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Inter-relationships among mosquito vectors, Plasmodium parasites, human ecology, and biotic and abiotic factors, drive malaria risk. Specifically, rural landscapes shaped by human activities have a great potential to increase the abundance of malaria vectors, putting many vulnerable people at risk. Understanding at which point the abundance of vectors increases in the landscape can help to design policies and interventions for effective and sustainable control. Using a dataset of adult female mosquitoes collected at 79 sites in malaria endemic areas in the Brazilian Amazon, this study aimed to (1) verify the association among forest cover percentage (PLAND), forest edge density (ED), and variation in mosquito diversity; and to (2) test the hypothesis of an association between landscape structure (i.e., PLAND and ED) and Nyssorhynchus darlingi (Root) dominance. Mosquito collections were performed employing human landing catch (HLC) (peridomestic habitat) and Shannon trap combined with HLC (forest fringe habitat). Nyssorhynchus darlingi abundance was used as the response variable in a generalized linear mixed model, and the Shannon diversity index (H') of the Culicidae community, PLAND, and the distance house-water drainage were used as predictors. Three ED categories were also used as random effects. A path analysis was used to understand comparative strengths of direct and indirect relationships among Amazon vegetation classes, Culicidae community, and Ny. darlingi abundance. Our results demonstrate that Ny. darlingi is negatively affected by H´ and PLAND of peridomestic habitat, and that increasing these variables (one-unit value at β0 = 768) leads to a decrease of 226 (P < 0.001) and 533 (P = 0.003) individuals, respectively. At the forest fringe, a similar result was found for H' (β1 = -218; P < 0.001) and PLAND (β1 = -337; P = 0.04). Anthropogenic changes in the Amazon vegetation classes decreased mosquito biodiversity, leading to increased Ny. darlingi abundance. Changes in landscape structure, specifically decreases in PLAND and increases in ED, led to Ny. darlingi becoming the dominant species, increasing malaria risk. Ecological mechanisms involving changes in landscape and mosquito species composition can help to understand changes in the epidemiology of malaria., Competing Interests: The authors have declared that no competing interests exist.

Background: The major Neotropical malaria vector, Anopheles darlingi, was reintroduced into the Iquitos, Loreto, Peru area during the early 1990s, where it displaced other anophelines and caused a major malaria epidemic. Since then, case numbers in Loreto have fluctuated, but annual increases have been reported since 2012., Methods: The population genetic structure of An. darlingi sampled before and after the introduction of long-lasting insecticidal nets (LLINs) was investigated to test the hypothesis of temporal population change (2006 vs. 2012). Current samples of An. darlingi were used to test the hypothesis of ecological adaptation to human modified (highway) compared with wild (riverine) habitat, linked to forest cover. In total, 693 An. darlingi from nine localities in Loreto, Peru area were genotyped using 13 microsatellite loci. To test the hypothesis of habitat differentiation in An. darlingi biting time patterns, HBR and EIR, four collections of An. darlingi from five localities (two riverine and three highway) were analysed., Results: Analyses of microsatellite loci from seven (2006) and nine settlements (2012-2014) in the Iquitos area detected two distinctive populations with little overlap, although it is unclear whether this population replacement event is associated with LLIN distribution or climate. Within the 2012-2014 population two admixed subpopulations, A and B, were differentiated by habitat, with B significantly overrepresented in highway, and both in near-equal proportions in riverine. Both subpopulations had a signature of expansion and there was moderate genetic differentiation between them. Habitat and forest cover level had significant effects on HBR, such that Plasmodium transmission risk, as measured by EIR, in peridomestic riverine settlements was threefold higher than in peridomestic highway settlements. HBR was directly associated with available host biomass rather than forest cover., Conclusions: A population replacement event occurred between 2006 and 2012-2014, concurrently with LLIN distribution and a moderate El Niño event, and prior to an increase in malaria incidence. The likely drivers of this replacement cannot be determined with current data. The present-day An. darlingi population is composed of two highly admixed subpopulations, which appear to be in an early stage of differentiation, triggered by anthropogenic alterations to local habitat.

The unprecedented global efforts for malaria elimination in the past decade have resulted in altered vectorial systems, vector behaviors, and bionomics. These changes combined with increasingly evident heterogeneities in malaria transmission require innovative vector control strategies in addition to the established practices of long-lasting insecticidal nets and indoor residual spraying. Integrated vector management will require focal and tailored vector control to achieve malaria elimination. This switch of emphasis from universal coverage to universal coverage plus additional interventions will be reliant on improved entomological monitoring and evaluation. In 2010, the National Institutes for Allergies and Infectious Diseases (NIAID) established a network of malaria research centers termed ICEMRs (International Centers for Excellence in Malaria Research) expressly to develop this evidence base in diverse malaria endemic settings. In this article, we contrast the differing ecology and transmission settings across the ICEMR study locations. In South America, Africa, and Asia, vector biologists are already dealing with many of the issues of pushing to elimination such as highly focal transmission, proportionate increase in the importance of outdoor and crepuscular biting, vector species complexity, and "sub patent" vector transmission., (© The American Society of Tropical Medicine and Hygiene.)

Background: Malaria transmission in the peri-Iquitos region of Amazonian Peru has been designated as seasonal and hypo-endemic with recently described hyper-endemic hotspots. Despite relatively recent distribution of long-lasting insecticidal bed nets (LLINs), malaria in Amazonian Peru persists and increased substantially in 2014 compared to previous years. Anopheles darlingi, identified as the main malaria vector, is known for its variable behaviour depending on locality and environment., Methods: To evaluate vector biology metrics in relation to seasonality and malaria transmission, mosquito collections were carried out in three localities in the peri-Iquitos region, Loreto, Peru in 2011-2012. Human landing catch (HLC) collection method, Shannon (SHA) and CDC trap types were compared for effectiveness in a neotropical setting. Abundance, human biting rate and entomological inoculation rate (EIR) were measured to provide an updated view of transmission patterns post-LLIN distribution., Results: HLC collected significantly more anopheline mosquitoes than SHA and CDC light traps. Anopheles darlingi was the most prevalent species in all three villages (84% overall). Biting patterns varied depending on trap type, season and village. EIR varied temporally (monthly) and spatially and the highest (2.52) occurred during the 2012 malaria outbreak in Cahuide. Unexpectedly there was a high infection rate (1.47 and 1.75) outside the normal malaria transmission season, coincident with a second local outbreak in Cahuide. The first identification of Anopheles dunhami and Anopheles oswaldoi C in Peru, using molecular markers, is also reported in this study., Conclusion: These data underscore the importance of HLC as the most meaningful collection method for measuring vector biology indices in this region. The highest monthly EIR provides additional evidence of seasonal transmission in riverine localities correlated with high river levels, and An. darlingi as the only contributor to transmission. The trend of an increase in outdoor-biting together with early-evening infected mosquitoes may undermine the effectiveness of LLINs as a primary malaria intervention.

We describe a simple method for detection of Plasmodium vivax and 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 with Plasmodium-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 cytochrome b-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.

The Colombian Pacific region is second nationally in number of malaria cases reported. This zone presents great ecological heterogeneity and Anopheles species diversity. However, little is known about the current spatial and temporal distribution of vector species. This study, conducted in three ecologically different localities of the Pacific region, aimed to evaluate the composition and distribution of Anopheles species and characterize transmission intensity. A total of 4,016 Anopheles mosquitoes were collected representing seven species. The composition and dominant species differed in each locality. Three species were infected with malaria parasites: Anopheles darlingi and An. calderoni were infected with Plasmodium falciparum and An. nuneztovari with Plasmodium vivax VK210 and VK247. Annual EIRs varied from 3.5-7.2 infective bites per year. These results confirm the importance of the primary vector An. nuneztovari in areas disturbed by human interventions, of An. darlingi in deforested margins of humid tropical rainforest and An. albimanus and the suspected vector An. calderoni in areas impacted by urbanization and large-scale palm oil agriculture close to the coast. This constitutes the first report in the Colombia Pacific region of naturally infected An. darlingi, and in Colombia of naturally infected An. calderoni. Further studies should evaluate the epidemiological importance of An. calderoni in the Pacific region.

This study was conducted to determine Anopheles species composition and their natural infectivity by human Plasmodium in 2 localities with the highest malaria transmission in San Jose del Guaviare, Guaviare, Colombia. A total of 1,009 Anopheles mosquitoes were collected using human landing catches during 8 months in 2010. Anopheles darlingi was the most abundant (83.2%) followed by An. albitarsis s.l. (8.6%), Anopheles braziliensis (3.8%), An. oswaldoi s.l. (1%), and An. rangeli (0.3%). Anopheles darlingi showed the highest human biting rate, and it was found naturally infected with Plasmodium vivax VK210 (0.119%) using enzyme-linked immunosorbent assays. All species were collected biting both indoors and outdoors. Anopheles darlingi showed biting activity overnight with an indoor peak between 1200-0100 h. Therefore, we recommend that malaria prevention strategies focus on 1) insecticide-treated nets to reduce human-vector contact when people are most exposed and unprotected; 2) accurate diagnoses; 3) adequate treatment for patients; 4) more timely epidemiological notification; and 5) improved entomological surveillance.

Anopheles darlingi Root is the most important malaria vector in the Amazonia region of South America. However, continuous propagation of An. darlingi in the laboratory has been elusive, limiting entomological, genetic/genomic, and vector-pathogen interaction studies of this mosquito species. Here, we report the establishment of an An. darlingi colony derived from wild-caught mosquitoes obtained in the northeastern Peruvian Amazon region of Iquitos in the Loreto Department. We show that the numbers of eggs, larvae, pupae, and adults continue to rise at least to the F6 generation. Comparison of feeding Plasmodium vivax ex vivo of F4 and F5 to F1 generation mosquitoes showed the comparable presence of oocysts and sporozoites, with numbers that corresponded to blood-stage asexual parasitemia and gametocytemia, confirming P. vivax vectorial capacity in the colonized mosquitoes. These results provide new avenues for research on An. darlingi biology and study of An. darlingi-Plasmodium interactions.

The invasive dengue vector Aedes aegypti has persisted for > 200 years in South Florida in the United States. We tested the hypotheses that Florida's landscape creates dispersal barriers and corridors and that long-distance human-aided dispersal structures populations of Ae. aegypti. We evaluated the phylogeography of 362 individuals from Florida's East and West Coasts with a 760-bp (418- and 342-bp fragments of ND5 and ND4, respectively) mitochondrial sequence. Populations from these two coasts were not significantly differentiated, suggesting that limited urbanization in central Florida is not a strong barrier to gene flow. Evidence for long-distance dispersal between Ft. Lauderdale and the West and Ft. Myers and the East indicates the importance of human-aided dispersal. West Coast populations showed no genetic differentiation, indicating that West Coast rivers and bays did not significantly impede gene flow. Phylogeographic analysis of haplotypes showed two distinct matrilines with no geographic patterns, suggesting multiple introductions or balancing selection.

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.

Seventy-six sites characterized in Amazonian Brazil revealed distinct habitat diversification by examining the environmental factors associated with the distribution and abundance of five anopheline species (Diptera: Culicidae) in the subgenus Nyssorhynchus. These included three members of the Albitarsis Complex, Anopheles oryzalimnetes, Anopheles marajoara, Anopheles janconnae; Anopheles triannulatus, and Anopheles goeldii. Anopheles janconnae abundance had a positive correlation to water flow and a negative relationship to sun exposure. Abundance of An. oryzalimentes was associated with water chemistry. Anopheles goeldii larvae were abundant in shaded, more saline waters. Anopheles marajoara and An. triannulatus were negatively associated with available resources, although An. marajoara also showed several local correlations. These analyses suggest An. triannulatus is a habitat generalist, An. oryzalimentes and An. janconnae are specialists, and An. marajoara and An. goeldii could not be easily classified either way. Correlations described herein provide testable hypotheses for future research and identifying habitats for vector control.

Background: The molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated., Methods: The mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches., Results: Each data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis., Conclusion: Combination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.

Anopheline specimens were collected in 2011 by human landing catch, Shannon and CDC traps from the malaria endemic localities of Santa Rosa and San Pedro in Madre de Dios Department, Peru. Most specimens were either Anopheles (Nyssorhynchus) benarrochi B or An. (Nys.) rangeli, confirmed by polymerase chain reaction-restriction fragment length polymorphism-internal transcribed spacer 2 (PCR-RFLP-ITS2) and, for selected individuals, ITS2 sequences. A few specimens from Lupuna, Loreto Department, northern Amazonian Peru, were also identified as An. benarrochi B. A statistical parsimony network using ITS2 sequences confirmed that all Peruvian An. benarrochi B analyzed were identical to those in GenBank from Putumayo, southern Colombia. Sequences of the mtDNA COI BOLD region of specimens from all three Peruvian localities were connected using a statistical parsimony network, although there were multiple mutation steps between northern and southern Peruvian sequences. A Bayesian inference of concatenated Peruvian sequences of ITS2 + COI detected a single clade with very high support for all An. benarrochi B except one individual from Lupuna that was excluded. No samples were positive for Plasmodium by CytB-PCR.

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.

We review previous studies on the genetic diversity of malaria vectors to highlight the major trends in population structure and demographic history. In doing so, we outline key information about molecular markers, sampling strategies and approaches to investigate the causes of genetic structure in Anopheles mosquitoes. Restricted gene flow due to isolation by distance and physical barriers to dispersal may explain the spatial pattern of current genetic diversity in some Anopheles species. Nonetheless, there is noteworthy disagreement among studies, perhaps due to variation in sampling methodologies, choice of molecular markers, and/or analytical approaches. More refined genealogical methods of population analysis allowing for the inclusion of the temporal component of genetic diversity facilitated the evaluation of the contribution of historical demographic processes to genetic structure. A common pattern of past unstable demography (i.e., historical fluctuation in the effective population size) by several Anopheles species, regardless of methodology (DNA markers), mosquito ecology (anthropophilic vs zoophilic), vector status (primary vs secondary) and geographical distribution, suggests that Pleistocene environmental changes were major drivers of divergence at population and species levels worldwide., (Copyright © 2011. Published by Elsevier B.V.)

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.

Cryptic species and lineages characterize Anopheles nuneztovari s.l. Gabaldón, an important malaria vector in South America. We investigated the phylogeographic structure across the range of this species with cytochrome oxidase subunit I (COI) mitochondrial DNA sequences to estimate the number of clades and levels of divergence. Bayesian and maximum-likelihood phylogenetic analyses detected four groups distributed in two major monophyletic clades (I and II). Samples from the Amazon Basin were clustered in clade I, as were subclades II-A and II-B, whereas those from Bolivia/Colombia/Venezuela were restricted to one basal subclade (II-C). These data, together with a statistical parsimony network, confirm results of previous studies that An. nuneztovari is a species complex consisting of at least two cryptic taxa, one occurring in Colombia and Venezuela and the another occurring in the Amazon Basin. These data also suggest that additional incipient species may exist in the Amazon Basin. Divergence time and expansion tests suggested that these groups separated and expanded in the Pleistocene Epoch. In addition, the COI sequences clearly separated An. nuneztovari s.l. from the closely related species An. dunhami Causey, and three new records are reported for An. dunhami in Amazonian Brazil. These findings are relevant for vector control programs in areas where both species occur. Our analyses support dynamic geologic and landscape changes in northern South America, and infer particularly active divergence during the Pleistocene Epoch for New World anophelines.

Vector systematics research is being transformed by the recent development of theoretical, experimental and analytical methods, as well as conceptual insights into speciation and reconstruction of evolutionary history. We review this progress using examples from the mosquito genus Anopheles. The conclusion is that recent progress, particularly in the development of better tools for understanding evolutionary history, makes systematics much more informative for vector control purposes, and has increasing potential to inform and improve targeted vector control programmes.

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.

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.

Background: Anopheles albimanus is an important malaria vector in some areas throughout its distribution in the Caribbean and the Pacific regions of Colombia, covering three biogeographic zones of the neotropical region, Maracaibo, Magdalena and Chocó., Methods: This study was conducted to estimate intra-population genetic diversity, genetic differentiation and demographic history of An. albimanus populations because knowledge of vector population structure is a useful tool to guide malaria control programmes. Analyses were based on mtDNA COI gene sequences and four microsatellite loci of individuals collected in eight populations from the Caribbean and the Pacific regions of Colombia., Results: Two distinctive groups were consistently detected corresponding to COI haplotypes from each region. A star-shaped statistical parsimony network, significant and unimodal mismatch distribution, and significant negative neutrality tests together suggest a past demographic expansion or a selective sweep in An. albimanus from the Caribbean coast approximately 21,994 years ago during the late Pleistocene. Overall moderate to low genetic differentiation was observed between populations within each region. However, a significant level of differentiation among the populations closer to Buenaventura in the Pacific region was observed. The isolation by distance model best explained genetic differentiation among the Caribbean region localities: Los Achiotes, Santa Rosa de Lima and Moñitos, but it could not explain the genetic differentiation observed between Turbo (Magdalena providence), and the Pacific region localities (Nuquí, Buenaventura, Tumaco). The patterns of differentiation in the populations from the different biogeographic provinces could not be entirely attributed to isolation by distance., Conclusion: The data provide evidence for limited past gene flow between the Caribbean and the Pacific regions, as estimated by mtDNA sequences and current gene flow patterns among An. albimanus populations as measured by MS loci which may be mainly influenced by semi-permeable natural barriers in each biogeographical region that lead to the genetic differences and effective population sizes detected. The relatively high genetic differentiation in the port city of Buenaventura may be the result of specific ecological conditions, human migration and activities and/or differences in effective population sizes. This knowledge could serve to evaluate and coordinate vector control strategies in these 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.

Background: Anopheles darlingi is the most important malaria vector in the Neotropics. An understanding of A. darlingi's 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., Results: We 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., Conclusion: We 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.

Anopheles gambiae Giles s.s. and Anopheles arabiensis Patton (Diptera: Culicidae) are major vectors of malaria in Nigeria. We used 1115 bp of the mitochondrial COI gene to assess their population genetic structures based on samples from across Nigeria (n = 199). The mtDNA neighbour-joining tree, based on F(ST) estimates, separated An. gambiae M and S forms, except that samples of An. gambiae M from Calabar clustered with all the An. gambiae S form. Anopheles arabiensis and An. gambiae could be combined into a single star-shaped, parsimonious haplotype network, and shared three haplotypes. Haplotype diversity values were high in An. arabiensis and An. gambiae S, and intermediate in An. gambiae M; all nucleotide diversities were relatively low. Taken together, patterns of haplotype diversity, the star-like genealogy of haplotypes, five of seven significant neutrality tests, and the violation of the isolation-by-distance model indicate population expansion in An. arabiensis and An. gambiae S, but the signal was weak in An. gambiae M. Selection is supported as an important factor shaping genetic structure in An. gambiae in Nigeria. There were two geographical subdivisions in An. arabiensis: one included all southern localities and all but two central localities; the other included all northern and two central localities. Re-analysing an earlier microsatellite dataset of An. arabiensis using a Bayesian method determined that there were two distinctive clusters, northern and southern, that were fairly congruent with the mtDNA subdivisions. There was a trend towards decreasing genetic diversity in An. arabiensis from the northern savannah to the southern rainforest that corroborated previous data from microsatellites and polytene chromosomes.

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 (R(2) = 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 (approximately 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.

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.

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.

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.

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.

Anopheles aquasalis Curry, a coastal malaria vector with a Neotropical distribution, was collected from Belém, Pará state, Brazil, and 500 adults per cage were maintained at the Instituto Evandro Chagas insectary at 26 -30 degrees C and 80 -90% RH, where they fed on a 10% domestic sugar solution and blood from white mice. Oviposition of the parental generation (P) occurred in fresh water in dark cups introduced into mosquito cages. After eclosion, 100 larvae per pan were reared in artesian well water (salinity 0.04 g liter(-10) and fed ground fish food until pupation. After force mating in the F1 generation, the eight subsequent generations were free mating. Mean larval mortality was < 1%, and the mean developmental time from eclosion to emergence was 7.7 d (F1), 7.6 d (F2), 8 d (F3), and 7.5 d (F4). The maximum daily production of pupae (from the fourth generation on) occurred on day 6 postoviposition.

Intragenomic heterogeneity of the internal transcribed spacer (ITS) array was investigated in Anopheles aquasalis Curry mosquitoes from two geographic locations in each of Brazil and Venezuela, and one in Suriname. Polymerase chain reaction-amplified copies of the ITS were cloned and sequenced. The length of the entire array ranged from 782 to 990 bp, with most variation due to microsatellite insertions in ITS1. We detected 40 different ITSL sequences and 15 different ITS2 sequences of the 71 to 72 clones examined. The sequence divergence within localities ranged from 0.002 to 0.043 for ITS1 and from 0 to 0.006 for ITS2. Point mutations were common to both spacer regions, but dinucleotide microsatellite repeats were restricted to ITS1. Sequences from neither ITS1 nor ITS2 had a diagnostic distribution or were informative in distinguishing these populations, providing additional support for the status of An. aquasalis as a single species.

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.

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.

Gene flow was investigated in Anopheles gambiae from eight localities that span the ecological zones of Nigeria (arid savanna zones in the north gradually turn into humid forest zones in the south). Genetic differentiation was measured over 10 microsatellite loci and, to determine any effects of selection, five loci were located within chromosome inversions and the other five were outside inversions. Over all loci, the largest estimates of differentiation were in comparisons between localities in the savanna vs. forest zones (range FST 0.024-0.087, Nm 2.6-10.1; RST 0.014-0.100, Nm 2.2-16.4). However, three loci located within inversions on chromosome II, whose frequencies varied clinically from north to south, were responsible for virtually all of the differentiation. When the three loci were removed, genetic distances across the remaining seven loci were markedly reduced even between localities in the forest and savanna zones (range FST 0.001-0.019, Nm 12.7-226.1) or no longer significant (P > 0.05) in the case of RST. Although tests of isolation by distance gave seemingly equivocal results, geographical distance does not appear to limit gene flow. These observations suggest that gene flow is extensive across the country but that selection on genes located within some inversions on chromosome II counters the homogenizing effects of gene flow.

The distribution of Anopheles gambiae and An. arabiensis across the ecological zones of Nigeria (arid savanna in the north gradually turns into humid forest in the south) was investigated. Results of the present study were compared to the distributions determined from samples of indoor-resting females reported by an earlier study over 20 years ago. Larvae were sampled in the rainy seasons of 1997 and 1999 from 24 localities, 10 of which were sampled in both years. Specimens were identified by the polymerase chain reaction method. Results showed that species composition changed significantly among the 10 localities in both years (chi2=13.62, P = 0.0002), but this change was significant in only four of the 10 localities. The identity of the prevalent (more abundant) species changed between 1997 and 1999 in only three of 10 localities. An. arabiensis was prevalent in several localities in the southern Guinea savanna, an area where it was virtually absent over 20 years ago. The data suggest that An. arabiensis has extend its range, although differences in sampling technique (larval sampling versus adult collection) can not be ruled out as a possible explanation.

Samples of the neotropical malaria vector Anopheles darlingi from Bolivia, Brazil, and Venezuela were analyzed to test for differences in mitochondrial haplotype frequencies. With the use of molecular variance components and F-statistics, significant genetic variability of An. darlingi was found apportioned primarily among populations within regions or within populations, with regions defined either as biomes (n = 5) or ecoregions (n = 2). The Mantel analysis resulted in a significant correlation [Prob (r) = 0.009] between genetic and geographic distances, evidence that these populations are genetically isolated by distance. Such isolation could reflect differences in phenotypes for factors affecting vector capacity.

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.

A genetic and morphologic survey of Anopheles darlingi populations collected from seven countries in Central and South America was performed to clarify the taxonomic status of this major malaria vector species in the Americas. Population genetics was based on three techniques including isozyme, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), and internal transcribed spacer 2 (ITS2) markers. The results of the isozyme analysis indicated moderate differences in the allele frequencies of three putative loci (glutamate oxalaoacetate transaminase-1, isocitrate dehydrogenase-1, and phosphoglucomutase) of the 31 analyzed. No fixed electromorphic differences separated the populations of An. darlingi, which showed little genetic divergence (Nei distances = 0.976-0.995). Fragments produced by RAPD-PCR demonstrated evidence of geographic partitioning and showed that all populations were separated by small genetic distances as measured with the 1 - S distance matrix. The ITS2 sequences for all samples were identical except for four individuals from Belize that differed by a three-base deletion (CCC). The morphologic study demonstrated that the Euclidean distances ranged from 0.02 to 0.14, with the highest value observed between populations from Belize and Bolivia. Based on these analyses, all the An. darlingi populations examined demonstrated a genetic similarity that is consistent with the existence of a single species and suggest that gene flow is occurring throughout the species' geographic range.

The field of medical entomology, by nature of its association with problems of human health, has been conservative in its application of molecular and computer technologies to systematic research. Recently, however, these methods have opened new interpretations for systematics of disease vectors. Medically important insects, particularly mosquitoes, are among those more thoroughly described by conventional taxonomy, and thereby provide a secure framework for testing congruencies with molecular data. In turn, molecular investigations have provided a stimulus to vector systematics in the discovery and delineation of cryptic species complexes, as well as providing new perspectives on relationships at higher taxonomic divisions. In this review, examples involving cladistic analysis, cytogenetics--in situ hybridization, isoenzymes, DNA sequencing, and restriction fragment polymorphism are drawn from the following taxa: Aedes communis; Aedes (Ochlerotatus) group G; Aedes (Stegomyia) species including A. aegypti, A. albopictus, and A. scutellaris group; Anopheles albitarsis, Anopheles dirus, Anopheles gambiae, Anopheles nuneztovari, Anopheles pseudopunctipennis, and Anopheles punctulatus groups; Culex pipiens and the Culex subgenus Melanoconion; and the tribe Sabethini.