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Background: Cryptic species complexes are common among anophelines. Previous phylogenetic analysis based on the complete mtDNA COI gene sequences detected paraphyly in the Neotropical malaria vector Anopheles marajoara. The "Folmer region" detects a single taxon using a 3% divergence threshold., Methods: To test the paraphyletic hypothesis and examine the utility of the Folmer region, genealogical trees based on a concatenated (white + 3' COI sequences) dataset and pairwise differentiation of COI fragments were examined. The population structure and demographic history were based on partial COI sequences for 294 individuals from 14 localities in Amazonian Brazil. 109 individuals from 12 localities were sequenced for the nDNA white gene, and 57 individuals from 11 localities were sequenced for the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2)., Results: Distinct A. marajoara lineages were detected by combined genealogical analysis and were also supported among COI haplotypes using a median joining network and AMOVA, with time since divergence during the Pleistocene (<100,000 ya). COI sequences at the 3' end were more variable, demonstrating significant pairwise differentiation (3.82%) compared to the more moderate 2.92% detected by the Folmer region. Lineage 1 was present in all localities, whereas lineage 2 was restricted mainly to the west. Mismatch distributions for both lineages were bimodal, likely due to multiple colonization events and spatial expansion (~798-81,045 ya). There appears to be gene flow within, not between lineages, and a partial barrier was detected near Rio Jari in Amapá state, separating western and eastern populations. In contrast, both nDNA data sets (white gene sequences with or without the retention of the 4th intron, and ITS2 sequences and length) detected a single A. marajoara lineage., Conclusions: Strong support for combined data with significant differentiation detected in the COI and absent in the nDNA suggest that the divergence is recent, and detectable only by the faster evolving mtDNA. A within subgenus threshold of >2% may be more appropriate among sister taxa in cryptic anopheline complexes than the standard 3%. Differences in demographic history and climatic changes may have contributed to mtDNA lineage divergence in A. marajoara.

Anopheles halophylus Silva-do-Nascimento & Lourenço-de-Oliveira was recently described using morphological and biological variants in specimens previously identified as Anopheles triannulatus (Neiva & Pinto). Because these two species occur in sympatry in central Brazil, we used allozymes to determine the extent of gene flow to confirm that they are different species. Of 11 allozyme loci analyzed, one (Mpi) was found to be diagnostic for An. halophylus and An. triannulatus, confirming their specific status. This locus revealed a second sibling species within An. triannulatus sensu lato. An. halophylus and the new undescribed species were confirmed using random amplified polymorphic DNA markers that showed moderate genetic divergence among these three sympatric and closely related taxa (D = 0.145-0.428). Moreover, this marker indicates that An. halophylus and the new species are more closely related to each other than either is to An. triannulatus.

Anopheles darlingi is the primary malaria vector in Latin America, and is especially important in Amazonian Brazil. Historically, control efforts have been focused on indoor house spraying using a variety of insecticides, but since the mid-1990s there has been a shift to patient treatment and focal insecticide fogging. Anopheles darlingi was believed to have been significantly reduced in a gold-mining community, Peixoto de Azevedo (in Mato Grosso State), in the early 1990s by insecticide use during a severe malaria epidemic. In contrast, although An. darlingi was eradicated from some districts of the city of Belem (the capital of Para State) in 1968 to reduce malaria, populations around the water protection area in the eastern district were treated only briefly. To investigate the population structure of An. darlingi including evidence for a population bottleneck in Peixoto, we analyzed eight microsatellite loci of 256 individuals from seven locations in Brazil: three in Amapa State, three in Para State, and one in Mato Grosso State. Allelic diversity and mean expected heterozygosity were high for all populations (mean number alleles/locus and H(E) were 13.5 and 0.834, respectively) and did not differ significantly between locations. Significant heterozygote deficits were associated with linkage disequilibrium, most likely due to either the Wahlund effect or selection. We found no evidence for a population bottleneck in Peixoto, possibly because the reduction was not extreme enough to be detected. Overall estimates of long-term N(e) varied from 92.4 individuals under the linkage disequilibrium model to infinity under the heterozygote excess model. Fixation indices and analysis of molecular variance demonstrated significant differentiation between locations north and south of the Amazon River, suggesting a degree of genetic isolation between them, attributed to isolation by distance.

Anopheles (Nyssorhynchus) marajoara is a proven primary vector of malaria parasites in Northeast Brazil, and An. deaneorum is a suspected vector in Western Brazil. Both are members of the morphologically similar Albitarsis Complex, which also includes An. albitarsis and an undescribed species, An. albitarsis "B". These four species were recognized and can be identified using random amplified polymorphic DNA (RAPD) markers, but various other methodologies also point to multiple species under the name An. albitarsis. We describe here a technique for identification of these species employing polymerase chain reaction (PCR) primers based on ribosomal DNA internal transcribed spacer 2 (rDNA ITS2) sequence. Since this method is based on known sequence it is simpler than the sometimes problematical RAPD-PCR. Primers were tested on samples previously identified using RAPD markers with complete correlation.

In a region of northeastern Amazonia, we find a species previously of minor importance, Anopheles marajoara, to be the principal malaria vector. In a total of five collections during 1996-97 in three replicated sites near the city of Macapá, Amapá state, this species occurs in much greater abundance compared with the presumed vector Anopheles darlingi. Also, a significantly higher proportion of An. marajoara is infected with malaria parasites, determined by the ELISA technique. This appears to be the result of increased abundance of An. marajoara due to alterations in land use, invasion of its primary breeding sites by human immigrants, and its anthropophilic behavior. This discovery highlights one of the challenges of Neotropical malaria control, namely that the targeting of specific vectors may be complicated by a changing mosaic of different locally important vectors and their interactions with human populations.

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was applied to samples from widespread populations of the poorly characterized Anopheles (Nyssorhynchus) albitarsis Lynch-Arribálzaga species complex, and 4 genetically differentiated species were distinguished. A screen of 65 random decamer oligonucleotide primers identified 12 primers, which produced 19 reproducible species-specific genetic markers and 4 markers common to 2 or more species. These markers were correlated in nearly all individuals of each species throughout the ranges sampled, including populations as far apart as 2,500 km. Each individual analyzed was from a different isofemale progeny brood, with associated morphological specimens. These specimens will facilitate studies to relate these species to previously reported chromosomal and enzymatic variation as well as to their feeding behavior and potential as malaria vectors. We hypothesize that 3 of the species have recognized valid names: An. (Nys.) albitarsis Lynch-Arribálzaga, An. (Nys.) marajoara Galvão and Damasceno, and An. (Nys.) deaneorum Rosa-Freitas, whereas the 4th is undescribed.