The Malaria Frontier Hypothesis (MFH) is the current model for predicting malaria emergence in the Brazilian Amazon. It has two important dimensions, ‘settlement time’ and ‘malaria incidence’, and its prediction are: malaria incidence peaks five years after the initiation of human settlement and declines towards zero after an estimated 10 years. Although MFH is currently accepted, it has been challenged recently. Herein, we described a novel method for estimating settlement timeline by using remote sensing technology integrated in an open-software geographic information system. Surprisingly, we found that of the majority of the rural settlements with high malaria incidence are more than 10 years old.

BACKGROUND Nyssorhynchus dunhami, a member of the Nuneztovari Complex, has been collected in Brazil, Colombia, and Peru and described as zoophilic. Although to date Ny. dunhami has not been documented to be naturally infected by Plasmodium, it is frequently misidentified as other Oswaldoi subgroup species that are local or regional malaria vectors. OBJECTIVES The current study seeks to verify the morphological identification of Nuneztovari Complex species collected in the peri-Iquitos region of Amazonian Peru, to determine their Plasmodium infection status, and to describe ecological characteristics of their larval habitats. METHODS We collected Ny. nuneztovari s.l. adults in 2011-2012, and Ny. nuneztovari s.l. larvae and adults in 2016-2017. When possible, samples were identified molecularly using cytochrome c oxidase subunit I (COI) barcode sequencing. Adult Ny. nuneztovari s.l. from 2011-2012 were tested for Plasmodium using real-time PCR. Environmental characteristics associated with Ny. nuneztovari s.l. larvae-positive water bodies were evaluated. FINDINGS We collected 590 Ny. nuneztovari s.l. adults and 116 larvae from eight villages in peri-Iquitos. Of these, 191 adults and 111 larvae were identified by COI sequencing; all were Ny. dunhami. Three Ny. dunhami were infected with P. falciparum, and one with P. vivax, all collected from one village on one night. Ny. dunhami larvae were collected from natural and artificial water bodies, and their presence was positively associated with other Anophelinae larvae and amphibians, and negatively associated with people living within 250m. MAIN CONCLUSIONS Of Nuneztovari Complex species, we identified only Ny. dunhami across multiple years in eight peri-Iquitos localities. This study is, to our knowledge, the first report of natural infection of molecularly identified Ny. dunhami with Plasmodium. We advocate the use of molecular identification methods in this region to monitor Ny. dunhami and other putative secondary malaria vectors to more precisely evaluate their importance in malaria transmission.

We describe a simple method for detection of Plasmodium vivaxand Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed withPlasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochromeb-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.

Malaria in La Guajira, the most northern state of Colombia, shows two different epidemiological patterns. Malaria is endemic in the municipality of Dibulla whereas in Riohacha it is characterised by sporadic outbreaks. This study aimed to establish whether differences in transmission patterns could be attributed to different vector species. The most abundant adult female species were Anopheles aquasalis, exclusive to Riohacha, and Anopheles darlingi, restricted to Dibulla. Anopheles mosquitoes were identified using morphology and the molecular markers internal transcribed spacer 2 and cytochrome c oxidase I. All specimens (n = 1,393) were tested by ELISA to determine natural infection rates with Plasmodium falciparum and Plasmodium vivax. An. darlingi was positive for P. vivax 210, with an infection rate of 0.355% and an entomological inoculation rate of 15.87 infective bites/person/year. Anopheles albimanus larvae were the most common species in Riohacha, found in temporary swamps; in contrast, in Dibulla An. darlingi were detected mainly in permanent streams. Distinctive species composition and larval habitats in each municipality may explain the differences in Plasmodium transmission and suggest different local strategies should be used for vector control.

Patterns of malaria cases were compared between the department of Meta and the municipality of Puerto Gaitán, Colombia, to examine temporal change in malaria from 2005-2010. During this time frame in Meta the mean ratio was 2.53; in contrast, in Puerto Gaitán it was 1.41, meaning that a surprisingly high proportion of Plasmodium falciparum cases were reported from this municipality. A detailed analysis of data from Puerto Gaitán for 2009 and 2010 detected a significant difference (χ2, p < 0.001) in the distribution of plasmodia, with Plasmodium vivax more prevalent in 2009 and P. falciparum in 2010. Males had the highest number of cases but there was no difference in the distribution of cases between sexes and years. In both years, for both sexes, people 16-40 accounted for the majority of cases (58.9% in 2009; 60.4% in 2010). There were significant differences in the distribution of both P. vivax (χ2, p < 0.01) and P. falciparum cases (χ2, p < 0.05) by geographic setting (urban vs. non-urban) between years. Urban cases of both P. vivax and P. falciparum are recorded in this study for the first time in Puerto Gaitán, possibly the result of area wide displacement and migration due to armed conflict.

To evaluate whether environmental heterogeneity contributes to the genetic heterogeneity in Anopheles triannulatus, larval habitat characteristics across the Brazilian states of Roraima and Pará and genetic sequences were examined. A comparison with Anopheles goeldii was utilised to determine whether high genetic diversity was unique to An. triannulatus. Student t test and analysis of variance found no differences in habitat characteristics between the species. Analysis of population structure of An. triannulatus and An. goeldii revealed distinct demographic histories in a largely overlapping geographic range. Cytochrome oxidase I sequence parsimony networks found geographic clustering for both species; however nuclear marker networks depicted An. triannulatus with a more complex history of fragmentation, secondary contact and recent divergence. Evidence of Pleistocene expansions suggests both species are more likely to be genetically structured by geographic and ecological barriers than demography. We hypothesise that niche partitioning is a driving force for diversity, particularly in An. triannulatus.

Introducción. El estudio de los aspectos de la biología de los mosquitos Anopheles spp.. fortalece la vigilancia entomológica. Objetivo. Determinar los aspectos de la biología y el comportamiento de las especies adultas del género Anopheles presentes en el área urbana de Puerto Carreño. Materiales y métodos. Se capturaron Anopheles spp.. silvestres que se habían posado en personas, en el intradomicilio y peridomicilio de viviendas ubicadas en el área urbana del municipio de Puerto Carreño (Vichada), entre las 18:00 y las 06:00 horas, durante dos noches consecutivas por mes y durante ocho meses. Se determinó la actividad de picadura para cada especie, la infección natural por Plasmodium falciparum y P. vivax VK247 y VK210 mediante la técnica ELISA, y se determinó la tasa de inoculación entomológica. Los individuos pertenecientes al complejo Albitarsis se determinaron mediante amplificación en cadena de la polimerasa del fragmento del gen white. Resultados. En orden de abundancia, se encontraron: An. darlingi (n=1.166), An. marajoara sensu stricto (n=152), An. braziliensis (n=59), An. albitarsis F (n=25), An. albitarsis sensu lato (n=16), An. argyritarsis (n=3) y An. oswaldoi sensu lato (n=2). Anopheles darlingi registró dos picos de actividad de picadura entre las 21:00-22:00 y las 5:00-06:00 horas en el peridomicilio y, entre las 21:00-22:00 y las 04:00-05:00 horas, en el intradomicilio. Esta especie se encontró naturalmente infectada por P. vivax VK210 y registró una tasa de inoculación entomológica de dos para el año. Anopheles marajoara s.s. se encontró naturalmente infectado por P. falciparum y registró una tasa de inoculación entomológica de 5 para el año, con un máximo de actividad de picadura entre las 18:00 y las 19:00 horas, tanto en el intradomicilio como en el peridomicilio. Conclusión. Es posible que exista transmisión de malaria en el área urbana de Puerto Carreño (Vichada) y An. darlingi y An. marajoara s.s. serían las especies incriminadas.

Introduction. The study of the biological aspects of Anopheles spp., strengthens the entomological surveillance. Objective. To determine biological aspects and behavior of adult Anopheles mosquitoes in the urban area of Puerto Carreño municipality, Vichada, Colombia. Materials and methods. Wild anophelines were collected landing on humans both indoors and outdoors between 18:00h and 06:00h for 50 min/h during two consecutive nights/month for eight months in the urban area of Puerto Carreño. The biting rate activity, the natural infection by Plasmodium falciparum and P. vivax VK247 and VK210 using ELISA, and the annual entomological inoculation rate were determined for each species. The members of the Albitarsis complex were determined by amplificacion of the white gene by polymerase chain reaction. Results. In order of abundance the species found were An. darlingi (n=1,166), An. marajoara sensu stricto (n=152), An. braziliensis (n=59), An. albitarsis F (n=25), An. albitarsis sensu lato (n=16), An. argyritarsis (n=3) and An. oswaldoi sensu lato (n=2). An. darlingi showed two activity peaks between 21:00 to 22:00 and 05:00 to 06:00 hours outdoors and between 21:00 to 22:00 and 04:00 to 05:00 indoors. Natural infection of this species was found with P. vivax VK210 and its annual entomological inoculation rate was 2. Natural infection of An marajoara sensu stricto with P. falciparum was found, with an annual entomological inoculation rate of 5 and a peak biting activity between 18:00 to 19:00 hrs both indoors and outdoors. Conclusion. Transmission of malaria in the urban area of Puerto Carreño, Vichada, can occur by An. darlingi and An. marajoara s.s.

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.

An understanding of the taxonomic status and vector distribution of anophelines is crucial in controlling malaria. Previous phylogenetic analyses have supported the description of six species of the Neotropical malaria vector Anopheles (Nyssorhynchus) albitarsis s.l. (Diptera: Culicidae): An. albitarsis, Anopheles deaneorum, Anopheles marajoara, Anopheles oryzalimnetes, Anopheles janconnae and An. albitarsis F. To evaluate the taxonomic status of An. albitarsis s.l. mosquitoes collected in various localities in the Colombian Caribbean region, specimens were analyzed using the complete mitochondrial DNA cytochrome oxidase I (COI) gene, the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) region and partial nuclear DNA white gene sequences. Phylogenetic analyses of the COI gene sequences detected a new lineage closely related to An. janconnae in the Caribbean region of Colombia and determined its position relative to the other members of the complex. However, the ITS2 and white gene sequences lacked sufficient resolution to support a new lineage closely related to An. janconnae or the An. janconnae clade. The possible involvement of this new lineage in malaria transmission in Colombia remains unknown, but its phylogenetic closeness to An. janconnae, which has been implicated in local malaria transmission in Brazil, is intriguing.

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.

El Grupo Oswaldoi, subgénero Nyssorhynchus (Diptera: Culicidae), incluye 16 especies. Algunas de estas especies presentan una alta variabilidad morfológica intraespecífica y similitud interespecie; lo anterior puede ocasionar dificultad en la identificación de la hembra al usar las claves morfológicas, como ocurre con Anopheles nuneztovari, considerado vector primario de malaria en Colombia. En este trabajo se comparó la utilidad de cinco claves morfológicas para la identificación de especímenes A. nuneztovari recolectados en la localidad de Puerto Anchica, Montelibano, Córdoba, Colombia. Se analizaron tres relaciones morfométricas y se confirmó la identificación morfológica utilizando la técnica PCR-RFLP basada en secuencias ITS2. El análisis de 41 hembras usando las claves, mostró que los especímenes A. nuneztovari presentaron sobrelapamiento morfológico con otras especies del Grupo Oswaldoi, como: A. rangeli, A. oswaldoi, A. evansae y A. benarrochi. El análisis molecular confirmó que todos los especímenes corresponden a A. nuneztovari. Dado que el uso de claves morfológicas continúa siendo la estrategia de elección para la identificación de anofelinos, los resultados sugieren que en caso de especies problemáticas, es conveniente confirmar la identificación mediante herramientas moleculares desarrolladas con el respaldo de la identificación de estadios inmaduros.

The Oswaldoi Group, subgenus Nyssorhynchus (Diptera: Culicidae), includes 16 species. Some of these species exhibit high intraspecific morphological variability and interspecific similarity; this can lead to difficulty in the identification of females by using morphological keys, as is the case with Anopheles nuneztovari, considered a primary malaria vector in Colombia. In this study we compared the usefulness of five morphological keys for the identification of A. nuneztovari specimens collected in the locality of Puerto Anchica, Montelibano, Córdoba, Colombia. Three morphometric ratios were analyzed and morphological identification results were confirmed using the PCR-RFLP technique based on ITS2 sequences. The analysis of 41 females using the keys showed that A. nuneztovari specimens presented overlap with other species of the Oswaldoi Group, such as A. rangeli, A. oswaldoi, A. evansae and A. benarrochi. Molecular analysis confirmed that all of the specimens corresponded to A. nuneztovari. Because the use of morphological keys continue to be a strategy of choice for the identification of anophelines, the results suggest that in the case of problematic species, it is convenient to confirm identification using molecular methods developed with the support of the identification of immature stages.

Malaria is a serious health problem in the states of Córdoba and Antioquia, Northwestern Colombia, where 64.4% of total Colombian cases were reported in 2007. Because little entomological information is available in this region, the aim of this work was to identify the Anopheles species composition and natural infectivity of mosquitoes distributed in seven localities with highest malaria transmission. A total of 1,768 Anopheles mosquitoes were collected using human landing catches from March 2007-July 2008. Ten species were identified; overall, Anopheles nuneztovari s.l. was the most widespread (62%) and showed the highest average human biting rates. There were six other species of the Nyssorhynchus subgenus: Anopheles albimanus (11.6%), Anopheles darlingi (9.8%), Anopheles braziliensis (6.6%), Anopheles triannulatus s.l. (3.5%), Anopheles albitarsis s.l. and Anopheles oswaldoi s.l. at < 1%; and three of the Anopheles subgenus: Anopheles punctimacula, Anopheles pseudopunctipennis s.l. and Anopheles neomaculipalpusat < 1% each. Two species from Córdoba, An. nuneztovari and An. darlingi, were found to be naturally infected by Plasmodium vivax VK247, as determined by ELISA and confirmed by nested PCR. All species were active indoors and outdoors. These results provide basic information for targeted vector control strategies in these localities.

The population genetic structure of Anopheles darlingi, the major human malaria vector in the Neotropics, was examined using seven microsatellite loci from nine localities in central and western Amazonian Brazil. High levels of genetic variability were detected (5-25 alleles per locus; H E = 0.519-0.949). There was deviation from Hardy-Weinberg Equilibrium for 59.79% of the tests due to heterozygote deficits, while the analysis of linkage disequilibrium was significant for only two of 189 (1.05%) tests, most likely caused by null alleles. Genetic differentiation (F ST = 0.001-0.095; Nm = 4.7-363.8) indicates that gene flow is extensive among locations < 152 km apart (with two exceptions) and reduced, but not absent, at a larger geographic scale. Genetic and geographic distances were significantly correlated (R² = 0.893, P < 0.0002), supporting the isolation by distance (IBD) model. The overall estimate of Ne was 202.4 individuals under the linkage disequilibrium model, and 8 under the heterozygote excess model. Analysis of molecular variance showed that nearly all variation (~ 94%) was within sample locations. The UPGMA phenogram clustered the samples geographically, with one branch including 5/6 of the state of Amazonas localities and the other branch the Acre, Rondônia, and remaining Amazonas localities. Taken together, these data suggest little genetic structure for An. darlingi from central and western Amazonian Brazil. These findings also imply that the IBD model explains nearly all of the differentiation detected. In practical terms, populations of An. darlingi at distances < 152 km should respond similarly to vector control measures, because of high gene flow.

Relato de coletas de anofelinos realizadas em Belém, Pará, Brasil, de 1995 a 2004, comparando os dados obtidos com os de levantamentos anteriores, feitos a partir da década de 1930. Nesses, vinte espécies haviam sido identificadas: Anopheles albitarsis s.l., An. aquasalis, An. argyritarsis, An. braziliensis, An. darlingi, An. eiseni, An. evansae, An. galvaoi, An. intermedius, An. kompi, An. mediopunctatus, An. nimbus, An. nuneztovari, An. oswaldoi, An. peryassui, An. punctimacula, An. shannoni, An. strodei, An. thomasi e An. triannulatus. Sete (An. argyritarsis, An. eiseni, An. galvaoi, An. kompi, An. nimbus, An. punctimacula e An. thomasi) não são agora registradas. A permanência de tantas outras espécies provavelmente decorre da preservação de áreas de mata no âmbito urbano. Duas delas são consideradas de importância vetorial (An. darlingi e An. aquasalis). Esta última continua sendo a de maior densidade nas coletas (46,26% dos adultos e 99,21% das larvas) e é a única registrada em todos os distritos administrativos. Existe, portanto, risco potencial de transmissão de malária em todo o município.

We present the results of anopheline captures in Belém, Pará, Brazil, from 1995-2004, and a comparison with captures from 1930-1999. In the earlier period, 20 species were identified: Anopheles albitarsis s.l., An. aquasalis, An. argyritarsis, An. braziliensis, An. darlingi, An. eiseni, An. evansae, An. galvaoi, An. intermedius, An. kompi, An. mediopunctatus, An. nimbus, An. nuneztovari, An. oswaldoi, An. peryassui, An. punctimacula, An. shannoni, An. strodei, An. thomasi, and An. triannulatus. Seven of these species were not found in 1995-2004 (An. argyritarsis, An. eiseni, An. galvaoi, An. kompi, An. nimbus, An. punctimacula, and An. thomasi). The persistence of so many species is probably due to the local preservation of forest areas. Two species are of vectorial importance (An. darlingi and An. aquasalis). An. aquasalis is still the most abundant species (46.26% of adults, 99.21% of larvae) and the only one detected in all capture sites. There is thus a potential risk of malaria transmission in the entire municipality of Belém.

Relato de coletas de anofelinos realizadas em Belém, Pará, Brasil, de 1995 a 2004, comparando os dados obtidos com os de levantamentos anteriores, feitos a partir da década de 1930. Nesses, vinte espécies haviam sido identificadas: Anopheles albitarsis s.l., An. aquasalis, An. argyritarsis, An. braziliensis, An. darlingi, An. eiseni, An. evansae, An. galvaoi, An. intermedius, An. kompi, An. mediopunctatus, An. nimbus, An. nuneztovari, An. oswaldoi, An. peryassui, An. punctimacula, An. shannoni, An. strodei, An. thomasi e An. triannulatus. Sete (An. argyritarsis, An. eiseni, An. galvaoi, An. kompi, An. nimbus, An. punctimacula e An. thomasi) não são agora registradas. A permanência de tantas outras espécies provavelmente decorre da preservação de áreas de mata no âmbito urbano. Duas delas são consideradas de importância vetorial (An. darlingi e An. aquasalis). Esta última continua sendo a de maior densidade nas coletas (46,26% dos adultos e 99,21% das larvas) e é a única registrada em todos os distritos administrativos. Existe, portanto, risco potencial de transmissão de malária em todo o município.

We present the results of anopheline captures in Belém, Pará, Brazil, from 1995-2004, and a comparison with captures from 1930-1999. In the earlier period, 20 species were identified: Anopheles albitarsis s.l., An. aquasalis, An. argyritarsis, An. braziliensis, An. darlingi, An. eiseni, An. evansae, An. galvaoi, An. intermedius, An. kompi, An. mediopunctatus, An. nimbus, An. nuneztovari, An. oswaldoi, An. peryassui, An. punctimacula, An. shannoni, An. strodei, An. thomasi, and An. triannulatus. Seven of these species were not found in 1995-2004 (An. argyritarsis, An. eiseni, An. galvaoi, An. kompi, An. nimbus, An. punctimacula, and An. thomasi). The persistence of so many species is probably due to the local preservation of forest areas. Two species are of vectorial importance (An. darlingi and An. aquasalis). An. aquasalis is still the most abundant species (46.26% of adults, 99.21% of larvae) and the only one detected in all capture sites. There is thus a potential risk of malaria transmission in the entire municipality of Belém.