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The role of reactive oxygen species in Anopheles aquasalis response to Plasmodium vivax infection.

ABSTRACT: 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.

SUBMITTER: Bahia AC

PROVIDER: S-EPMC3575503 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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The role of reactive oxygen species in Anopheles aquasalis response to Plasmodium vivax infection.

Bahia Ana C AC Oliveira José Henrique M JH Kubota Marina S MS Araújo Helena R C HR Lima José B P JB Ríos-Velásquez Claudia Maria CM Lacerda Marcus Vinícius G MV Oliveira Pedro L PL Traub-Csekö Yara M YM Pimenta Paulo F P PF

PloS one 20130218 2

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 ve ...[more]

PMID: 23441231

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Project description:BackgroundElimination of malaria depends on mastering transmission and understanding the biological basis of Plasmodium infection in the vector. The first mosquito organ to interact with the parasite is the midgut and its transcriptomic characterization during infection can reveal effective antiplasmodial responses able to limit the survival of the parasite. The vector response to Plasmodium vivax is not fully characterized, and its specificities when compared with other malaria parasites can be of fundamental interest for specific control measures.MethodsExperimental infections were performed using a membrane-feeding device. Three groups were used: P. vivax-blood-fed, blood-fed on inactivated gametocytes, and unfed mosquitoes. Twenty-four hours after feeding, the mosquitoes were dissected and the midgut collected for transcriptomic analysis using RNAseq. Nine cDNA libraries were generated and sequenced on an Illumina HiSeq2500. Readings were checked for quality control and analysed using the Trinity platform for de novo transcriptome assembly. Transcript quantification was performed and the transcriptome was functionally annotated. Differential expression gene analysis was carried out. The role of the identified mechanisms was further explored using functional approaches.ResultsForty-nine genes were identified as being differentially expressed with P. vivax infection: 34 were upregulated and 15 were downregulated. Half of the P. vivax-related differentially expressed genes could be related to autophagy; therefore, the effect of the known inhibitor (wortmannin) and activator (spermidine) was tested on the infection outcome. Autophagic activation significantly reduced the intensity and prevalence of infection. This was associated with transcription alterations of the autophagy regulating genes Beclin, DRAM and Apg8.ConclusionsOur data indicate that P. vivax invasion of An. aquasalis midgut epithelium triggers an autophagic response and its activation reduces infection. This suggests a novel mechanism that mosquitoes can use to fight Plasmodium infection.

Project description:The 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.To 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 ?g/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.IVM 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.In 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.

Project description:BackgroundThe colonization of mosquitoes susceptible to Plasmodium vivax via direct membrane feeding assay (DMFA) has the potential to significantly advance our knowledge of P. vivax biology, vector-parasite interaction and transmission-blocking vaccine research. Anopheles darlingi and Anopheles deaneorum are important vectors of malaria in the Western Brazilian Amazon. Since 2018, well-established colonies of these species have been maintained in order to mass produce mosquitoes destined for P. vivax infection. Plasmodium susceptibility was confirmed when the colonies were established, but susceptibility needs to be maintained for these colonies to remain good models for pathogen transmission. Thus, the susceptibility was assessed of colonized mosquitoes to P. vivax isolates circulating in the Western Amazon.MethodsLaboratory-reared mosquitoes from F10-F25 generations were fed on P. vivax blood isolates via DMFA. Susceptibility was determined by prevalence and intensity of infection as represented by oocyst load seven days after blood feeding, and sporozoite load 14 days after blood feeding. The effect of infection on mosquito survival was evaluated from initial blood feeding until sporogonic development and survival rates were compared between mosquitoes fed on infected and uninfected blood. Correlation was calculated between gametocytaemia and prevalence/intensity of infection, and between oocyst and sporozoite load.ResultsSignificant differences were found in prevalence and intensity of infection between species. Anopheles darlingi showed a higher proportion of infected mosquitoes and higher oocyst and sporozoite intensity than An. deaneorum. Survival analysis showed that An. deaneorum survival decreased drastically until 14 days post infection (dpi). Plasmodium vivax infection decreased survival in both species relative to uninfected mosquitoes. No correlation was observed between gametocytaemia and prevalence/intensity of infection, but oocyst and sporozoite load had a moderate to strong correlation.ConclusionsColonized An. darlingi make excellent subjects for modelling pathogen transmission. On the other hand, An. deaneorum could serve as a model for immunity studies due the low susceptibility under current colonized conditions. In the application of DMFA, gametocyte density is not a reliable parameter for predicting mosquito infection by P. vivax, but oocyst intensity should be used to schedule sporozoite experiments.

Project description:INTRODUCTION:Individuals exposed to malaria infections for a long time develop immune responses capable of blocking Plasmodium transmission to mosquito vectors, potentially limiting parasite spreading in nature. Development of a malaria TB vaccine requires a better understanding of the mechanisms and main effectors responsible for transmission blocking (TB) responses. The lack of an in vitro culture system for Plasmodium vivax has been an important drawback for development of a standardized method to assess TB responses to this parasite. This study evaluated host, vector, and parasite factors that may influence Anopheles mosquito infection in order to develop an efficient and reliable assay to assess the TB immunity. METHODS/PRINCIPAL FINDINGS:A total of 94 P. vivax infected patients were enrolled as parasite donors or subjects of direct mosquito feeding in two malaria endemic regions of Colombia (Tierralta, and Buenaventura). Parasite infectiousness was assessed by membrane feeding assay or direct feeding assay using laboratory reared Anopheles mosquitoes. Infection was measured by qPCR and by microscopically examining mosquito midguts at day 7 for the presence of oocysts. Best infectivity was attained in four day old mosquitoes fed at a density of 100 mosquitos/cage. Membrane feeding assays produced statistically significant better infections than direct feeding assays in parasite donors; cytokine profiles showed increased IFN-?, TNF and IL-1 levels in non-infectious individuals. Mosquito infections and parasite maturation were more reliably assessed by PCR compared to microscopy. CONCLUSIONS:We evaluated mosquito, parasite and host factors that may affect the outcome of parasite transmission as measured by artificial membrane feeding assays. Results have led us to conclude that: 1) optimal mosquito infectivity occurs with mosquitoes four days after emergence at a cage density of 100; 2) mosquito infectivity is best quantified by PCR as it may be underestimated by microscopy; 3) host cellular immune response did not appear to significantly affect mosquito infectivity; and 4) no statistically significant difference was observed in transmission between mosquitoes directly feeding on humans and artificial membrane feeding assays.

Dataset Information

The role of reactive oxygen species in Anopheles aquasalis response to Plasmodium vivax infection.

Publications

The role of reactive oxygen species in Anopheles aquasalis response to Plasmodium vivax infection.

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