Claudia M Ríos-Velásquez

The JAK-STAT pathway controls Plasmodium vivax load in early stages of Anopheles aquasalis infection

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.

Distribution of dengue vectors in neighborhoods with different urbanization types of Manaus, state of Amazonas, Brazil

Aedes aegypti and Ae. albopictus are vectors of dengue viruses, which cause endemic disease in the city of Manaus, capital of the state of Amazonas, Brazil. More than 53 thousand cases have been registered in this city since the first epidemic in 1998. We evaluated the hypothesis that different ecological conditions result in different patterns of vector infestation in Manaus, by measuring the infestation level in four neighborhoods with different urbanization patterns, during the rainy (April), dry (August), and transitional (November) seasons. Ae. aegypti predominated throughout the study areas and sampling periods, representing 86% of all specimens collected in oviposition traps. High frequencies of houses positive for both species were observed in all studied sites, with Ae. aegypti present in more than 84% of the houses in all seasons. Ae. albopictus, on the other hand, showed more spatial and temporal variation in abundance. We found no association between infestation level and house traits. This study highlights the homogeneity of dengue vector distribution in Manaus.

Trichomycetes (Zygomycota) in the digestive tract of arthropods in Amazonas, Brazil

Eight species of Harpellales and three species of Eccrinales (Zygomycota: Trichomycetes) were found associated with the digestive tract of arthropods from terrestrial and aquatic environments in the central Amazon region of Brazil. New species of Harpellales include: Harpella amazonica, Smittium brasiliense, Genistellospora tropicalis in Simuliidae larvae and Stachylina paucispora in Chironomidae larvae. Axenic cultures of S. brasiliense were obtained. Probable new species of Enterobryus (Eccrinales), Harpella, and Stachylina (Harpellales) are described but not named. Also reported are the previously known species of Eccrinales, Passalomyces compressus and Leidyomyces attenuatus in adult Coleoptera (Passalidae), and Smittium culisetae and Smittium aciculare (Harpellales) in Culicidae and Simuliidae larvae, respectively. Comments on the distribution of some of these fungi and their hosts in the Neotropics are provided.

The Role of Reactive Oxygen Species in Anopheles aquasalis Response to Plasmodium vivax Infection

Malaria affects millions of people worldwide and hundreds of thousands of people each year in Brazil. The mosquito Anopheles aquasalis is an important vector of Plasmodium vivax, the main human malaria parasite in the Americas. Reactive oxygen species (ROS) have been shown to have a role in insect innate immune responses as a potent pathogen-killing agent. We investigated the mechanisms of free radicals modulation after A. aquasalis infection with P. vivax. ROS metabolism was evaluated in the vector by studying expression and activity of three key detoxification enzymes, one catalase and two superoxide dismutases (SOD3A and SOD3B). Also, the involvement of free radicals in the mosquito immunity was measured by silencing the catalase gene followed by infection of A. aquasalis with P. vivax. Catalase, SOD3A and SOD3B expression in whole A. aquasalis were at the same levels of controls at 24 h and upregulated 36 h after ingestion of blood containing P. vivax. However, in the insect isolated midgut, the mRNA for these enzymes was not regulated by P. vivax infection, while catalase activity was reduced 24 h after the infectious meal. RNAi-mediated silencing of catalase reduced enzyme activity in the midgut, resulted in increased P. vivax infection and prevalence, and decreased bacterial load in the mosquito midgut. Our findings suggest that the interactions between A. aquasalis and P. vivax do not follow the model of ROS-induced parasite killing. It appears that P. vivax manipulates the mosquito detoxification system in order to allow its own development. This can be an indirect effect of fewer competitive bacteria present in the mosquito midgut caused by the increase of ROS after catalase silencing. These findings provide novel information on unique aspects of the main malaria parasite in the Americas interaction with one of its natural vectors.

Experimental Plasmodium vivax infection of key Anopheles species from the Brazilian Amazon

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.

Lipophosphoglycans from Leishmania amazonensis Strains Display Immunomodulatory Properties via TLR4 and Do Not Affect Sand Fly Infection

The immunomodulatory properties of lipophosphoglycans (LPG) from New World species of Leishmania have been assessed in Leishmania infantum and Leishmania braziliensis, the causative agents of visceral and cutaneous leishmaniasis, respectively. This glycoconjugate is highly polymorphic among species with variation in sugars that branch off the conserved Gal(β1,4)Man(α1)-PO4 backbone of repeat units. Here, the immunomodulatory activity of LPGs from Leishmania amazonensis, the causative agent of diffuse cutaneous leishmaniasis, was evaluated in two strains from Brazil. One strain (PH8) was originally isolated from the sand fly and the other (Josefa) was isolated from a human case. The ability of purified LPGs from both strains was investigated during in vitro interaction with peritoneal murine macrophages and CHO cells and in vivo infection with Lutzomyia migonei. In peritoneal murine macrophages, the LPGs from both strains activated TLR4. Both LPGs equally activate MAPKs and the NF-κB inhibitor p-IκBα, but were not able to translocate NF-κB. In vivo experiments with sand flies showed that both stains were able to sustain infection in L. migonei. A preliminary biochemical analysis indicates intraspecies variation in the LPG sugar moieties. However, they did not result in different activation profiles of the innate immune system. Also those polymorphisms did not affect infectivity to the sand fly.

An overview of malaria transmission from the perspective of Amazon Anopheles vectors

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.

A deep insight into the sialotranscriptome of the mosquito, Psorophora albipes

BackgroundPsorophora mosquitoes are exclusively found in the Americas and have been associated with transmission of encephalitis and West Nile fever viruses, among other arboviruses. Mosquito salivary glands represent the final route of differentiation and transmission of many parasites. They also secrete molecules with powerful pharmacologic actions that modulate host hemostasis, inflammation, and immune response. Here, we employed next generation sequencing and proteome approaches to investigate for the first time the salivary composition of a mosquito member of the Psorophora genus. We additionally discuss the evolutionary position of this mosquito genus into the Culicidae family by comparing the identity of its secreted salivary compounds to other mosquito salivary proteins identified so far.ResultsIllumina sequencing resulted in 13,535,229 sequence reads, which were assembled into 3,247 contigs. All families were classified according to their in silico-predicted function/ activity. Annotation of these sequences allowed classification of their products into 83 salivary protein families, twenty (24.39%) of which were confirmed by our subsequent proteome analysis. Two protein families were deorphanized from Aedes and one from Ochlerotatus, while four protein families were described as novel to Psorophora genus because they had no match with any other known mosquito salivary sequence. Several protein families described as exclusive to Culicines were present in Psorophora mosquitoes, while we did not identify any member of the protein families already known as unique to Anophelines. Also, the Psorophora salivary proteins had better identity to homologs in Aedes (69.23%), followed by Ochlerotatus (8.15%), Culex (6.52%), and Anopheles (4.66%), respectively.ConclusionsThis is the first sialome (from the Greek sialo = saliva) catalog of salivary proteins from a Psorophora mosquito, which may be useful for better understanding the lifecycle of this mosquito and the role of its salivary secretion in arboviral transmission.

Anthropic effects on sand fly (Diptera: Psychodidae) abundance and diversity in an Amazonian rural settlement, Brazil

Sand flies (Diptera: Psychodidae) are responsible for the transmission of protozoan parasites that cause leishmaniases. They are found predominantly in forests, but some species exploit environments that have been subject to deforestation and subsequent human colonization. Studies conducted in Brazil over the past 30 years show that some species are adapting to peri-urban and urban settings. We evaluated sand fly diversity and abundance in the rural settlement of Rio Pardo, Presidente Figueiredo Municipality, Amazonas State, Brazil. Settlement households were divided into four categories. These categories were determined by the human population density and the degree of deforestation in the immediate area. We used CDC light traps to sample the area surrounding 24 households (6 households in each category). Samples were taken on six occasions during September-November 2009 and June-August 2010. A total of 3074 sand fly specimens were collected, including 1163 females and 1911 males. These were classified into 13 genera and 52 species. The greatest abundance of sand flies and the greatest richness of species were observed in areas where human population density was highest. Our results show that changes in the human occupancy and vegetation management in rural settlements may affect the population dynamics and distribution of sand fly species, thereby affecting the local transmission of cutaneous leishmaniases.

Seasonal dynamics of Aedes aegypti (Diptera: Culicidae) in the northernmost state of Brazil: a likely port-of-entry for dengue virus 4

Roraima is the northernmost state of Brazil, bordering both Venezuela and Guyana. Appropriate climate and vector conditions for dengue transmission together with its proximity to countries where all four dengue serotypes circulate make this state, particularly the capital Boa Vista, strategically important for dengue surveillance in Brazil. Nonetheless, few studies have addressed the population dynamics of Aedes aegypti in Boa Vista. In this study, we report temporal and spatial variations in Ae. aegypti population density using ovitraps in two highly populated neighbourhoods; Centro and Tancredo Neves. In three out of six surveys, Ae. aegypti was present in more than 80% of the sites visited. High presence levels of this mosquito suggest ubiquitous human exposure to the vector, at least during part of the year. The highest infestation rates occurred during the peak of the rainy seasons, but a large presence was also observed during the early dry season (although with more variation among years). Spatial distribution of positive houses changed from a sparse and local pattern to a very dense pattern during the dry-wet season transition. These results suggest that the risk of dengue transmission and the potential for the new serotype invasions are high for most of the year.