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

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against <i>Plasmodium vivax</i> in the South American vectors <i>Anopheles aquasalis</i> and <i>Anopheles darlingi</i>

<div><p>Background</p><p>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 <i>Plasmodium</i> transmission. <i>Anopheles aquasalis</i> and <i>Anopheles darlingi</i> are competent vectors for <i>Plasmodium vivax</i>, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America.</p><p>Methods</p><p>To determine the IVM susceptibility against <i>P</i>. <i>vivax</i> in <i>An</i>. <i>aquasalis</i> and <i>An</i>. <i>darlingi</i>, ivermectin were mixed in <i>P</i>. <i>vivax</i> infected blood: (<b>1)</b> Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (<b>2</b>) 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) (<b>3</b>) Mosquitoes infected with <i>P</i>. <i>vivax</i> and after 4 days was provided with IVM plasma collected 4 hours post-treatment (<b>4</b>) <i>P</i>. <i>vivax</i>-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 <i>ex vivo</i> effects of IVM against asexual blood-stage <i>P</i>. <i>vivax</i> was evaluated.</p><p>Results</p><p>IVM significantly reduced the prevalence of <i>An</i>. <i>aquasalis</i> that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In <i>An</i>. <i>darlingi</i> to 4 hours and 1 day. The <i>An</i>. <i>aquasalis</i> mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in <i>An</i>. <i>darlingi</i> 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 <i>P</i>. <i>vivax</i> patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. <i>P</i>. <i>vivax</i> asexual development was considerably inhibited by mIVM at four-fold dilutions.</p><p>Conclusion</p><p>In conclusion, whole blood spiked with IVM reduced the infection rate of <i>P</i>. <i>vivax</i> in <i>An</i>. <i>aquasalis</i> and <i>An</i>. <i>darlingi</i>, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual <i>P</i>. <i>vivax</i> development. These findings support that ivermectin may be used to decrease <i>P</i>. <i>vivax</i> transmission.</p></div

Ivermectin powdered affects <i>Plasmodium vivax</i> development in <i>Anopheles aquasalis</i>.

<p>(A) The proportion of <i>P</i>. <i>vivax</i> infected mosquitoes is presented as a mean and standard deviation. (B) The infection intensity is presented as the number of oocysts per a single midgut (black dots) and the black lines represent the mean and standard deviation. (C) Mosquito mortality rate to 7 days post-feeding is presented as mean and standard deviation. Data from 5 independent experiments are presented in A-C. Asterisks (*) represent significant difference (P < 0.05) in relation to the control.</p

Plasmatic ivermectin and malaria drugs have a direct effect on <i>Plasmodium vivax</i> infection in <i>Anopheles aquasalis</i>.

<p><i>An</i>. <i>aquasalis</i> female mosquitos were fed with unprocessed blood samples from <i>P</i>. <i>vivax</i> patients at time 0 (control) or 4 hours after different treatment regimens intake or with erythrocytes from <i>P</i>. <i>vivax</i> patients’ blood samples (before treatment) reconstituted to a hematocrit of 40% with the respective plasma after 4 hours of different treatment regimens intake. (A) The proportion of <i>P</i>. <i>vivax</i> infected mosquitos are presented as mean and standard deviation. Black bars represent the unprocessed blood and the grey bars represent the reconstitute samples. (B) The intensity of infection is presented as the number of oocysts per a single mosquito midgut (dots), black dots represent the unprocessed blood and the grey dots represent the reconstituted blood samples, and the black lines represent the median and standard error blood. Asterisks (*) represent significant difference (P < 0.05) in relation to the control.</p

Schematic outline of the experiments of membrane feeding.

<p>Four experiments were performed to determine the transmission blocking potential of IVM in either <i>An</i>. <i>aquasalis</i> or <i>An</i>. <i>darlingi</i>. Abbreviations: Ivermectin (IVM), powdered ivermectin (pIVM), metabolized ivermectin (mIVM), chloroquine (CQ), primaquine (PQ).</p

Metabolized ivermectin impairs asexual <i>P</i>. <i>vivax</i> maturation.

<p>Five <i>P</i>. <i>vivax</i> samples containing more than 80% of parasites on ring stage were evaluated. Nine different concentrations of pIVM were added to <i>P</i>. <i>vivax</i> culture. Moreover, plasma from healthy volunteers collected before (0 hours) and after 4 hours of IVM intake was added in 4 different serial dilutions (1:2) with complete medium. A drug free condition was used as the control. The number of schizonts in 200 asexual parasites was evaluated for each condition and the maturation in relation to control were determined. Asterisks (*) represents significant differences (P < 0.05) in relation to control by Kruskal-Wallis test.</p

Ivermectin impairs <i>Plasmodium vivax</i> development in <i>Anopheles aquasalis</i> and <i>Anopheles darlingi</i> when administered in a double feed 4 days after infection.

<p><i>An</i>. <i>aquasalis</i> and <i>An</i>. <i>darlingi</i> female mosquitos were fed <i>P</i>. <i>vivax</i> blood samples. Four days after infection, two groups of mosquitos received a second uninfected blood meal with mIVM plasma from control (0 hours) or 4 hours after IVM intake. (A) The proportion of <i>P</i>. <i>vivax</i> infected <i>An</i>. <i>aquasalis</i> and (B) <i>An</i>. <i>darlingi</i> are presented as the mean and standard deviation. (C and D) The intensity of infection is presented as the number of oocysts per midgut (dots) and the black lines represent the median and standard error. Data from 5 independent experiments are presented. Asterisks (*) represent significant difference (P < 0.05) in relation to control.</p

Ivermectin metabolized affects <i>Plasmodium vivax</i> development in <i>Anopheles aquasalis</i> and <i>Anopheles darlingi</i>.

<p><i>An</i>. <i>aquasalis</i> (A,C,E) and <i>An</i>. <i>darlingi</i> (B,D,F) mosquitoes were fed <i>P</i>. <i>vivax</i> with mIVM collected at various time points (0 and 4 hours; 1, 5, 10 and 14 days). (A) and (B) Proportion of <i>P</i>. <i>vivax</i> infected mosquitoes are presented as mean and standard deviation. (C) and (D) The intensity of infection is presented as the number of oocysts per a single midgut (black dots) and the black lines represent the median and standard deviation. (E) and (F) The mosquito mortality rate is presented as mean and standard deviation. Data from 5 independent experiments are presented. Asterisks (*) represent significant difference (P < 0.05) in relation to the control.</p

<i>Ex vivo</i> activity of ivermectin and chloroquine against <i>P</i>. <i>vivax</i> isolates from Manaus- AM, Brazil.

<p><i>Ex vivo</i> activity of ivermectin and chloroquine against <i>P</i>. <i>vivax</i> isolates from Manaus- AM, Brazil.</p