNA haplotype diversity and five of six tests for equilibrium implicate demographic expansion in the South American populations as the historical structure, but mismatch distribution depicts populations at mutation drift equilibrium (MDE). In South America, the departure from equilibrium was consistent with an expansion that occurred during the Pleistocene.To analyze the genetic relatedness and phylogeographic structure of Anopheles darlingi from 19 localities throughout Central and South America, we used a minimum spanning network, diversity measures, differentiation, neutrality tests, and mismatch distribution with mitochondrial cytochrome oxidase subunit I (COI) sequences. All the Central American haplotypes were separated by seven mutational steps from the South American haplotypes and the FST distance-based neighbor-joining tree showed a primary division between Central and South America, evidence for a putative gene pool division. More ancestral and diverse haplotypes were found in Amazonian and southern Brazil populations, suggesting that Central American populations may have originated in South America. The patterns of the mtDNA haplotype diversity and five of six tests for equilibrium implicate demographic expansion in the South American populations as the historical structure, but mismatch distribution depicts populations at mutation drift equilibrium (MDE). In South America, the departure from equilibrium was consistent with an expansion that occurred during the Pleistocene.

Malaria Vectors, Epidemiology, and the Re-Emergence of Anopheles darlingi in Belém, Pará, Brazil

Abstract An evaluation of malaria transmission and epidemiology in the Amazonian city of Belém over the last 70 years shows that (1) Anopheles darlingi, reported to be eradicated in 1968, reappeared in the mid 1990s, with a marked increase in abundance between 1997 to 1999 in two of three districts sampled; (2) An. darlingi and An. aquasalis are each implicated in current malaria transmission in different districts of the city; (3) mosquito species diversity (in Anopheles subgenus Nyssorhynchus) has increased from two in the 1930s to six in the 1940s to 10 in the 1990s; (4) there is no overall correlation between malaria case incidence and human population size from 1940 to 1996 in Belém; (5) however, the total number of malaria cases has increased significantly since the late 1970s and over the short term from 1993 to 1999; and (6) interestingly, the short term increases are due solely to cases of Plasmodium vivax infection; cases of P. falciparum malaria are declining (significantly for Pará state only). The reappearance of An. darlingi may be a result of the continued expansion of Belém into the surrounding forest in the 1990s. In the absence of preventative measures, we predict an increase in local outbreaks of malaria in the DAENT and DAICO districts where the population sizes of An. darlingi are increasing.

Intragenomic heterogeneity of a ribosomal DNA spacer (ITS2) varies regionally in the neotropical malaria vector Anopheles nuneztovari (Diptera: Culicidae)

We investigated intragenomic heterogeneity of ITS2 within twenty individual Anopheles nuneztovari (subgenus Nyssorhynchus) from five geographical localities in the neotropics (three from Brazil and one each from Colombia and Venezuela) by cloning and sequencing PCR‐amplified copies of this spacer. Intragenomic heterogeneity was observed in thirteen of twenty mosquitoes of both sexes from all localities. As estimated by uncorrected P, however, mean sequence divergence was greater in mosquitoes from Brazil (PR = 0.0100, BL = 0.0196, AB = 0.0182) than in those from Venezuela (SO = 0.0026) or Colombia (SI = 0.0078). Sequence divergence per genome was significantly higher in mosquitoes from Brazil than in those from SO and SI. In fact, divergence among ITS2 variants within single mosquitoes from the Brazilian localities was often as great as that between localities. Similarly, the number of variants per mosquito was significantly greater in Brazil (maximum of six variants) than in both SO and SI (maximum of two variants). These results indicate that homogenization of ITS2 has proceeded to a greater extent in SO and SI than in the localities in Brazil. The differences in intragenomic heterogeneity between both SO and SI versus the Brazilian localities probably reflect either differences in population‐level processes such as gene flow and genetic drift, or the fact that these localities may represent two or more cryptic species, as suggested by other studies.

The importance of Anopheles albitarsis E and An. darlingi in human malaria transmission in Boa Vista, state of Roraima, Brazil

In several districts of Boa Vista, state of Roraima, Brazil we found Anopheles (Nyssorhynchus) albitarsis E to be the primary vector of human malaria parasites, and during 2001-2002 it was significantly more abundant than An. darlingi (p < 0.001). Other species sampled were An. (Nys.) braziliensis, An. (Ano.) peryassui, An. (Nys.) nuneztovari, An. (Nys.) oswaldoi s.l., and An. (Nys.) triannulatus. As determined by the ELISA technique An. darlingi had a higher overall infection rate (2.1%) compared with An. albitarsis E (1.2%). However a marginally higher proportion of An. albitarsis E was infected with Plasmodium vivax compared with An. darlingi, and the An. albitarsis E biting index was also much higher These results suggest the importance of An. albitarsis E in malaria transmission in a savannah ecoregion of northern Amazonian Brazil, and reconfirm the importance of An. darlingi even if at lower abundance.

Infection of New- and Old-World Aedes albopictus (Diptera: Culicidae) by the Intracellular Parasite Wolbachia: Implications for Host Mitochondrial DNA Evolution

Abstract Wolbachia are cytoplasmically inherited, endosymbiotic bacteria known to infect a wide variety of arthropods. Polymerase chain reaction (PCR) amplification of the Wolbachia surface protein (wsp) gene was used to assay the infection of geographically disparate populations of Aedes albopictus (Skuse) by Wolbachia. Nine North American, four South American, one Hawaiian, and four Old World populations of A. albopictus were all doubly infected with both the wAlbA and wAlbB strains of Wolbachia. A 365-bp region of the wAlbA wsp gene was sequenced from seven geographically disparate host populations, and all sequences were identical. Similarly, a 474-bp region of the wAlbB wsp gene was sequenced from the same populations, and all sequences were identical. These results suggest a role for Wolbachia infection in causing the previously established pattern of low mitochondrial DNA variability, but average nuclear gene diversity, within and among populations of A. albopictus.

Complete mtDNA genomes of Anopheles darlingi and an approach to anopheline divergence time

BackgroundThe complete sequences of the mitochondrial genomes (mtDNA) of members of the northern and southern genotypes of Anopheles (Nyssorhynchus) darlingi were used for comparative studies to estimate the time to the most recent common ancestor for modern anophelines, to evaluate differentiation within this taxon, and to seek evidence of incipient speciation.MethodsThe mtDNAs were sequenced from mosquitoes from Belize and Brazil and comparative analyses of structure and base composition, among others, were performed. A maximum likelihood approach linked with phylogenetic information was employed to detect evidence of selection and a Bayesian approach was used to date the split between the subgenus Nyssorhynchus and other Anopheles subgenera.ResultsThe comparison of mtDNA sequences within the Anopheles darlingi taxon does not provide sufficient resolution to establish different units of speciation within the species. In addition, no evidence of positive selection in any protein-coding gene of the mtDNA was detected, and purifying selection likely is the basis for this lack of diversity. Bayesian analysis supports the conclusion that the most recent ancestor of Nyssorhynchus and Anopheles+Cellia was extant ~94 million years ago.ConclusionAnalyses of mtDNA genomes of Anopheles darlingi do not provide support for speciation in the taxon. The dates estimated for divergence among the anopheline groups tested is in agreement with the geological split of western Gondwana (95 mya), and provides additional support for explaining the absence of Cellia in the New World, and Nyssorhynchus in the Afro-Eurasian continents.

Natural infectivity of Anopheles species from the Pacific and Atlantic Regions of Colombia

Malaria is an important public health problem in Colombia. Among the major vectors in Colombia, Anopheles albimanus is recognized for its importance on the Pacific Coast where it is the predominant species; it is also found in the Atlantic Coast, although its vectorial role in this region is not clear. We examined the occurrence of An. albimanus in four localities of the Pacific and three of the Atlantic Coast. Morphological identification of problematic specimens was confirmed by a molecular assay. All identified mosquitoes at these sites, including An. albimanus, were also tested for malaria parasite infection. From 12,189 anophelines collected, 6370 were from the Pacific Coast, and corresponded to 99% An. albimanus, 0.8% Anopheles neivai, and three other species at <0.2%. From the Atlantic Coast we identified 5819 specimens with 61% An. albimanus, 36% Anopheles triannulatus s.l. and five other species at <2%. In both coasts, species present at lower percentages included several incriminated as vectors in neighboring countries. Six Pacific Coast specimens were infected with malaria parasites: four An. albimanus, two with Plasmodium vivax VK247, one with P. vivax VK210 and one with Plasmodium falciparum; two An. neivai with P. falciparum. Our data support the continued predominance of An. albimanus in the Pacific Coast, and demonstrate that this species is the most abundant in the Atlantic Coast as well.

The biogeography and population genetics of neotropical vector species

Phylogenetic and population genetic data support the Pliocene or Pleistocene divergences of the co-distributed hematophagous insect vectors, the sand fly Lutzomyia longipalpis s.l., the mosquitoes Anopheles darlingi and A. albitarsis s.l., and the triatomines Rhodnius prolixus and R. robustus. We examined patterns of divergence and distribution in relation to three hypotheses of neotropical diversification: Miocene/Pliocene marine incursion, Pliocene/Pleistocene riverine barriers and Pleistocene refugia. Only R. prolixus has a pattern concordant with the refugia hypothesis, and R. robustus conforms to the marine incursion predictions. A. darlingi partially fits the refugia hypothesis. For L. longipalpis s.l. and A. albitarsis s.l., elements of both incursion and refugia hypotheses seem to fit, suggesting perhaps an interaction of factors determining their distribution patterns.

Microsatellite data suggest significant population structure and differentiation within the malaria vector Anopheles darlingi in Central and South America

BackgroundAnopheles darlingi is the most important malaria vector in the Neotropics. An understanding of A. darlingis population structure and contemporary gene flow patterns is necessary if vector populations are to be successfully controlled. We assessed population genetic structure and levels of differentiation based on 1,376 samples from 31 localities throughout the Peruvian and Brazilian Amazon and Central America using 5–8 microsatellite loci.ResultsWe found high levels of polymorphism for all of the Amazonian populations (mean RS = 7.62, mean HO = 0.742), and low levels for the Belize and Guatemalan populations (mean RS = 4.3, mean HO = 0.457). The Bayesian clustering analysis revealed five population clusters: northeastern Amazonian Brazil, southeastern and central Amazonian Brazil, western and central Amazonian Brazil, Peruvian Amazon, and the Central American populations. Within Central America there was low non-significant differentiation, except for between the populations separated by the Maya Mountains. Within Amazonia there was a moderate level of significant differentiation attributed to isolation by distance. Within Peru there was no significant population structure and low differentiation, and some evidence of a population expansion. The pairwise estimates of genetic differentiation between Central America and Amazonian populations were all very high and highly significant (FST = 0.1859 – 0.3901, P < 0.05). Both the DA and FST distance-based trees illustrated the main division to be between Central America and Amazonia.ConclusionWe detected a large amount of population structure in Amazonia, with three population clusters within Brazil and one including the Peru populations. The considerable differences in Ne among the populations may have contributed to the observed genetic differentiation. All of the data suggest that the primary division within A. darlingi corresponds to two white gene genotypes between Amazonia (genotype 1) and Central America, parts of Colombia and Venezuela (genotype 2), and are in agreement with previously published mitochondrial COI gene sequences interpreted as incipient species. Overall, it appears that two main factors have contributed to the genetic differentiation between the population clusters: physical distance between the populations and the differences in effective population sizes among the subpopulations.