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.

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.

Brazilian Aedes aegypti from Amazonas is highly permissive to monoinfection and coinfection with dengue virus and Zika virus (ZIKV) and capable of cotransmitting both arboviruses by bite to vertebrate hosts. Coinfection strongly influences vector competence, favoring transmission of ZIKV.

Zika is a re-emerging infection that has been considered a major threat to global public health. Currently at least 100 countries are at risk of Zika virus (ZIKV) transmission. Aedes aegypti is the main mosquito vector in the Americas. This vector is exposed to, and interacts symbiotically with a variety of microorganisms in its environment, which may result in the formation of a lifetime association. Here, the unknown effect that ZIKV exerts on the dynamic bacterial community harbored by this mosquito vector was investigated using a metagenomic analysis of its microbiota. Groups of Ae. aegypti were experimentally fed on sugar, blood and blood mixed with ZIKV, and held for 3 to 7 days after blood meal and eggs development respectively. The infected groups were processed by qPCR to confirm the presence of ZIKV. All groups were analyzed by metagenomics (Illumina Hiseq Sequencing) and 16S rRNA amplicon sequences were obtained to create bacterial taxonomic profiles. A core microbiota and exclusive bacterial taxa were identified that incorporate 50.5% of the predicted reads from the dataset, with 40 Gram-negative and 9 Gram-positive families. To address how ZIKV invasion may disturb the ecological balance of the Ae. aegypti microbiota, a CCA analysis coupled with an explanatory matrix was performed to support the biological interpretation of shifts in bacterial signatures. Two f-OTUs appeared as potential biomarkers of ZIKV infection: Rhodobacteraceae and Desulfuromonadaceae. Coincidentally, both f-OTUs were exclusively present in the ZIKV-infected blood-fed and ZIKVinfected gravid groups. In conclusion, this study shows that bacterial symbionts act as biomarkers of the insect physiological states and how they respond as a community when ZIKV invades Ae. aegypti. Basic knowledge of local haematophagous vectors and their associated microbiota is relevant when addressing transmission of vector-borne infectious diseases in their regional surroundings.PLOS ONE | https://doi.org/10.1371/journal.pone

BackgroundThe mosquito resistance to the insecticides threatens malaria control efforts, potentially becoming a major public health issue. Alternative methods like ivermectin (IVM) administration to humans has been suggested as a possible vector control to reduce Plasmodium transmission. Anopheles aquasalis and Anopheles darlingi are competent vectors for Plasmodium vivax, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America.MethodsTo determine the IVM susceptibility against P. vivax in An. aquasalis and An. darlingi, ivermectin were mixed in P. vivax infected blood: (1) Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (2) Plasma (0 hours, 4 hours, 1 day, 5, 10 and 14 days) was collected from healthy volunteers after to administer a single oral dose of IVM (200 μg/kg) (3) Mosquitoes infected with P. vivax and after 4 days was provided with IVM plasma collected 4 hours post-treatment (4) P. vivax-infected patients were treated with various combinations of IVM, chloroquine, and primaquine and plasma or whole blood was collected at 4 hours. Seven days after the infective blood meal, mosquitoes were dissected to evaluate oocyst presence. Additionally, the ex vivo effects of IVM against asexual blood-stage P. vivax was evaluated.ResultsIVM significantly reduced the prevalence of An. aquasalis that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In An. darlingi to 4 hours and 1 day. The An. aquasalis mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in An. darlingi only to 4 hours and 1 day. The double fed meal with mIVM by the mosquitoes has a considerable impact on the proportion of infected mosquitoes for 7 days post-feeding. The oocyst infection prevalence and intensity were notably reduced when mosquitoes ingested blood from P. vivax patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. P. vivax asexual development was considerably inhibited by mIVM at four-fold dilutions.ConclusionIn conclusion, whole blood spiked with IVM reduced the infection rate of P. vivax in An. aquasalis and An. darlingi, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual P. vivax development. These findings support that ivermectin may be used to decrease P. vivax transmission.

Whole mitogenome sequences (mtDNA) have been exploited for insect ecology studies, using them as molecular markers to reconstruct phylogenies, or to infer phylogeographic relationships and gene flow. Recent Anopheles phylogenomic studies have provided information regarding the time of deep lineage divergences within the genus. Here we report the complete 15,393 bp mtDNA sequences of Anopheles aquasalis , a Neotropical human malaria vector. When comparing its structure and base composition with other relevant and available anopheline mitogenomes, high similarity and conserved genomic features were observed. Furthermore, 22 mtDNA sequences comprising anopheline and Dipteran sibling species were analyzed to reconstruct phylogenies and estimate dates of divergence between taxa. Phylogenetic analysis using complete mtDNA sequences suggests that A . aquasalis diverged from the Anopheles albitarsis complex ~28 million years ago (MYA), and ~38 MYA from Anopheles darlingi . Bayesian analysis suggests that the most recent ancestor of Nyssorhynchus and Anopheles + Cellia was extant ~83 MYA, corroborating current estimates of ~79–100 MYA. Additional sampling and publication of African, Asian, and North American anopheline mitogenomes would improve the resolution of the Anopheles phylogeny and clarify early continental dispersal routes.

BackgroundPlasmodium vivax is predominant in the Amazon region, and enhanced knowledge of its development inside a natural vector, Anopheles aquasalis, is critical for future strategies aimed at blocking parasite development. The peritrophic matrix (PM), a chitinous layer produced by the mosquito midgut in response to blood ingestion, is a protective barrier against pathogens. Plasmodium can only complete its life-cycle, and consequently be transmitted to a new host, after successfully passing this barrier. Interestingly, fully engorged mosquitoes that had a complete blood meal form a thicker, well-developed PM than ones that feed in small amounts. The amount of red blood cells (RBC) in the blood meal directly influences the production of digestive enzymes and can protect parasites from being killed during the meal digestion. A specific study interrupting the development of the PM associated with the proteolytic activity inhibition, and distinct RBC concentrations, during the P. vivax infection of the New World malaria vector An. aquasalis is expected to clarify whether these factors affect the parasite development.ResultsAbsence of PM in the vector caused a significant reduction in P. vivax infection. However, the association of chitinase with trypsin inhibitor restored infection rates to those of mosquitoes with a structured PM. Also, only the ingestion of trypsin inhibitor by non-chitinase treated mosquitoes increased the infection intensity. Moreover, the RBC concentration in the infected P. vivax blood meal directly influenced the infection rate and its intensity. A straight correlation was observed between RBC concentrations and infection intensity.ConclusionsThis study established that there is a balance between the PM role, RBC concentration and digestive enzyme activity influencing the establishment and development of P. vivax infection inside An. aquasalis. Our results indicate that the absence of PM in the midgut facilitates digestive enzyme dispersion throughout the blood meal, causing direct damage to P. vivax. On the other hand, high RBC concentrations support a better and thick, well-developed PM and protect P. vivax from being killed. Further studies of this complex system may provide insights into other details of the malaria vector response to P. vivax infection.

BackgroundThe leishmaniases are a group of diseases caused by protozoans of the genus Leishmania, which are transmitted by the bite of phlebotomine sand flies. In the New World, Lutzomyia longipalpis is the most important vector of visceral leishmaniasis and is a proven vector for Leishmania infantum chagasi in Brazil. During development within the vector, Leishmania can interact with a variety of microorganisms such as fungi and bacteria. The presence of bacteria in the midgut of sand flies can influence the development and survival of the parasite.ResultsThe bacteria-targeted metagenomic analysis revealed different community compositions between the distinct physiological stages of those tested. The amplicon-oriented metagenomic profiling revealed 64 bacterial genera and 46 families. By crossing the taxa indices from each experimental condition a core composed of 6 genera was identified (Enterobacter, Serratia, Stenotrophomonas, Enhydrobacter, Pseudomonas and Chryseobacterium).ConclusionsThe observed dynamic nature of the bacterial community expands the knowledge pertaining to the tripartite host-microbiota-pathogen interactions. Further studies addressing how laboratory and field collected communities differ are critical to successfully develop control strategies based on bacterial symbionts and paratransgenesis, as already tested in other arthropod vectors.Electronic supplementary materialThe online version of this article (10.1186/s13071-017-2593-7) contains supplementary material, which is available to authorized users.