Increased insulin signaling in the Anopheles stephensi fat body regulates metabolism and enhances the host response to both bacterial challenge and Plasmodium falciparum infection.

In vertebrates and invertebrates, the insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) cascade is highly conserved and plays a vital role in many different physiological processes. Among the many tissues that respond to IIS in mosquitoes, the fat body has a central role in metabolism, lifespan, reproduction, and innate immunity. We previously demonstrated that fat body specific expression of active Akt, a key IIS signaling molecule, in adult Anopheles stephensi and Aedes aegypti activated the IIS cascade and extended lifespan. Additionally, we found that transgenic females produced more vitellogenin (Vg) protein than non-transgenic mosquitoes, although this did not translate into increased fecundity. These results prompted us to further examine how IIS impacts immunity, metabolism, growth and development of these transgenic mosquitoes. We observed significant changes in glycogen, trehalose, triglycerides, glucose, and protein in young (3-5 d) transgenic mosquitoes relative to non-transgenic sibling controls, while only triglycerides were significantly changed in older (18 d) transgenic mosquitoes. More importantly, we demonstrated that enhanced fat body IIS decreased both the prevalence and intensity of Plasmodium falciparum infection in transgenic An. stephensi. Additionally, challenging transgenic An. stephensi with Gram-positive and Gram-negative bacteria altered the expression of several antimicrobial peptides (AMPs) and two anti-Plasmodium genes, nitric oxide synthase (NOS) and thioester complement-like protein (TEP1), relative to non-transgenic controls. Increased IIS in the fat body of adult female An. stephensi had little to no impact on body size, growth or development of progeny from transgenic mosquitoes relative to non-transgenic controls. This study both confirms and expands our understanding of the critical roles insulin signaling plays in regulating the diverse functions of the mosquito fat body.

Enhanced transmission of malaria parasites to mosquitoes in a murine model of type 2 diabetes.

BACKGROUND: More than half of the world's population is at risk of malaria and simultaneously, many malaria-endemic regions are facing dramatic increases in the prevalence of type 2 diabetes. Studies in murine malaria models have examined the impact of malaria infection on type 2 diabetes pathology, it remains unclear how this chronic metabolic disorder impacts the transmission of malaria. In this report, the ability type 2 diabetic rodents infected with malaria to transmit parasites to Anopheles stephensi mosquitoes is quantified. METHODS: The infection prevalence and intensity of An. stephensi mosquitoes that fed upon control or type 2 diabetic C57BL/6 db/db mice infected with either lethal Plasmodium berghei NK65 or non-lethal Plasmodium yoelii 17XNL murine malaria strains were determined. Daily parasitaemias were also recorded. RESULTS: A higher percentage of mosquitoes (87.5 vs 61.5 % for P. yoelii and 76.9 vs 50 % for P. berghei) became infected following blood feeding on Plasmodium-infected type 2 diabetic mice compared to mosquitoes that fed on infected control animals, despite no significant differences in circulating gametocyte levels. CONCLUSIONS: These results suggest that type 2 diabetic mice infected with malaria are more efficient at infecting mosquitoes, raising the question of whether a similar synergy exists in humans.

Reactive oxygen species-dependent cell signaling regulates the mosquito immune response to Plasmodium falciparum.

Reactive oxygen species (ROS) have been implicated in direct killing of pathogens, increased tissue damage, and regulation of immune signaling pathways in mammalian cells. Available research suggests that analogous phenomena affect the establishment of Plasmodium infection in Anopheles mosquitoes. We have previously shown that provision of human insulin in a blood meal leads to increased ROS levels in Anopheles stephensi. Here, we demonstrate that provision of human insulin significantly increased parasite development in the same mosquito host in a manner that was not consistent with ROS-induced parasite killing or parasite escape through damaged tissue. Rather, our studies demonstrate that ROS are important mediators of both the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling branches of the mosquito insulin signaling cascade. Further, ROS alone can directly activate these signaling pathways and this activation is growth factor specific. Our data, therefore, highlight a novel role for ROS as signaling mediators in the mosquito innate immune response to Plasmodium parasites.

Both hemolytic anemia and malaria parasite-specific factors increase susceptibility to Nontyphoidal Salmonella enterica serovar typhimurium infection in mice.

Severe pediatric malaria is an important risk factor for developing disseminated infections with nontyphoidal Salmonella serotypes (NTS). While recent animal studies on this subject are lacking, early work suggests that an increased risk for developing systemic NTS infection during malaria is caused by hemolytic anemia, which leads to reduced macrophage microbicidal activity. Here we established a model for oral Salmonella enterica serotype Typhimurium challenge in mice infected with Plasmodium yoelii nigeriensis. Initial characterization of this model showed that 5 days after coinoculation, P. yoelii nigeriensis infection increased the recovery of S. Typhimurium from liver and spleen by approximately 1,000-fold. The increased bacterial burden could be only partially recapitulated by antibody-mediated hemolysis, which increased the recovery of S. Typhimurium from liver and spleen by 10-fold. These data suggested that both hemolysis and P. yoelii nigeriensis-specific factors contributed to the increased susceptibility to S. Typhimurium. The mechanism by which hemolysis impaired resistance to S. Typhimurium was further investigated. In vitro, S. Typhimurium was recovered 24 h after infection of hemophagocytic macrophages in 2-fold-higher numbers than after infection of mock-treated macrophages, making it unlikely that reduced macrophage microbicidal activity was solely responsible for hemolysis-induced immunosuppression during malaria. Infection with P. yoelii nigeriensis, but not antibody-mediated hemolysis, reduced serum levels of interleukin-12p70 (IL-12p70) in response to S. Typhimurium challenge. Collectively, studies establishing a mouse model for this coinfection suggest that multiple distinct malaria-induced immune defects contribute to increased susceptibility to S. Typhimurium.

Transfection and mutagenesis of target genes in mosquito cells by locked nucleic acid-modified oligonucleotides.

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development (1). From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in (2)). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice (3,4). We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

MAPK ERK signaling regulates the TGF-beta1-dependent mosquito response to Plasmodium falciparum.

Malaria is caused by infection with intraerythrocytic protozoa of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Although a variety of anti-parasite effector genes have been identified in anopheline mosquitoes, little is known about the signaling pathways that regulate these responses during parasite development. Here we demonstrate that the MEK-ERK signaling pathway in Anopheles is controlled by ingested human TGF-beta1 and finely tunes mosquito innate immunity to parasite infection. Specifically, MEK-ERK signaling was dose-dependently induced in response to TGF-beta1 in immortalized cells in vitro and in the A. stephensi midgut epithelium in vivo. At the highest treatment dose of TGF-beta1, inhibition of ERK phosphorylation increased TGF-beta1-induced expression of the anti-parasite effector gene nitric oxide synthase (NOS), suggesting that increasing levels of ERK activation negatively feed back on induced NOS expression. At infection levels similar to those found in nature, inhibition of ERK activation reduced P. falciparum oocyst loads and infection prevalence in A. stephensi and enhanced TGF-beta1-mediated control of P. falciparum development. Taken together, our data demonstrate that malaria parasite development in the mosquito is regulated by a conserved MAPK signaling pathway that mediates the effects of an ingested cytokine.