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The increase in malaria transmission in the Amazon region motivated vector control units of the Ministry of Health of Ecuador and Peru to investigate Anopheles (Diptera: Culicidae) species present in transmission hotspots. Mosquitoes were collected using prokopack aspirators and CDC light traps (Ecuador) and human landing catch in Peru. In Ecuador, 84 Anopheles were captured from Pastaza, Morona Santiago, and Orellana provinces and identified morphologically [An. (An.) apicimacula Dyar and Knab, An. (Nys.) near benarrochi, An. (Nys.) near oswaldoi, An. (Nys.) near strodei, An. (An.) nimbus (Theobald, 1902), and An. (Nyssorhynchus) sp.]. In Peru, 1,150 Anopheles were collected in Andoas District. A subsample of 166 specimens was stored under silica and identified as An. near oswaldoi, An. darlingi, and An. (An.) mattogrossensis Lutz and Neiva. COI barcode region sequences were obtained for 137 adults (107 from Peru, 30 from Ecuador) identified by ITS2 PCR-RFLP as An. benarrochi Gabaldon, Cova Garcia, and Lopez and retained in the final analysis. Haplotypes from the present study plus An. benarrochi B GenBank sequences grouped separately from Brazilian An. benarrochi GenBank sequences by 44 mutation steps, indicating that the present study specimens were An. benarrochi B. Our findings confirm the presence of An. benarrochi B in Ecuador and reported here for the first time from the Amazonian provinces of Orellana and Morona Santiago. Furthermore, we confirm that the species collected in Andoas District in the Datem del Maranon Province, Peru, is An. benarrochi B, and we observed that it is highly anthropophilic. Overall, the known distribution of An. benarrochi B has been extended and includes southern Colombia, much of Peru and eastern Ecuador.

Background Malaria remains an important public health problem in Peru where incidence has been increasing since 2011. Of over 55,000 cases reported in 2017, Plasmodium vivax was the predominant species (76%), with P. falciparum responsible for the remaining 24%. Nyssorhynchus darlingi (previously Anopheles darlingi) is the main vector in Amazonian Peru, where hyperendemic Plasmodium transmission pockets have been found. Mazán district has pronounced spatial heterogeneity of P. vivax malaria. However, little is known about behavior, ecology or seasonal dynamics of Ny. darlingi in Mazán. This study aimed to gather baseline information about bionomics of malaria vectors and transmission risk factors in a hyperendemic malaria area of Amazonian Peru. Methods To assess vector biology metrics, five surveys (two in the dry and three in the rainy season), including collection of sociodemographic information, were conducted in four communities in 2016–2017 on the Napo (Urco Miraño, URC; Salvador, SAL) and Mazán Rivers (Visto Bueno, VIB; Libertad, LIB). Human-biting rate (HBR), entomological inoculation rate (EIR) and human blood index (HBI) were measured to test the hypothesis of differences in entomological indices of Ny. darlingi between watersheds. A generalized linear mixed effect model (GLMM) was constructed to model the relationship between household risk factors and the EIR. Results Nyssorhynchus darlingi comprised 95% of 7117 Anophelinae collected and its abundance was significantly higher along the Mazán River. The highest EIRs (3.03–4.54) were detected in March and June in URC, LIB and VIB, and significantly more Ny. darlingi were infected outdoors than indoors. Multivariate analysis indicated that the EIR was >12 times higher in URC compared with SAL. The HBI ranged from 0.42–0.75; humans were the most common blood source, followed by Galliformes and cows. There were dramatic differences in peak biting time and malaria incidence with similar bednet coverage in the villages. Conclusions Nyssorhynchus darlingi is the predominant contributor to malaria transmission in the Mazán District, Peru. Malaria risk in these villages is higher in the peridomestic area, with pronounced heterogeneities between and within villages on the Mazán and the Napo Rivers. Spatiotemporal identification and quantification of the prevailing malaria transmission would provide new evidence to orient specific control measures for vulnerable or at high risk populations. Electronic supplementary material The online version of this article (10.1186/s13071-019-3619-0) contains supplementary material, which is available to authorized users.

In Amazonian Peru, the primary malaria vector, Nyssorhynchus darlingi (formerly Anopheles darlingi), is difficult to target using standard vector control methods because it mainly feeds and rests outdoors. Larval source management could be a useful supplementary intervention, but to determine its feasibility, more detailed studies on the larval ecology of Ny. darlingi are essential. We conducted a multi-level study of the larval ecology of Anophelinae mosquitoes in the peri-Iquitos region of Amazonian Peru, examining the environmental characteristics of the larval habitats of four species, comparing the larval microbiota among species and habitats, and placing Ny. darlingi larval habitats in the context of spatial heterogeneity in human malaria transmission. We collected Ny. darlingi, Nyssorhynchus rangeli (formerly Anopheles rangeli), Nyssorhynchus triannulatus s.l. (formerly Anopheles triannulatus s.l.), and Nyssorhynchus sp. nr. konderi (formerly Anopheles sp. nr. konderi) from natural and artificial water bodies throughout the rainy and dry seasons. We found that, consistent with previous studies in this region and in Brazil, the presence of Ny. darlingi was significantly associated with water bodies in landscapes with more recent deforestation and lower light intensity. Nyssorhynchus darlingi presence was also significantly associated with a lower vegetation index, other Anophelinae species, and emergent vegetation. Though they were collected in the same water bodies, the microbial communities of Ny. darlingi larvae were distinct from those of Ny. rangeli and Ny. triannulatus s.l., providing evidence either for a species-specific larval microbiome or for segregation of these species in distinct microhabitats within each water body. We demonstrated that houses with more reported malaria cases were located closer to Ny. darlingi larval habitats; thus, targeted control of these sites could help ameliorate malaria risk. The co-occurrence of Ny. darlingi larvae in water bodies with other putative malaria vectors increases the potential impact of larval source management in this region., Author summary The standard methods used to combat mosquitoes that transmit malaria, long-lasting insecticide treated nets and indoor residual spraying, target mosquitoes that bite people indoors and rest indoors after biting. In Amazonian Peru, the major malaria vector, Nyssorhynchus darlingi (formerly Anopheles darlingi), is known to bite and rest mostly outdoors and to feed on animals as well as humans. Therefore, additional methods are needed to control this species, such as targeting immature mosquitoes in water bodies using chemical or biological larvicides or environmental modification. To determine whether this is feasible, more ecological information about Ny. darlingi larval habitats in this region is needed. In this study, we found that Ny. darlingi were more likely to be collected from water bodies in more deforested areas, and in the presence of other species of mosquitoes that transmit malaria. We characterized the bacteria detected in three species of mosquito larvae, and found that the species of mosquito, and not the water body, determined which bacteria were identified. As we found that houses with more malaria cases were located closer to Ny. darlingi larval habitats, management of larval habitats may be an effective method to reduce the risk of malaria in this region.

The relationship between deforestation and malaria in Amazonian Brazil is complex, and a deeper understanding of this relationship is required to inform effective control measures in this region. Here, we are particularly interested in characterizing the impact of land use and land cover change on the genetics of the major regional vector of malaria, Nyssorhynchus darlingi (Root). We used nextera-tagmented, Reductively Amplified DNA (nextRAD) genotyping-by-sequencing to genotype 164 Ny. darlingi collected from 16 collection sites with divergent forest cover levels in seven municipalities in four municipality groups that span the state of Amazonas in northwestern Amazonian Brazil: Sao Gabriel da Cachoeira, Presidente Figueiredo, four municipalities in the area around Cruzeiro do Sul, and Labrea. Using a dataset of 5,561 Single Nucleotide Polymorphisms (SNPs), we investigated the genetic structure of these Ny. darlingi populations with a combination of model- and non-model-based analyses. We identified weak to moderate genetic differentiation among the four municipality groups. There was no evidence for microgeographic genetic structure of Ny. darlingi among forest cover levels within the municipality groups, indicating that there may be gene flow across areas of these municipalities with different degrees of deforestation. Additionally, we conducted an environmental association analysis using two outlier detection methods to determine whether individual SNPs were associated with forest cover level without affecting overall population genetic structure. We identified 14 outlier SNPs, and investigated functions associated with their proximal genes, which could be further characterized in future studies.

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 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.

Anopheles darlingi, the main malaria vector in the Neotropics, has been considered to be highly anthropophilic. However, many behavioral aspects of this species remain unknown, such as the range of blood-meal sources. Barrier screens were used to collect resting Anopheles darlingi mosquitoes from 2013 to 2015 in three riverine localities (Lupuna, Cahuide and Santa Emilia) in Amazonian Peru. Overall, the Human Blood Index (HBI) ranged from 0.58–0.87, with no significant variation among years or sites. Blood-meal analysis revealed that humans are the most common blood source, followed by avian hosts (Galliformes-chickens and turkeys), and human/Galliforme mixed-meals. The Forage Ratio and Selection Index both show a strong preference for Galliformes over humans in blood-fed mosquitoes. Our data show that 30% of An. darlingi fed on more than one host, including combinations of dogs, pigs, goats and rats. There appears to be a pattern of host choice in An. darlingi, with varying proportions of mosquitoes feeding only on humans, only on Galliformes and some taking mixed-meals of blood (human plus Galliforme), which was detected in the three sites in different years, indicating that there could be a structure to these populations based on blood-feeding preferences. Mosquito age, estimated in two localities, Lupuna and Cahuide, ranged widely between sites and years. This variation may reflect the range of local environmental factors that influence longevity or possibly potential changes in the ability of the mosquito to transmit the parasite. Of 6,204 resting An. darlingi tested for Plasmodium infection, 0.42% were infected with P. vivax. This study provides evidence for the first time of the usefulness of barrier screens for the collection of blood-fed resting mosquitoes to calculate the Human Blood Index (HBI) and other blood-meal sources in a neotropical malaria endemic setting., Author summary Anopheles darlingi is the major malaria vector in the Amazon. This species has been commonly described as highly anthropophilic throughout its geographic range, although little is known about its feeding preferences. Scant information is available regarding the origin of An. darlingi blood-meals. In the context of malaria elimination programs, the Human Blood Index (HBI) may provide crucial information regarding mosquito-human contact related to transmission dynamics. Additionally, collection of resting An. darlingi is challenging, mainly because the resting behavior of this species has not been well characterized. Our study, conducted from 2013–2015 in three localities in Loreto Department in the Peruvian Amazon, showed for the first time the efficacy of the barrier screen methodology for collecting recently blood-fed An. darlingi in a neotropical setting for the purpose of identifying the source of their blood-meals. Our data show that An. darlingi feeds on humans, Galliformes, dogs, pigs and goats, and that 30% of the mosquitoes fed on more than one type of host. Despite this opportunistic feeding behavior, however, An. darlingi is primarily anthropophilic. We hypothesize that mosquito population structure is associated with feeding preferences, which may affect the pattern of malaria transmission in the area.

American martens (Martes americana) in the northern Lower Peninsula of Michigan (NLP) represent a re-introduced and isolated population that is the southern-most distributed marten population in eastern North America. During 2005–2006 we conducted a radiotelemetry and track-survey study of martens in the NLP. We estimated home-range size and patterns of habitat use in order to develop a spatial model to predict distribution of marten habitat. Marten home ranges were comprised of more (>75%) upland deciduous and upland mixed forest compared to the landscape, which included ≤60% of these cover types. Within core areas, martens selected upland conifer stands. Our Penrose habitat model identified

Interest in larval source management (LSM) as an adjunct intervention to control and eliminate malaria transmission has recently increased mainly because long-lasting insecticidal nets (LLINs) and indoor residual spray (IRS) are ineffective against exophagic and exophilic mosquitoes. In Amazonian Peru, the identification of the most productive, positive water bodies would increase the impact of targeted mosquito control on aquatic life stages. The present study explores the use of unmanned aerial vehicles (drones) for identifying Nyssorhynchus darlingi (formerly Anopheles darlingi) breeding sites with high-resolution imagery (~0.02m/pixel) and their multispectral profile in Amazonian Peru. Our results show that high-resolution multispectral imagery can discriminate a profile of water bodies where Ny. darlingi is most likely to breed (overall accuracy 86.73%- 96.98%) with a moderate differentiation of spectral bands. This work provides proof-of-concept of the use of high-resolution images to detect malaria vector breeding sites in Amazonian Peru and such innovative methodology could be crucial for LSM malaria integrated interventions., Author summary The most efficient malaria vector in the Latin American region is Nyssorhynchus darlingi (formerly Anopheles darlingi). In Amazonian Peru, where malaria is endemic, Ny. darlingi feeds both indoors and outdoors (endophagy, exophagy), depending on the local environment, and rests outdoors (exophily). LLINs and IRS, the most common tools employed for vector control, target endophagic and endophilic mosquitoes. Thus, they are only partially effective against Ny. darlingi. Control of the aquatic stages of vector mosquitoes, larval source management (LSM), targets the most productive breeding sites nearest to human habitation. In four riverine communities, we used drones with high-resolution imagery as a key initial step to analyze water bodies within the estimated flight range of Ny. darlingi, ~ 1 km. We found distinctive spectral profiles for water bodies that were positive versus negative for Ny. darlingi. The methodology and analysis reported here provide the basis for testing whether LSM can be combined successfully with LLINs and IRS to contribute to the elimination of transmission in malaria hotspots in the Amazon.

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.

The process of colonizing any arthropod species, including vector mosquitoes, necessarily involves adaptation to laboratory conditions. The adaptation and evolution of colonized mosquito populations needs consideration when such colonies are used as representative models for pathogen transmission dynamics. A recently established colony of Anopheles darlingi, the primary malaria vector in Amazonian South America, was tested for genetic diversity and bottleneck after 21 generations, using microsatellites. As expected, laboratory An. darlingi had fewer private and rare alleles (frequency < 0.05), decreased observed heterozygosity, and more common alleles (frequency > 0.50), but no significant evidence of a bottleneck, decrease in total alleles, or increase in inbreeding compared with field specimens (founder population). Low-moderate differentiation between field and laboratory populations was detected. With these findings, and the documented inherent differences between laboratory and field populations, results of pathogen transmission studies using this An. darlingi colony need to be interpreted cautiously.

Made available in DSpace on 2015-12-07T15:35:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2015. Added 1 bitstream(s) on 2015-12-07T15:53:22Z : No. of bitstreams: 1 PMC4587789.pdf: 2864459 bytes, checksum: 70ff70ae2e9c46ecea32b315b8b2a5b7 (MD5) 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. 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. 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. 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. Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY, USA Wadsworth Center, New York State Department of Health, Griffin Laboratory, 5669 State Farm Road, Building 1, Room 101, Slingerlands 12159, NY, USA Division of Infectious Diseases, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Asociación Benéfica PRISMA, Iquitos, Peru Universidade Estadual Paulista, São Paulo, Brazil Instituto de Medicine Tropical “Alexander von Humboldt”, Universidad Peruana Cayetano Heredia, Lima, Peru. Universidade Estadual Paulista, São Paulo, Brazil.

Accurate taxonomic identification of highland mosquito species may be complicated because of the lack of comprehensive regional morphological keys and taxonomic specialists, particularly for mosquitoes of medical or ecological importance. We applied a multi-locus approach to explore the diversity of genera/species collected, to define the Molecular Operational Taxonomic Units (MOTUs) and to perform phylogenetic clustering. Twenty MOTUs and three single sequences were revealed from 78 concatenated cox1 + ITS2 sequences, and the species name was allocated for five of these. This study provides molecular taxonomic information of culicid fauna present in high Andean mountain ecosystems in Antioquia, Colombia. However, future morphological and integrative taxonomic studies should be conducted to achieve the specific identity of all detected MOTUs.

Anopheles punctimacula s.l. is a regional malaria vector in parts of Central America, but its role in transmission is controversial due to its unresolved taxonomic status. Two cryptic species, An. malefactor and An. calderoni , have been previously confused with this taxon, and evidence for further genetic differentiation has been proposed. In the present study we collected and morphologically identified adult female mosquitoes of An. punctimacula s.l. from 10 localities across Panama and one in Costa Rica. DNA sequences from three molecular regions, the three prime end of the mitochondrial cytochrome c oxidase I gene (3′ COI ), the Barcode region in the five prime end of the COI (5′ COI ), and the rDNA second internal transcribed spacer (ITS2) were used to test the hypothesis of new molecular lineages within An. punctimacula s.l. Phylogenetic analyses using the 3′ COI depicted six highly supported molecular lineages (A–F), none of which was An. malefactor . In contrast, phylogenetic inference with the 5′ COI demonstrated paraphyly. Tree topologies based on the combined COI regions and ITS2 sequence data supported the same six lineages as the 3′ COI alone. As a whole this evidence suggests that An. punctimacula s.l. comprises two geographically isolated lineages, but it is not clear whether these are true species. The phylogenetic structure of the An. punctimacula cluster as well as that of other unknown lineages (C type I vs C type II; D vs E) appears to be driven by geographic partition, because members of these assemblages did not overlap spatially. We report An. malefactor for the first time in Costa Rica, but our data do not support the presence of An. calderoni in Panama.

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 Bo rAn .( Nys.) rangeli, confirmed by polymerase chain reaction-restriction frag- ment 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.

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.

We investigated the historical demography of Anopheles albimanus using mosquitoes from five countries and three different DNA regions, the mitochondrial cytochrome oxidase subunit I gene (COI), the single copy nuclear white gene and the ribosomal internal transcribed spacer two (ITS2). All the molecular markers supported the taxonomic status of a single species of An. albimanus. Furthermore, agreement between the COI and the white genes suggested a scenario of Pleistocene geographic fragmentation (i.e., population contraction) and subsequent range expansion across southern Central America.