Anopheles Gambiae Immune Gene Variants Associated with Natural Plasmodium Infection

Molecular and Biochemical Parasitology. Apr, 2003  |  Pubmed ID: 12706800

Mammalian Transforming Growth Factor Beta1 Activated After Ingestion by Anopheles Stephensi Modulates Mosquito Immunity

Infection and Immunity. Jun, 2003  |  Pubmed ID: 12761076

During the process of bloodfeeding by Anopheles stephensi, mammalian latent transforming growth factor beta1 (TGF-beta1) is ingested and activated rapidly in the mosquito midgut. Activation may involve heme and nitric oxide (NO), agents released in the midgut during blood digestion and catalysis of L-arginine oxidation by A. stephensi NO synthase (AsNOS). Active TGF-beta1 persists in the mosquito midgut to extended times postingestion and is recognized by mosquito cells as a cytokine. In a manner analogous to the regulation of vertebrate inducible NO synthase and malaria parasite (Plasmodium) infection in mammals by TGF-beta1, TGF-beta1 regulates AsNOS expression and Plasmodium development in A. stephensi. Together, these observations indicate that, through conserved immunological cross talk, mammalian and mosquito immune systems interface with each other to influence the cycle of Plasmodium development.

Transforming Growth Factor-betas and Related Gene Products in Mosquito Vectors of Human Malaria Parasites: Signaling Architecture for Immunological Crosstalk

Molecular Immunology. Aug, 2004  |  Pubmed ID: 15302159

The participation of a divergent mosquito transforming growth factor-beta (TGF-beta) and mammalian TGF-beta1 in the Anopheles stephensi response to malaria parasite development [Infect. Genet. Evol. 1 (2001) 131-141; Infect. Immun. 71 (2003) 3000-3009] suggests that a network of Anopheles TGF-beta ligands and signaling pathways figure prominently in immune defense of this important vector group. To provide a basis for identifying the roles of these proteins in Anopheles innate immunity, we identified six predicted TGF-beta ligand-encoding genes in the Anopheles gambiae genome, including two expressed, diverged copies of 60A, the first evidence of ligand gene duplication outside of chordates. In addition to five predicted type I and II receptors, we identified three Smad genes in the A. gambiae genome that would be predicted to support both TGF-beta/Activin- and bone morphogenetic protein (BMP)-like signaling. All three Smad genes are expressed in an immunocompetent A. stephensi cell line and in the A. stephensi midgut epithelium, confirming that a conserved signaling architecture is in place to support signaling by divergent exogenous and endogenous TGF-beta superfamily proteins.

Cross-talk Between Nitric Oxide and Transforming Growth Factor-beta1 in Malaria

Current Molecular Medicine. Nov, 2004  |  Pubmed ID: 15579025

Malaria has re-emerged as a global health problem, leading to an increased focus on the cellular and molecular biology of the mosquito Anopheles and the parasite Plasmodium with the goal of identifying novel points of intervention in the parasite life cycle. Anti-parasite defenses mounted by both mammalian hosts and Anopheles can suppress the growth of Plasmodium. Nonetheless, the parasite is able to escape complete elimination in vivo, perhaps by thwarting or co-opting these mechanisms for its own survival, as do numerous other pathogens. Among the defense systems used by the mammalian host against Plasmodium is the synthesis of nitric oxide (NO), catalyzed by an inducible NO synthase (iNOS). Nitric oxide produced by the action of an inducible Anopheles stephensi NO synthase (AsNOS) may be central to the anti-parasitic arsenal of this mosquito. In mammals, iNOS can be modulated by members of the transforming growth factor-beta (TGF-beta) cytokine superfamily. Transforming growth factor-beta is produced as an inactive precursor that is activated following dissociation of certain inhibitory proteins, a process that can be promoted by reaction products of NO as well as by hemin. Ingestion by Anopheles of blood containing Plasmodium initiates parasite development, blood digestion which results in the accumulation of hematin (hemin) in the insect midgut, and induction of both AsNOS and TGF-beta-like (As60A) gene expression in the midgut epithelium. Active mammalian TGF-beta1 can be detected in the A. stephensi midgut up to 48h post-ingestion and latent TGF-beta1 can be activated by midgut components in vitro, a process that is potentiated by NO and that may involve hematin. Further, mammalian TGF-beta1 is perceived as a cytokine by A. stephensi cells in vitro and can alter Plasmodium development in vivo. Bloodfeeding by Anopheles, therefore, results in a juxtaposition of evolutionarily conserved mosquito and mammalian TGF-beta superfamily homologs that may influence transmission dynamics of Plasmodium in endemic regions.

Induction of Nitric Oxide Synthase in Anopheles Stephensi by Plasmodium Falciparum: Mechanism of Signaling and the Role of Parasite Glycosylphosphatidylinositols

Infection and Immunity. May, 2005  |  Pubmed ID: 15845481

Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h postinfection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that adversely impact parasite development. In mammals, P. falciparum glycosylphosphatidylinositols (PfGPIs) can induce NOS expression in immune and endothelial cells and are sufficient to reproduce the major effects of parasite infection. These effects are mediated in part by mimicry of insulin signaling by PfGPIs. In this study, we demonstrate that PfGPIs can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi Akt/protein kinase B and a pathway involving DSOR1, a mitogen-activated protein kinase kinase, and an extracellular signal-regulated kinase. However, despite the involvement of kinases that are also associated with insulin signaling in A. stephensi cells, signaling by P. falciparum and by PfGPIs is distinctively different from signaling by insulin. Therefore, although mimicry of insulin by PfGPIs appears to be restricted to mammalian hosts of P. falciparum, the conservation of PfGPIs as a prominent parasite-derived signal of innate immunity can now be extended to include Anopheles mosquitoes, indicating that parasite signaling of innate immunity is conserved in mosquito and mammalian cells.

A Mosquito 2-Cys Peroxiredoxin Protects Against Nitrosative and Oxidative Stresses Associated with Malaria Parasite Infection

Free Radical Biology & Medicine. Mar, 2006  |  Pubmed ID: 16540402

Malaria parasite infection in anopheline mosquitoes induces nitrosative and oxidative stresses that limit parasite development, but also damage mosquito tissues in proximity to the response. Based on these observations, we proposed that cellular defenses in the mosquito may be induced to minimize self-damage. Specifically, we hypothesized that peroxiredoxins (Prxs), enzymes known to detoxify reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS), protect mosquito cells. We identified an Anopheles stephensi 2-Cys Prx ortholog of Drosophila melanogaster Prx-4783, which protects fly cells against oxidative stresses. To assess function, AsPrx-4783 was overexpressed in D. melanogaster S2 and in A. stephensi (MSQ43) cells and silenced in MSQ43 cells with RNA interference before treatment with various ROS and RNOS. Our data revealed that AsPrx-4783 and DmPrx-4783 differ in host cell protection and that AsPrx-4783 protects A. stephensi cells against stresses that are relevant to malaria parasite infection in vivo, namely nitric oxide (NO), hydrogen peroxide, nitroxyl, and peroxynitrite. Further, AsPrx-4783 expression is induced in the mosquito midgut by parasite infection at times associated with peak nitrosative and oxidative stresses. Hence, whereas the NO-mediated defense response is toxic to both host and parasite, AsPrx-4783 may shift the balance in favor of the mosquito.

Evidence for Transmission of Plasmodium Vivax Among a Duffy Antigen Negative Population in Western Kenya

The American Journal of Tropical Medicine and Hygiene. Oct, 2006  |  Pubmed ID: 17038676

We present evidence that a parasite with characteristics of Plasmodium vivax is being transmitted among Duffy blood group-negative inhabitants of Kenya. Thirty-two of 4,901 Anopheles gambiae and An. funestus (0.65%) collected in Nyanza Province were ELISA positive for the P. vivax circumsporozoite protein VK 247. All positives were found late in the rainy season, when An. funestus predominated, and disproportionately many were found at a single village. A P. vivax specific sequence of the SSU rRNA gene was amplified from three of six ELISA-positive mosquitoes. Erythrocytes from 31 children, including 9 microscopically diagnosed as infected with P. vivax, were negative by flow cytometry for the Fy3 or Fy6 epitopes, which indicate Duffy blood group expression. A DNA fragment specific for the C terminus of the gene for P. vivax merozoite surface protein 1 (MSP-1) was amplified from the blood of four of these children and subsequently sequenced from two.

Nitric Oxide Metabolites Induced in Anopheles Stephensi Control Malaria Parasite Infection

Free Radical Biology & Medicine. Jan, 2007  |  Pubmed ID: 17157200

Malaria parasite infection in anopheline mosquitoes is limited by inflammatory levels of nitric oxide metabolites. To assess the mechanisms of parasite stasis or toxicity, we investigated the biochemistry of these metabolites within the blood-filled mosquito midgut. Our data indicate that nitrates, but not nitrites, are elevated in the Plasmodium-infected midgut. Although levels of S-nitrosothiols do not change with infection, blood proteins are S-nitrosylated after ingestion by the mosquito. In addition, photolyzable nitric oxide, which can be attributed to metal nitrosyls, is elevated after infection and, based on the abundance of hemoglobin, likely includes heme iron nitrosyl. The persistence of oxyhemoglobin throughout blood digestion and changes in hemoglobin conformation in response to infection suggest that hemoglobin catalyzes the synthesis of nitric oxide metabolites in a reducing environment. Provision of urate, a potent reductant and scavenger of oxidants and nitrating agents, as a dietary supplement to mosquitoes increased parasite infection levels relative to allantoin-fed controls, suggesting that nitrosative and/or oxidative stresses negatively impact developing parasites. Collectively, our results reveal a unique role for nitric oxide in an oxyhemoglobin-rich environment. In contrast to facilitating oxygen delivery by hemoglobin in the mammalian vasculature, nitric oxide synthesis in the blood-filled mosquito midgut drives the formation of toxic metabolites that limit parasite development.

The Insulin Signaling Cascade from Nematodes to Mammals: Insights into Innate Immunity of Anopheles Mosquitoes to Malaria Parasite Infection

Developmental and Comparative Immunology. 2007  |  Pubmed ID: 17161866

As revealed over the past 20 years, the insulin signaling cascade plays a central role in regulating immune and oxidative stress responses that affect the life spans of mammals and two model invertebrates, the nematode Caenorhabitis elegans and the fruit fly Drosophila melanogaster. In mosquitoes, insulin signaling regulates key steps in egg maturation and immunity and likely affects aging, although the latter has yet to be examined in detail. Reproduction, immunity and aging critically influence the capacity of mosquitoes to effectively transmit malaria parasites. Current work has demonstrated that molecules from the invading parasite and the blood meal elicit functional responses in female mosquitoes that are regulated through the insulin signaling pathway or by cross-talk with interacting pathways. Defining the details of these regulatory interactions presents significant challenges for future research, but will increase our understanding of mosquito/malaria parasite transmission and of the conservation of insulin signaling as a key regulatory nexus in animal biology.

Induction of Nitric Oxide Synthase and Activation of Signaling Proteins in Anopheles Mosquitoes by the Malaria Pigment, Hemozoin

Infection and Immunity. Aug, 2007  |  Pubmed ID: 17526741

Anopheles stephensi, a major vector for malaria parasite transmission, responds to Plasmodium infection by synthesis of inflammatory levels of nitric oxide (NO), which can limit parasite development in the midgut. We have previously shown that Plasmodium falciparum glycosylphosphatidylinositols (PfGPIs) can induce A. stephensi NO synthase (AsNOS) expression in the midgut epithelium in vivo in a manner similar to the manner in which cytokines and NO are induced by PfGPIs in mammalian cells. In mosquito cells, signaling by PfGPIs and P. falciparum merozoites is mediated through Akt/protein kinase B (Akt/PKB), the mitogen-activated protein kinase kinase DSOR1, and extracellular signal-regulated kinase (ERK). In mammalian cells, a second parasite factor, malaria pigment or hemozoin (Hz), signals NOS induction through ERK- and nuclear factor kappa B-dependent pathways and has been demonstrated to be a novel proinflammatory ligand for Toll-like receptor 9. In this study, we demonstrate that Hz can also induce AsNOS gene expression in immortalized A. stephensi and Anopheles gambiae cell lines in vitro and in A. stephensi midgut tissue in vivo. In mosquito cells, Hz signaling is mediated through transforming growth factor beta-associated kinase 1, Akt/PKB, ERK, and atypical protein kinase C zeta/lambda. Our results show that Hz is a prominent parasite-derived signal for Anopheles and that signaling pathways activated by PfGPIs and Hz have both unique and shared components. Together with our previous findings, our data indicate that parasite signaling of innate immunity is conserved in mosquito and mammalian cells.

Discrimination of Seven Anopheles Species from San Pedro De Uraba, Antioquia, Colombia, by Polymerase Chain Reaction-restriction Fragment Length Polymorphism Analysis of Its Sequences

The American Journal of Tropical Medicine and Hygiene. Jul, 2007  |  Pubmed ID: 17620632

Accurate identification of anopheline species is essential for vector incrimination and implementation of appropriate control strategies. Several anopheline species are considered important malaria vectors in Colombia; however, species determination is complicated by cryptic morphology and intra-individual variation. We describe polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer 2 (ITS2) sequences for differentiation of seven Anopheles species collected in a locality in Antioquia, Colombia, with high levels of malaria transmission. Each of these seven species can be identified by unique AluI PCR-RFLP restriction patterns. Comparisons of morphologic identification with molecular identification of voucher specimens confirmed species designation for 886 wild-caught anophelines. This new method can be used as a diagnostic tool for discrimination of anopheline species of medical importance in this region, some of which have overlapping morphologic characters and for conducting complementary studies where rapid and accurate identification of large numbers of specimens is needed.

Low Levels of Mammalian TGF-beta1 Are Protective Against Malaria Parasite Infection, a Paradox Clarified in the Mosquito Host

Experimental Parasitology. Feb, 2008  |  Pubmed ID: 17920060

Nitric oxide (NO), derived from catalysis of inducible NO synthase (iNOS), limits malaria parasite growth in mammals. Transforming growth factor (TGF)-beta1 suppresses iNOS in cells in vitro as well as in vivo in mice, but paradoxically severe malaria in humans is associated with low levels of TGF-beta1. We hypothesized that this paradox is a universal feature of infection and occurs in the mosquito Anopheles stephensi, an invertebrate host for Plasmodium that also regulates parasite development with inducible NO synthase (AsNOS). We show that exogenous human TGF-beta1 dose-dependently regulates mosquito AsNOS expression and that parasite killing by low dose TGF-beta1 depends on AsNOS catalysis. Furthermore, induction of AsNOS expression by TGF-beta1 is regulated by NO synthesis. These results suggest that TGF-beta1 plays similar roles during parasite infection in mammals and mosquitoes and that this role is linked to the effects of TGF-beta1 on inducible NO synthesis.

Insulin Regulates Aging and Oxidative Stress in Anopheles Stephensi

The Journal of Experimental Biology. Mar, 2008  |  Pubmed ID: 18281336

Observations from nematodes to mammals indicate that insulin/insulin-like growth factor signaling (IIS) regulates lifespan. As in other organisms, IIS is conserved in mosquitoes and signaling occurs in multiple tissues. During bloodfeeding, mosquitoes ingest human insulin. This simple observation suggested that exogenous insulin could mimic the endogenous hormonal control of aging in mosquitoes, providing a new model to examine this phenomenon at the organismal and cellular levels. To this end, female Anopheles stephensi mosquitoes were maintained on diets containing human insulin provided daily in sucrose or three times weekly by artificial bloodmeal. Regardless of delivery route, mosquitoes provided with insulin at 1.7 x 10(-4) and 1.7 x 10(-3) micromol l(-1), doses 0.3-fold and 3.0-fold higher than non-fasting blood levels, died at a faster rate than controls. In mammals, IIS induces the synthesis of reactive oxygen species and downregulates antioxidants, events that increase oxidative stress and that have been associated with reduced lifespan. Insulin treatment of mosquito cells in vitro induced hydrogen peroxide synthesis while dietary supplementation reduced total superoxide dismutase (SOD) activity and manganese SOD activity relative to controls. The effects of insulin on mortality were reversed when diets were supplemented with manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable SOD mimetic agent, suggesting that insulin-induced mortality was due to oxidative stress. In addition, dietary insulin activated Akt/protein kinase B and extracellular signal-regulated kinase (ERK) in the mosquito midgut, suggesting that, as observed in Caenorhabditis elegans, the midgut may act as a 'signaling center' for mosquito aging.

Natural Infectivity of Anopheles Species from the Pacific and Atlantic Regions of Colombia

Acta Tropica. Aug, 2008  |  Pubmed ID: 18554564

Malaria is an important public health problem in Colombia. Among the major vectors in Colombia, Anopheles albimanus is recognized for its importance on the Pacific Coast where it is the predominant species; it is also found in the Atlantic Coast, although its vectorial role in this region is not clear. We examined the occurrence of An. albimanus in four localities of the Pacific and three of the Atlantic Coast. Morphological identification of problematic specimens was confirmed by a molecular assay. All identified mosquitoes at these sites, including An. albimanus, were also tested for malaria parasite infection. From 12,189 anophelines collected, 6370 were from the Pacific Coast, and corresponded to 99% An. albimanus, 0.8% Anopheles neivai, and three other species at <0.2%. From the Atlantic Coast we identified 5819 specimens with 61% An. albimanus, 36% Anopheles triannulatus s.l. and five other species at <2%. In both coasts, species present at lower percentages included several incriminated as vectors in neighboring countries. Six Pacific Coast specimens were infected with malaria parasites: four An. albimanus, two with Plasmodium vivax VK247, one with P. vivax VK210 and one with Plasmodium falciparum; two An. neivai with P. falciparum. Our data support the continued predominance of An. albimanus in the Pacific Coast, and demonstrate that this species is the most abundant in the Atlantic Coast as well.

Metabolic Pathways in Anopheles Stephensi Mitochondria

The Biochemical Journal. Oct, 2008  |  Pubmed ID: 18588503

No studies have been performed on the mitochondria of malaria vector mosquitoes. This information would be valuable in understanding mosquito aging and detoxification of insecticides, two parameters that have a significant impact on malaria parasite transmission in endemic regions. In the present study, we report the analyses of respiration and oxidative phosphorylation in mitochondria of cultured cells [ASE (Anopheles stephensi Mos. 43) cell line] from A. stephensi, a major vector of malaria in India, South-East Asia and parts of the Middle East. ASE cell mitochondria share many features in common with mammalian muscle mitochondria, despite the fact that these cells are of larval origin. However, two major differences with mammalian mitochondria were apparent. One, the glycerol-phosphate shuttle plays as major a role in NADH oxidation in ASE cell mitochondria as it does in insect muscle mitochondria. In contrast, mammalian white muscle mitochondria depend primarily on lactate dehydrogenase, whereas red muscle mitochondria depend on the malate-oxaloacetate shuttle. Two, ASE mitochondria were able to oxidize proline at a rate comparable with that of alpha-glycerophosphate. However, the proline pathway appeared to differ from the currently accepted pathway, in that oxoglutarate could be catabolized completely by the tricarboxylic acid cycle or via transamination, depending on the ATP need.

Plasmodium Development in the Mosquito: Biology Bottlenecks and Opportunities for Mathematical Modeling

Trends in Parasitology. Aug, 2008  |  Pubmed ID: 18603475

Quantitative analyses of malaria parasite development are necessary to assess the efficacy of control measures. Such analyses in the mammalian host have been difficult to implement, lagging behind the use of antiparasitic drugs, vaccine development and transmission-blocking strategies. Even less is known about the genetic, environmental and other factors that impact sporogony in the mosquito host. Here, we summarize current knowledge and review a first attempt to model sporogonic development quantitatively.

MAPK ERK Signaling Regulates the TGF-beta1-dependent Mosquito Response to Plasmodium Falciparum

PLoS Pathogens. Apr, 2009  |  Pubmed ID: 19343212

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.

Population Structure Analyses and Demographic History of the Malaria Vector Anopheles Albimanus from the Caribbean and the Pacific Regions of Colombia

Malaria Journal. 2009  |  Pubmed ID: 19922672

Anopheles albimanus is an important malaria vector in some areas throughout its distribution in the Caribbean and the Pacific regions of Colombia, covering three biogeographic zones of the neotropical region, Maracaibo, Magdalena and Chocó.

Species Composition and Natural Infectivity of Anthropophilic Anopheles (Diptera: Culicidae) in the States of Córdoba and Antioquia, Northwestern Colombia

Memórias Do Instituto Oswaldo Cruz. Dec, 2009  |  Pubmed ID: 20140372

Malaria is a serious health problem in the states of Córdoba and Antioquia, Northwestern Colombia, where 64.4% of total Colombian cases were reported in 2007. Because little entomological information is available in this region, the aim of this work was to identify the Anopheles species composition and natural infectivity of mosquitoes distributed in seven localities with highest malaria transmission. A total of 1,768 Anopheles mosquitoes were collected using human landing catches from March 2007-July 2008. Ten species were identified; overall, Anopheles nuneztovari s.l. was the most widespread (62%) and showed the highest average human biting rates. There were six other species of the Nyssorhynchus subgenus: Anopheles albimanus (11.6%), Anopheles darlingi (9.8%), Anopheles braziliensis (6.6%), Anopheles triannulatus s.l. (3.5%), Anopheles albitarsis s.l. and Anopheles oswaldoi s.l. at < 1%; and three of the Anopheles subgenus: Anopheles punctimacula, Anopheles pseudopunctipennis s.l. and Anopheles neomaculipalpusat < 1% each. Two species from Córdoba, An. nuneztovari and An. darlingi, were found to be naturally infected by Plasmodium vivax VK247, as determined by ELISA and confirmed by nested PCR. All species were active indoors and outdoors. These results provide basic information for targeted vector control strategies in these localities.

Translational Systems Approaches to the Biology of Inflammation and Healing

Immunopharmacology and Immunotoxicology. Jun, 2010  |  Pubmed ID: 20170421

Inflammation is a complex, non-linear process central to many of the diseases that affect both developed and emerging nations. A systems-based understanding of inflammation, coupled to translational applications, is therefore necessary for efficient development of drugs and devices, for streamlining analyses at the level of populations, and for the implementation of personalized medicine. We have carried out an iterative and ongoing program of literature analysis, generation of prospective data, data analysis, and computational modeling in various experimental and clinical inflammatory disease settings. These simulations have been used to gain basic insights into the inflammatory response under baseline, gene-knockout, and drug-treated experimental animals for in silico studies associated with the clinical settings of sepsis, trauma, acute liver failure, and wound healing to create patient-specific simulations in polytrauma, traumatic brain injury, and vocal fold inflammation; and to gain insight into host-pathogen interactions in malaria, necrotizing enterocolitis, and sepsis. These simulations have converged with other systems biology approaches (e.g., functional genomics) to aid in the design of new drugs or devices geared towards modulating inflammation. Since they include both circulating and tissue-level inflammatory mediators, these simulations transcend typical cytokine networks by associating inflammatory processes with tissue/organ impacts via tissue damage/dysfunction. This framework has now allowed us to suggest how to modulate acute inflammation in a rational, individually optimized fashion. This plethora of computational and intertwined experimental/engineering approaches is the cornerstone of Translational Systems Biology approaches for inflammatory diseases.

Reframing Critical Needs in Vector Biology and Management of Vector-borne Disease

PLoS Neglected Tropical Diseases. 2010  |  Pubmed ID: 20186276

Analysis of Wolbachia Strains Associated with Conotrachelus Nenuphar (Coleoptera: Curculionidae) in the Eastern United States

Environmental Entomology. Apr, 2010  |  Pubmed ID: 20388268

We studied the distribution patterns of Wolbachia infection associated with plum curculio strains in eight states of the eastern United States. The presence of the Wolbachia-specific gene wsp identified infections of this endosymbiont in 97.8% of the 93 samples tested. Three distinct Wolbachia strains were identified. The strains wCne1 (593 bp) and wCne2 (593 bp) were 97% identical, and their sequences were both 84% identical with wCne3 (590 bp). BLASTN searches through GenBank showed strong similarities between the wsp sequences of the three strains compared with Wolbachia sequenced from other hosts. Degree of similarity with sequences in other Wolbachia strains is discussed. Polymerase chain reaction-restriction fragment length polymorphism was used for superinfection detection. Of 93 samples, 15 (16.1%), 21 (22.6%), 19 (20.4%), and 36 (38.7%) samples were infected by wCne1, wCne2, wCne1 + 2, and wCne3, respectively. Only two (2.2%) samples had no infection. The wCne3 strain was always present as a single infection. Wolbachia strains approximate the distribution of plum curculio strains: northern strain infected with wCne1 and wCne2 strains in supergroup B, and southern strain infected with wCne3 strain in supergroup A, with the mid-Atlantic region as the convergence area. Based on haplotype distribution of plum curculio mitochondrial cytochrome oxidase I, there was a closer relation of the mid-southern plum curculio clade to the far-southern clade than to the northern clade. However, Wolbachia symbionts in mid-southern plum curculio are more closely related to those in northern plum curculio than to those in far-southern plum curculio. The relationship of Wolbachia infection with reproductive incompatibility between plum curculio populations was also discussed.

Identification of Three Single Nucleotide Polymorphisms in Anopheles Gambiae Immune Signaling Genes That Are Associated with Natural Plasmodium Falciparum Infection

Malaria Journal. 2010  |  Pubmed ID: 20540770

Laboratory studies have demonstrated that a variety of immune signaling pathways regulate malaria parasite infection in Anopheles gambiae, the primary vector species in Africa.

Microgeographic Genetic Variation of the Malaria Vector Anopheles Darlingi Root (Diptera: Culicidae) from Cordoba and Antioquia, Colombia

The American Journal of Tropical Medicine and Hygiene. Jul, 2010  |  Pubmed ID: 20595475

Anopheles darlingi is an important vector of Plasmodium spp. in several malaria-endemic regions of Colombia. This study was conducted to test genetic variation of An. darlingi at a microgeographic scale (approximately 100 km) from localities in Córdoba and Antioquia states, in western Colombia, to better understand the potential contribution of population genetics to local malaria control programs. Microsatellite loci: nuclear white and cytochrome oxidase subunit I (COI) gene sequences were analyzed. The northern white gene lineage was exclusively distributed in Córdoba and Antioquia and shared COI haplotypes were highly represented in mosquitoes from both states. COI analyses showed these An. darlingi are genetically closer to Central American populations than southern South American populations. Overall microsatellites and COI analysis showed low to moderate genetic differentiation among populations in northwestern Colombia. Given the existence of high gene flow between An. darlingi populations of Córdoba and Antioquia, integrated vector control strategies could be developed in this region of Colombia.

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles Stephensi Mosquitoes

PLoS Pathogens. 2010  |  Pubmed ID: 20664791

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60-99%. Of those mosquitoes that were infected, we observed a 75-99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18-20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.

Correction: Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles Stephensi Mosquitoes

PLoS Pathogens. 2010  |  Pubmed ID: 20714345

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60-99%. Of those mosquitoes that were infected, we observed a 75-99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18-20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.

A New MtDNA COI Gene Lineage Closely Related to Anopheles Janconnae of the Albitarsis Complex in the Caribbean Region of Colombia

Memórias Do Instituto Oswaldo Cruz. Dec, 2010  |  Pubmed ID: 21225199

An understanding of the taxonomic status and vector distribution of anophelines is crucial in controlling malaria. Previous phylogenetic analyses have supported the description of six species of the Neotropical malaria vector Anopheles (Nyssorhynchus) albitarsis s.l. (Diptera: Culicidae): An. albitarsis, Anopheles deaneorum, Anopheles marajoara, Anopheles oryzalimnetes, Anopheles janconnae and An. albitarsis F. To evaluate the taxonomic status of An. albitarsis s.l. mosquitoes collected in various localities in the Colombian Caribbean region, specimens were analyzed using the complete mitochondrial DNA cytochrome oxidase I (COI) gene, the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2) region and partial nuclear DNA white gene sequences. Phylogenetic analyses of the COI gene sequences detected a new lineage closely related to An. janconnae in the Caribbean region of Colombia and determined its position relative to the other members of the complex. However, the ITS2 and white gene sequences lacked sufficient resolution to support a new lineage closely related to An. janconnae or the An. janconnae clade. The possible involvement of this new lineage in malaria transmission in Colombia remains unknown, but its phylogenetic closeness to An. janconnae, which has been implicated in local malaria transmission in Brazil, is intriguing.

Both Hemolytic Anemia and Malaria Parasite-specific Factors Increase Susceptibility to Nontyphoidal Salmonella Enterica Serovar Typhimurium Infection in Mice

Infection and Immunity. Apr, 2010  |  Pubmed ID: 20100860

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.

Evaluation of a PCR-RFLP-ITS2 Assay for Discrimination of Anopheles Species in Northern and Western Colombia

Acta Tropica. May, 2011  |  Pubmed ID: 21345325

Anopheles mosquitoes are routinely identified using morphological characters of the female that often lead to misidentification due to interspecies similarity and intraspecies variability. The aim of this work was to evaluate the applicability of a previously developed PCR-RFLP-ITS2 assay for accurate discrimination of anophelines in twelve localities spanning three Colombian malaria epidemiological regions: Atlantic Coast, Pacific Coast, and Uraba-Bajo Cauca-Alto Sinu region. The evaluation of the stability of the PCR-RFLP patterns is required since variability of the ITS2 has been documented and may produce discrepancies in the patterns previously reported. The assay was used to evaluate species assignation of 939 mosquitoes identified by morphology. Strong agreement between the morphological and molecular identification was found for species Anopheles albimanus, Anopheles aquasalis, Anopheles darlingi and Anopheles triannulatus s.l. (p≥0.05, kappa=1). However, disagreement was found for species Anopheles nuneztovari s.l., Anopheles neomaculipalpus, Anopheles apicimacula and Anopheles punctimacula (p≤0.05; kappa ranging from 0.33 to 0.80). The ITS2-PCR-RFLP assay proved valuable for discriminating anopheline species of northern and western Colombia, especially those with overlapping morphology in the Oswaldoi Group.

Holographic Pixel Super-resolution in Portable Lensless On-chip Microscopy Using a Fiber-optic Array

Lab on a Chip. Apr, 2011  |  Pubmed ID: 21365087

We report a portable lensless on-chip microscope that can achieve <1 µm resolution over a wide field-of-view of ∼ 24 mm(2) without the use of any mechanical scanning. This compact on-chip microscope weighs ∼ 95 g and is based on partially coherent digital in-line holography. Multiple fiber-optic waveguides are butt-coupled to light emitting diodes, which are controlled by a low-cost micro-controller to sequentially illuminate the sample. The resulting lensfree holograms are then captured by a digital sensor-array and are rapidly processed using a pixel super-resolution algorithm to generate much higher resolution holographic images (both phase and amplitude) of the objects. This wide-field and high-resolution on-chip microscope, being compact and light-weight, would be important for global health problems such as diagnosis of infectious diseases in remote locations. Toward this end, we validate the performance of this field-portable microscope by imaging human malaria parasites (Plasmodium falciparum) in thin blood smears. Our results constitute the first-time that a lensfree on-chip microscope has successfully imaged malaria parasites.

A New Role for an Old Antimicrobial: Lysozyme C-1 Can Function to Protect Malaria Parasites in Anopheles Mosquitoes

PloS One. 2011  |  Pubmed ID: 21573077

Plasmodium requires an obligatory life stage in its mosquito host. The parasites encounter a number of insults while journeying through this host and have developed mechanisms to avoid host defenses. Lysozymes are a family of important antimicrobial immune effectors produced by mosquitoes in response to microbial challenge.

Insulin-like Peptides in the Mosquito Anopheles Stephensi: Identification and Expression in Response to Diet and Infection with Plasmodium Falciparum

General and Comparative Endocrinology. Sep, 2011  |  Pubmed ID: 21703270

Insulin-like peptides (ILPs) regulate a multitude of biological processes, including metabolism and immunity to infection, and share similar structural motifs across widely divergent taxa. Insulin/insulin-like growth factor signaling (IIS) pathway elements are similarly conserved. We have shown that IIS regulates reproduction, innate immunity, and lifespan in female Anopheles stephensi, a major mosquito vector of human malaria. To further explore IIS regulation of these processes, we identified genes encoding five ILPs in this species and characterized their expression in tissues. Antisera to ILP homologs in Anopheles gambiae were used to identify cellular sources in An. stephensi females by immunocytochemistry. We analyzed tissue-specific ILP transcript expression in young and older females, in response to different feeding regimens, and in response to infection with Plasmodiumfalciparum with quantitative reverse transcriptase-PCR assays. While some ILP transcript changes were evident in older females and in response to blood feeding, significant changes were particularly notable in response to hormonal concentrations of ingested human insulin and to P. falciparum infection. These changes suggest that ILP secretion and action may be similarly responsive in Plasmodium-infected females and potentially alter metabolism and innate immunity.

Evidence for an Increased Risk of Transmission of Simian Immunodeficiency Virus and Malaria in a Rhesus Macaque Coinfection Model

Journal of Virology. Nov, 2011  |  Pubmed ID: 21917966

In sub-Saharan Africa, HIV-1 infection frequently occurs in the context of other coinfecting pathogens, most importantly, Mycobacterium tuberculosis and malaria parasites. The consequences are often devastating, resulting in enhanced morbidity and mortality. Due to the large number of confounding factors influencing pathogenesis in coinfected people, we sought to develop a nonhuman primate model of simian immunodeficiency virus (SIV)-malaria coinfection. In sub-Saharan Africa, Plasmodium falciparum is the most common malaria parasite and is responsible for most malaria-induced deaths. The simian malaria parasite Plasmodium fragile can induce clinical symptoms, including cerebral malaria in rhesus macaques, that resemble those of P. falciparum infection in humans. Thus, based on the well-characterized rhesus macaque model of SIV infection, this study reports the development of a novel rhesus macaque SIV-P. fragile coinfection model to study human HIV-P. falciparum coinfection. Using this model, we show that coinfection is associated with an increased, although transient, risk of both HIV and malaria transmission. Specifically, SIV-P. fragile coinfected macaques experienced an increase in SIV viremia that was temporarily associated with an increase in potential SIV target cells and systemic immune activation during acute parasitemia. Conversely, primary parasitemia in SIV-P. fragile coinfected animals resulted in higher gametocytemia that subsequently translated into higher oocyst development in mosquitoes. To our knowledge, this is the first animal model able to recapitulate the increased transmission risk of both HIV and malaria in coinfected humans. Therefore, this model could serve as an essential tool to elucidate distinct immunological, virological, and/or parasitological parameters underlying disease exacerbation in HIV-malaria coinfected people.

Mitochondrial NAD+-dependent Malic Enzyme from Anopheles Stephensi: a Possible Novel Target for Malaria Mosquito Control

Malaria Journal. 2011  |  Pubmed ID: 22029897

Anopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized A. stephensi (ASE) cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development.

The Mitogen-activated Protein Kinome from Anopheles Gambiae: Identification, Phylogeny and Functional Characterization of the ERK, JNK and P38 MAP Kinases

BMC Genomics. 2011  |  Pubmed ID: 22111877

Anopheles gambiae is the primary mosquito vector of human malaria parasites in sub-Saharan Africa. To date, three innate immune signaling pathways, including the nuclear factor (NF)-kappaB-dependent Toll and immune deficient (IMD) pathways and the Janus kinase/signal transducers and activators of transcription (Jak-STAT) pathway, have been extensively characterized in An. gambiae. However, in addition to NF-kappaB-dependent signaling, three mitogen-activated protein kinase (MAPK) pathways regulated by JNK, ERK and p38 MAPK are critical mediators of innate immunity in other invertebrates and in mammals. Our understanding of the roles of the MAPK signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted.

Molecular Evidence for a Single Taxon, Anopheles Nuneztovari S.l., from Two Endemic Malaria Regions in Colombia

Memórias Do Instituto Oswaldo Cruz. Dec, 2011  |  Pubmed ID: 22241127

To elucidate the Anopheles nuneztovari s.l. taxonomic status at a microgeographic level in four malaria endemic localities from Antioquia and Córdoba, Colombia, fragments of the cytochrome oxidase subunit I (COI) and the white gene were used. The COI analysis showed low genetic differentiation with fixation index (F(ST)) levels between -0.02-0.137 and Nm values between 3-∞, indicating the presence of high gene flow among An. nuneztovari s.l. populations from the four localities. The COI network showed a single most common haplotype, type 1 (n = 55), present in all localities, as the likely ancestral haplotype. Analysis of the white gene showed that An. nuneztovari s.l. populations from both departments grouped with haplotypes 19 and 20, which are part of lineage 3 reported previously. The results of the present study suggest that An. nuneztovari s.l. is a single taxon in the area of the present study.

Portable and Cost-effective Pixel Super-resolution On-chip Microscope for Telemedicine Applications

Conference Proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference. Aug, 2011  |  Pubmed ID: 22256247

We report a field-portable lensless on-chip microscope with a lateral resolution of <1μm and a large field-of-view of ∼24mm(2). This microscope is based on digital in-line holography and a pixel super-resolution algorithm to process multiple lensfree holograms and obtain a single high-resolution hologram. In its compact and cost-effective design, we utilize 23 light emitting diodes butt-coupled to 23 multi-mode optical fibers, and a simple optical filter, with no moving parts. Weighing only ∼95 grams, we demonstrate the performance of this field-portable microscope by imaging various objects including human malaria parasites in thin blood smears.

Reactive Oxygen Species-dependent Cell Signaling Regulates the Mosquito Immune Response to Plasmodium Falciparum

Antioxidants & Redox Signaling. Mar, 2011  |  Pubmed ID: 21126166

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.