Human intervention in the Brazilian Amazon region promotes contacts between humans and vectors that may favor the propagation of anopheline mosquitoes and the spread of malaria in the absence of planning and infrastructure to control this disease. Vector ecology studies were carried out to determine the risk areas. These data should help in designing appropriate malaria control measures. Data from 14 different regions are reported. Vectors are able to adapt to different environments, which made it necessary to study each area. The parameters studied were Anopheles breeding sites, species distribution, incidence, feeding preferences, hours of maximum activity of adult mosquitoes, seasonality, resting places, and the presence of Plasmodium. Species complexes were also studied. Anopheles darlingi may be responsible for maintaining malaria in human populations in this region. A reduction in the population density of A. darlingi in a particular geographic area can sometimes cause the disappearance of malaria. This species feeds at night but has a peak of activity at the beginning of the evening and another at dawn. Other species are mainly crepuscular and all anophelines demonstrated pronounced exophilia. The timing of feeding activities was found to vary in areas altered by human intervention and also depended on the time of the year and climatic conditions. The larvae were more abundant in the rivers with a less acidic pH and rural areas showed the highest larval index.

SUMMARYVarious species of Anopheles (Nyssorhynchus) were studied in the Amazon with the objective of determining their importance as malaria vectors. Of the 33 known Anopheles species occurring in the Amazon, only 9 were found to be infected with Plasmodium. The different species of this subgenus varied both in diversity and density in the collection areas. The populations showed a tendency towards lower density and diversity in virgin forest than in areas modified by human intervention.The principal vector, An. darlingi, is anthropophilic with a continuous activity cycle lasting the entire night but peaking at sunset and sunrise. These species (Nyssorhynchus) are peridomiciliary, entering houses to feed on blood and immediately leaving to settle on nearby vegetation.Anopheles nuneztovari proved to be zoophilic, crepuscular and peridomiciliary. These habits may change depending on a series of external factors, especially those related to human activity.There is a possibility that sibling species exist in the study area and they are being studied with reference to An. darlingi, An. albitarsis and An. nuneztovari. The present results do not suggest the existence of subpopulations of An. darlingi in the Brazilian Amazon.

BackgroundThe complete sequences of the mitochondrial genomes (mtDNA) of members of the northern and southern genotypes of Anopheles (Nyssorhynchus) darlingi were used for comparative studies to estimate the time to the most recent common ancestor for modern anophelines, to evaluate differentiation within this taxon, and to seek evidence of incipient speciation.MethodsThe mtDNAs were sequenced from mosquitoes from Belize and Brazil and comparative analyses of structure and base composition, among others, were performed. A maximum likelihood approach linked with phylogenetic information was employed to detect evidence of selection and a Bayesian approach was used to date the split between the subgenus Nyssorhynchus and other Anopheles subgenera.ResultsThe comparison of mtDNA sequences within the Anopheles darlingi taxon does not provide sufficient resolution to establish different units of speciation within the species. In addition, no evidence of positive selection in any protein-coding gene of the mtDNA was detected, and purifying selection likely is the basis for this lack of diversity. Bayesian analysis supports the conclusion that the most recent ancestor of Nyssorhynchus and Anopheles+Cellia was extant ~94 million years ago.ConclusionAnalyses of mtDNA genomes of Anopheles darlingi do not provide support for speciation in the taxon. The dates estimated for divergence among the anopheline groups tested is in agreement with the geological split of western Gondwana (95 mya), and provides additional support for explaining the absence of Cellia in the New World, and Nyssorhynchus in the Afro-Eurasian continents.

Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ∼100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector–human and vector–parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.

In the Americas, areas with a high risk of malaria transmission are mainly located in the Amazon Forest, which extends across nine countries. One keystone step to understanding the Plasmodium life cycle in Anopheles species from the Amazon Region is to obtain experimentally infected mosquito vectors. Several attempts to colonise Ano- pheles species have been conducted, but with only short-lived success or no success at all. In this review, we review the literature on malaria transmission from the perspective of its Amazon vectors. Currently, it is possible to develop experimental Plasmodium vivax infection of the colonised and field-captured vectors in laboratories located close to Amazonian endemic areas. We are also reviewing studies related to the immune response to P. vivax infection of Anopheles aquasalis, a coastal mosquito species. Finally, we discuss the importance of the modulation of Plasmodium infection by the vector microbiota and also consider the anopheline genomes. The establishment of experimental mosquito infections with Plasmodium falciparum, Plasmodium yoelii and Plasmodium berghei parasites that could provide interesting models for studying malaria in the Amazonian scenario is important. Understanding the molecular mechanisms involved in the development of the parasites in New World vectors is crucial in order to better determine the interaction process and vectorial competence.

BackgroundAnopheles darlingi is the major malaria vector in countries located in the Amazon region. Anopheles aquasalis and Anopheles albitarsis s.l. are also proven vectors in this region. Anopheles nuneztovari s.l. and Anopheles triannulatus s.l. were found infected with Plasmodium vivax; however, their status as vectors is not yet well defined. Knowledge of susceptibility of Amazon anopheline populations to Plasmodium infection is necessary to better understand their vector capacity. Laboratory colonization of An. darlingi, the main Amazon vector, has proven to be difficult and presently An. aquasalis is the only available autonomous colony.MethodsLarvae of An. darlingi, An. albitarsis s.l., An. nuneztovari s.l. and An. triannulatus s.l. were collected in the field and reared until adult stage. Adults of An. aquasalis were obtained from a well-established colony. Mosquitoes were blood-fed using a membrane-feeding device containing infected blood from malarial patients.The infection of the distinct Anopheles species was evaluated by the impact variance of the following parameters: (a) parasitaemia density; (b) blood serum inactivation of the infective bloodmeal; (c) influence of gametocyte number on infection rates and number of oocysts. The goal of this work was to compare the susceptibility to P. vivax of four field-collected Anopheles species with colonized An. aquasalis.ResultsAll Anopheles species tested were susceptible to P. vivax infection, nevertheless the proportion of infected mosquitoes and the infection intensity measured by oocyst number varied significantly among species. Inactivation of the blood serum prior to mosquito feeding increased infection rates in An. darlingi and An. triannulatus s.l., but was diminished in An. albitarsis s.l. and An. aquasalis. There was a positive correlation between gametocyte density and the infection rate in all tests (Z = −8.37; p < 0.001) but varied among the mosquito species. Anopheles albitarsis s.l., An. aquasalis and An. nuneztovari s.l. had higher infection rates than An. darlingi.ConclusionAll field-collected Anopheles species, as well as colonized An. aquasalis are susceptible to experimental P. vivax infections by membrane feeding assays. Anopheles darlingi, An. albitarsis s.l. and An. aquasalis are very susceptible to P. vivax infection. However, colonized An. aquasalis mosquitoes showed the higher infection intensity represented by infection rate and oocyst numbers. This study is the first to characterize experimental development of Plasmodium infections in Amazon Anopheles vectors and also to endorse that P. vivax infection of colonized An. aquasalis is a feasible laboratory model.

BackgroundPopulation-based studies conducted in Latin America have shown a high proportion of asymptomatic and submicroscopic malarial infections. Considering efforts aiming at regional elimination, it is important to investigate the role of this asymptomatic reservoir in malaria transmission in peri-urban areas. This study aimed to estimate the prevalence of Plasmodium spp. and gametocyte burden on symptomatic and asymptomatic infections in the Brazilian Amazon.ResultsTwo cross-sectional household surveys (CS) were conducted including all inhabitants in a peri-urban area of Manaus, western Amazonas State, Brazil. Malaria parasites were detected by light microscopy (LM) and qPCR. Sexual stages of Plasmodium spp. were detected by LM and RT-qPCR. A total of 4083 participants were enrolled during the two surveys. In CS1, the prevalence of Plasmodium vivax infections was 4.3% (86/2010) by qPCR and 1.6% (32/2010) by LM. Fifty percent (43/86) of P. vivax infected individuals (qPCR) carried P. vivax gametocytes. In CS2, 3.4% (70/2073) of participants had qPCR-detectable P. vivax infections, of which 42.9% (30/70) of infections were gametocyte positive. The P. vivax parasite density was associated with gametocyte carriage (P < 0.001). Sixty-seven percent of P. vivax infected individuals and 53.4% of P. vivax gametocyte carriers were asymptomatic.ConclusionsThis study confirms a substantial proportion of asymptomatic and submicroscopic P. vivax infections in the study area. Most asymptomatic individuals carried gametocytes and presented low asexual parasitemia. This reservoir actively contributes to malaria transmission in the Brazilian Amazon, underscoring a need to implement more efficient control and elimination strategies.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2787-7) contains supplementary material, which is available to authorized users.

Malaria affects 300 million people worldwide every year and 450,000 in Brazil. In coastal areas of Brazil, the main malaria vector is Anopheles aquasalis, and Plasmodium vivax is responsible for the majority of malaria cases in the Americas. Insects possess a powerful immune system to combat infections. Three pathways control the insect immune response: Toll, IMD, and JAK-STAT. Here we analyze the immune role of the A. aquasalis JAK-STAT pathway after P. vivax infection. Three genes, the transcription factor Signal Transducers and Activators of Transcription (STAT), the regulatory Protein Inhibitors of Activated STAT (PIAS) and the Nitric Oxide Synthase enzyme (NOS) were characterized. Expression of STAT and PIAS was higher in males than females and in eggs and first instar larvae when compared to larvae and pupae. RNA levels for STAT and PIAS increased 24 and 36 hours (h) after P. vivax challenge. NOS transcription increased 36 h post infection (hpi) while this protein was already detected in some midgut epithelial cells 24 hpi. Imunocytochemistry experiments using specific antibodies showed that in non-infected insects STAT and PIAS were found mostly in the fat body, while in infected mosquitoes the proteins were found in other body tissues. The knockdown of STAT by RNAi increased the number of oocysts in the midgut of A. aquasalis. This is the first clear evidence for the involvement of a specific immune pathway in the interaction of the Brazilian malaria vector A. aquasalis with P. vivax, delineating a potential target for the future development of disease controlling strategies.