Endogenously-expressed NH2-terminus of circumsporozoite protein interferes with sporozoite invasion of mosquito salivary glands

Abstract\ud \ud Background\ud The circumsporozoite protein is the most abundant polypeptide expressed by sporozoites, the malaria parasite stage capable of infecting humans. Sporozoite invasion of mosquito salivary glands prior to transmission is likely mediated by a receptor/ligand-like interaction of the parasites with the target tissues, and the amino (NH2)-terminal portion of CSP is involved in this interaction but not the TSR region on the carboxyl (C)-terminus. Peptides based on the NH2-terminal domain could compete with the parasites for the salivary gland receptors and thus inhibit penetration.\ud \ud \ud Methods\ud Peptides based on the NH2-terminus and TSR domains of the CSP from avian or human malaria parasites, Plasmodium gallinaceum and Plasmodium falciparum, respectively, were expressed endogenously in mosquito haemolymph using a transient (Sindbis virus-mediated) or stable (piggyBac-mediated transgenesis) system.\ud \ud \ud Results\ud Transient endogenous expression of partial NH2-terminus peptide from P. falciparum CSP in P. gallinaceum-infected Aedes aegypti resulted in a reduced number of sporozoites in the salivary glands. When a transgenic approach was used to express a partial CSP NH2-terminal domain from P. gallinaceum the number of sporozoites in the salivary glands did not show a difference when compared to controls. However, a significant difference could be observed when mosquitoes with a lower infection were analysed. The same result could not be observed with mosquitoes endogenously expressing peptides based on the TSR domain from either P. gallinaceum or P. falciparum.\ud \ud \ud \ud Conclusion\ud These results support the conclusion that CSP partial NH2-terminal domain can be endogenously expressed to promote a competition for the receptor used by sporozoites to invade salivary glands, and they could be used to block this interaction and reduce parasite transmission. The same effect cannot be obtained with peptides based on the TSR domain.We thank Neuza Saraiva Fernandes and Ediane Saraiva Fernandes for technical\ud assistance and Alexandre Santos de Moura for sample sequencing. This work\ud was supported by FAPESP (Process#04/00889-9 and#00/12138-7). AAJ was\ud supported in part by an award from the National Institute of Allergy and Infec‑\ud tious Diseases (USA) (R37AI029746)

Microplusin expression in Aedes aegypti: evaluation of effect on Plasmodium gallinaceum.

A transmissão de parasitas da malária por mosquitos vetores é dependente do desenvolvimento bem sucedido das formas infectantes de Plasmodium sp., especialmente os esporozoítas, que são as formas que infectam o hospedeiro vertebrado. A manipulação genética de mosquitos vetores tem sido uma estratégia alternativa na tentativa de controle da malária. Um componente extremamente importante desta estratégia é a escolha de uma molécula efetora capaz de reduzir a transmissão do patógeno. Microplusina é um peptídeo antimicrobiano rico em cisteína, originalmente descrito como um componente antimicrobiano da hemolinfa e dos ovos de carrapato bovino Rhipicephalus (Boophilus) microplus. Testes anteriores utilizando o modelo experimental mosquito Aedes aegypti infectado por Plasmodium gallinaceum mostraram que a microplusina é altamente tóxico para esporozoítas de Plasmodium gallinaceum em concentração relativamente baixa, sem apresentar toxicidade aos mosquitos vetores Aedes aegypti. Nosso objetivo foi analisar a expressão da microplusina e seu efeito na infecção de P. gallinaceum em mosquitos transgênicos. Obtivemos quatro linhagens através da integração de um transgene contendo a região promotora do gene da vitelogenina de Ae. aegypti, peptídeo sinal maltase-like I de Ae. aegypti e a sequência codificadora da microplusina (PMOS [3xP3-EGFP-AeVg Micro]). A atividade anti esporozoítas da microplusina expressa pelos mosquitos transgênicos mostrou diferença significante as linhagens. O desenho de novas moléculas utilizando como molde moléculas efetoras existentes e testadas, possibilitará o aperfeiçoamento da expressão de genes exógenos em mosquitos transgênicos, tornando-os refratários ao parasita.Transmission of malaria parasites by mosquito vectors is dependent on the successful development of Plasmodium sp. infective forms, particularly the sporozoites, which are the forms that enter the vertebrate host. The genetic manipulation of mosquito vectors has been a strategy for malaria control. An extremely important component of this strategy is the effector molecule of choice which reduces parasite transmission. Microplusin is a cysteine-rich antimicrobial peptide originally described as an hemolymph and eggs antimicrobial component of the cattle tick Boophilus microplus. Previous tests using the experimental model Plasmodium gallinaceum infected Aedes aegypti showed that microplusin is highly toxic to P. gallinaceum sporozoites in relatively low concentration, without showing toxicity to the mosquito vector A. aegypti. Our goal was to analyze transgenic mosquitoes expressing microplusin and its effect on infection of P. gallinaceum. We obtained four lines through the integration of transgene that containing the promoter region of the A. aegypti vitelogenin gene, the maltase-like I signal peptide of A. aegypti and microplusin coding sequence (pMos[3xP3-EGFPAeVg-Micro]). The activity anti sporozoites microplusin expressed by transgenic mosquitoes showed significant differences between strains. The design of effector molecules using information from existing and tested molecules as template will enable the improvement of the expression of foreign genes in transgenic mosquitoes, making them resistant to the parasite

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development

Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites. (C) 2010 Elsevier Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Ministério da Saúde MTC/DECITMinistério da Saúde MTC/DECITConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Anti-Plasmodium Activity of Angiotensin II and Related Synthetic Peptides

Plasmodium species are the causative agents of malaria, the most devastating insect-borne parasite of human populations. Finding and developing new drugs for malaria treatment and prevention is the goal of much research. Angiotensins I and II (ang I and ang II) and six synthetic related peptides designated Vaniceres 1-6 (VC1-VC6) were assayed in vivo and in vitro for their effects on the development of the avian parasite, Plasmodium gallinaceum. Ang II and VC5 injected into the thoraces of the insects reduced mean intensities of infection in the mosquito salivary glands by 88 % and 76%, respectively. Although the mechanism(s) of action is not completely understood, we have demonstrated that these peptides disrupt selectively the P.gallinaceum cell membrane. Additionally, incubation in vitro of sporozoites with VC5 reduced the infectivity of the parasites to their vertebrate host. VC5 has no observable agonist effects on vertebrates, and this makes it a promising dru

Evans Blue as a Simple Method to Discriminate Mosquitoes’ Feeding Choice on Small Laboratory Animals

<div><p>Background</p><p>Temperature, humidity, vision, and particularly odor, are external cues that play essential roles to mosquito blood feeding and oviposition. Entomological and behavioral studies employ well-established methods to evaluate mosquito attraction or repellency and to identify the source of the blood meal. Despite the efficacy of such methods, the costs involved in the production or acquisition of all parts, components and the chemical reagents involved are unaffordable for most researchers from poor countries. Thus, a simple and relatively low-cost method capable of evaluating mosquito preferences and the blood volume ingested is desirable.</p><p>Principal Findings</p><p>By using Evans blue (EB) vital dye and few standard laboratory supplies, we developed and validated a system capable of evaluating mosquito’s choice between two different host sources of blood. EB-injected and PBS-injected mice submitted to a number of situations were placed side by side on the top of a rounded recipient covered with tulle fabric and containing <i>Aedes aegypti</i> mosquitoes. Homogenates from engorged mosquitoes clearly revealed the blood source (EB- or PBS-injected host), either visually or spectrometrically. This method was able to estimate the number of engorded mosquitoes, the volume of blood ingested, the efficacy of a commercial repellent and the attractant effects of black color and human sweat.</p><p>Significance</p><p>Despite the obvious limitations due to its simplicity and to the dependence of a live source of blood, the present method can be used to assess a number of host variables (diet, aging, immunity, etc) and optimized for several aspects of mosquito blood feeding and vector-host interactions. Thus, it is proposed as an alternative to field studies, and it could be used for initial screenings of chemical compound candidates for repellents or attractants, since it replicates natural conditions of exposure to mosquitoes in a laboratory environment.</p></div

Effect of coat color and fresh human sweat on attraction of <i>A. aegypti</i> to mice.

<p><i>A</i>. Proportion of mosquitoes that chose the C57BL/6 mouse (black coat). <i>B.</i> Proportion of mosquitoes that chose sweat-applied mice (EB-injected or PBS-injected). The bars represent the 95% confidence interval.</p

Presence of EB is detected either visually or spectrophotometrically in blood samples or in homogenates of engorged mosquitoes.

<p><i>A.</i> Visual comparison of blood dilutions from PBS- and EB-injected mice (20 and 200 mg/kg). <i>B.</i> Absorbance comparison of blood from PBS- and EB-injected mice at 540 and 620 nm. <i>C.</i> Linear regression of blood volume (X axis) <i>versus</i> absorbance (Y axis) at 540 nm and 620 nm from PBS- and EB-injected mice. <i>D.</i> Experimental scheme and typical visual profile of homogenates from <i>A. aegypti</i> mosquitoes fed on PBS-injected (red) or EB-injected (blue) mice. <i>E.</i> Absorbance profiles of homogenates from individual <i>A. aegypti</i> mosquitoes fed on PBS- or EB-injected mice at 540 nm and 620 nm.</p

Basic laboratory reagents and supplies needed to evaluate/discriminate the blood source from engorged mosquitoes.

<p>Materials are: <i>1.</i> Tulle fabric; <i>2.</i> Rounded container; <i>3.</i> Glass beaker containing distilled water; <i>4.</i> 96-well plate, flat bottom; <i>5.</i> 1 mL syringe with needle; <i>6.</i> plastic pestle; <i>7.</i> 1.7 mL microcentrifuge tube; <i>8.</i> 20–200 µL universal tip; <i>9.</i> 15 mL tube; <i>10.</i> EB solution; <i>11.</i> 20–200 µL pipette.</p

EB model is useful to evaluate the activity of a commercial repellent against <i>A. aegypti</i> mosquitoes.

<p><i>A</i> and <i>D</i>. Experimental scheme and typical visual profile of homogenate from <i>A. aegypti</i> mosquitoes fed on PBS-injected (red) and EB-injected (blue) mice that received a commercial repellent before exposure to mosquitoes. <i>B</i> and <i>E</i>. Proportion of mosquitoes that chose the PBS and EB mice without repellent, respectively. The bars represent the 95% confidence interval. <i>C</i> and <i>F</i>. Boxplots of the blood volumes from engorged mosquitoes fed on PBS- or EB-injected mice (2.5–97.5 percentile). Dark dots represent the outliers.</p

EB presence in blood circulation does not affect <i>A. aegypti</i> attractiveness to mice.

<p><i>A</i>. Experimental scheme and typical visual profile of homogenates from <i>A. aegypti</i> mosquitoes fed on PBS-injected (red) and EB-injected (blue) mice. <i>B.</i> Proportion of mosquitoes that chose the EB-injected mouse. The bars represent the 95% confidence interval. <i>C.</i> Boxplots of the blood volumes from engorged mosquitoes fed on PBS- or EB-injected mice (2.5–97.5 percentile). Dark dots represent the outliers.</p