Malaria transmission in the Southern Colombian state of Putumayo continues despite the absence of traditional vector species, except for the presence of Anopheles darlingi near the southeastern border with the state of Amazonas. In order to facilitate malaria vector incrimination in Putumayo, 2445 morphologically identified Anopheles females were tested for natural infection of Plasmodium vivax by ELISA. Specimens tested included An. apicimacula (n = 2), An. benarrochi B (n = 1617), An. darlingi (n = 29), An. mattogrossensis (n = 7), An. neomaculipalpus (n = 7), An. oswaldoi (n = 362), An. peryassui (n = 1), An. punctimacula (n = 1), An. rangeli (n = 413), and An. triannulatus (n = 6). Despite being overwhelmingly the most anthropophilic species in the region and comprising 66.1% of the mosquitoes tested, An. benarrochi B was not shown to be a vector. Thirty-five An. rangeli and one An. oswaldoi were naturally infected with P. vivax VK210. Sequence data were generated for the nuclear second internal transcriber space region of 31 of these 36 vivax positive mosquitoes (86.1%) to confirm their morphological identification. An. oswaldoi is known to be a species complex in Latin America, but its internal taxonomy remains unresolved. Herein we show that the An. oswaldoi found in the state of Putumayo is genetically similar to specimens from the state of Amapá in Brazil and from the Ocama region in the state of Amazonas in Venezuela, and that this form harbors natural infections of P. vivax. That An. rangeli and this member of the An. oswaldoi complex are incriminated as malaria vectors in Putumayo, is a novel finding of significance for malaria control in Southern Colombia, and possibly in other areas of Latin America.

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4%. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.

The most important vectors of human Plasmodium in the neotropics belong to the subgenus Nyssorhynchus. These species are generally sympatric in terms of their geographical distributions. Some are difficult to identify based solely on examination of adult females using the available morphological keys, in these cases examination of immature stages and male genitalia is required to make correct determinations. However, in epidemiological studies it is necessary to identify the species of adult females which are found near humans, i.e. in studies of malaria transmission or evaluation of control measures. The purpose of the present study was to evaluate the discrimination of adult females of different species of Nyssorhynchus isolated mainly from Southern Colombia (department of Putumayo), using morphometric analysis. Adult females were obtained after rearing larvae collected in natural breeding places and from the progeny of females collected on humans. The morphological characteristics of the immature stages allowed the identification of four species of the subgroup Oswaldoi from Southern Colombia: Anopheles rangeli Gabaldon, Cova Garcia & Lopez, An. oswaldoi (Peryassu), An. benarrochi Gabaldon, Cova Garcia & Lopez and An. triannulatus (Neiva & Pinto). The species An. nuneztovari (Gabaldon) from the Northwest of Colombia was included for comparison. Morphometric analysis allowed differentiation of the females of all species to a confidence level approaching 90% using principal components analysis of 10 wing and leg variables, followed by canonical variate analysis of the first four principal components. We conclude that morphometrics may represent a useful taxonomic tool for this group and that its use should be further studied.