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Extracellular matrices in diverse biological systems are cross-linked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that a peroxidase, secreted by the Anopheles gambiae midgut, and dual oxidase form a dityrosine network that decreases gut permeability to immune elicitors. This network protects the microbiota by preventing activation of epithelial. immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses. 10.1126/science.1184008

We performed a forward genetic screen, using Drosophila as a surrogate mosquito, to identify host factors required for the growth of the avian malaria parasite, Plasmodium gallinaceum. We identified 18 presumed loss-of-function mutants that reduced the growth of the parasite in flies. Presumptive mutation sites were identified in 14 of the mutants on the basis of the insertion site of a transposable element. None of the identified genes have been previously implicated in innate immune responses or interactions with Plasmodium. The functions of five Anopheles gambiae homologs were tested by using RNAi to knock down gene function followed by measuring the growth of the rodent parasite, Plasmodium berghei. Loss of function of four of these genes in the mosquito affected Plasmodium growth, suggesting that Drosophila can be used effectively as a surrogate mosquito to identify relevant host factors in the mosquito.

The mosquito Anopheles gambiae is a primary vector of Plasmodium parasites in Africa. The effect of aging on reproductive output in A. gambiae females from three strains that differ in their ability to melanize Plasmodium and in their systemic levels of hydrogen peroxide ([H.sub.2][O.sub.2]), a reactive oxygen species (ROS), was analyzed. The number of eggs oviposited after the first blood meal decreases with age in all strains; however, this decline was much more pronounced in the G3 (unselected) and R (refractory to Plasmodium infection) strains than in the S (highly susceptible to Plasmodium) strain. Reduction of ROS levels in G3 and R females by administration of antioxidants reversed this age-related decline in fecundity. The S and G3 strains were fixed for two functionally different catalase alleles that differ at the second amino acid position (Ser2Trp). Biochemical analysis of recombinant proteins revealed that the Trp isoform has lower specific activity and higher [K.sub.m] than the Ser isoform, indicating that the former is a less efficient enzyme. The Trp-for-Ser substitution appears to destabilize the functional tetrameric form of the enzyme. Both alleles are present in the R strain, and Ser/Ser females had significantly higher fecundity than Trp/Trp females. Finally, a systemic reduction in catalase activity by dsRNA-mediated knockdown significantly reduced the reproductive output of mosquito females, indicating that catalase plays a central role in protecting the oocyte and early embryo from ROS damage. insect | malaria | Plasmodium | reproduction | single-nucleotide polymorphism

In vivo responses of midgut epithelial cells to ookinete invasion of three different vector-parasite combinations, Aedes aegypti-Plasmodium gallinaceum, Anopheles stephensi-Plasmodium berghei, and A. stephensi-P. gallinaceum, were directly compared by using enzymatic markers and immunofluorescence stainings. Our studies indicate that, in A. aegypti and A. stephensi ookinetes traverse the midgut via an intracellular route and inflict irreversible damage to the invaded cells. These two mosquito species differ, however, in their mechanisms of epithelial repair. A. stephensi detaches damaged cells by an actin-mediated budding-off mechanism when invaded by either P. berghei or P. gallinaceum. In A. aegypti, the midgut epithelium is repaired by a unique actin cone zipper mechanism that involves the formation of a cone-shaped actin aggregate at the base of the cell that closes sequentially, expelling the cellular contents into the midgut lumen as it brings together healthy neighboring cells. Invasion of A. stephensi by P. berghei induced expression of nitric oxide synthase and peroxidase activities, which mediate tyrosine nitration. These enzymes and nitrotyrosine, however, were not induced in the other two vector-parasite combinations examined. These studies indicate that the epithelial responses of different mosquito-parasite combinations are not universal. The implications of these observations to validate animal experimental systems that reflect the biology of natural vectors of human malarias are discussed. Anopheles stephensi | malaria | ookinete | invasion | Plasmodium berghei

The exploitation of insect galectins, which are associated with embryonic developments by parasites for survival in their insect hosts, is shown. PpGalec, a tandem repeat galectin expressed in the midgut of the sand fly Phlebotomus papatasi, is used by Leishmania major as a receptor for mediating specific binding to the insect midgut, an event crucial for parasite survival and accounts for species-specific vector competence.

Malaria transmission depends on the competence of some Anopheles mosquitoes to sustain Plasmodium development (susceptibility). A genetically selected refractory strain of Anopheles gambiae blocks Plasrnodium development, melanizing, and encapsulating the parasite in a reaction that begins with tyrosine oxidation, and involves three quantitative trait loci. Morphological and microarray mRNA expression analysis suggest that the refractory and susceptible strains have broad physiological differences, which ate related to the production and detoxification of reactive oxygen species. Physiological studies corroborate that the refractory strain is in a chronic state of oxidative stress, which is exacerbated by blood feeding, resulting in increased steady-state levels of reactive oxygen species, which favor melanization of parasites as well as Sephadex beads.

Comparison of the genomes and proteomes of the two diptera Anopheles gambiae and Drosophila melanogaster, which diverged about 250 million years ago, reveals considerable similarities. However, numerous differences are also observed; some of these must reflect the selection and subsequent adaptation associated with different ecologies and life strategies. Almost haft of the genes in both genomes are interpreted as orthologs and show an average sequence identity of about 56%, which is slightly lower than that observed between the orthologs of the pufferfish and human (diverged about 450 million years ago). This indicates that these two insects diverged considerably faster than vertebrates. Aligned sequences reveal that orthologous genes have retained only haft of their intron/exon structure, indicating that intron gains or losses have occurred at a rate of about one per gene per 125 million years. Chromosomal arms exhibit significant remnants of homology between the two species, although only 34% of the genes colocalize in small 'microsyntenic' dusters, and major interarm transfers as well as intra-arm shuffling of gene order are detected., The fruit fly Drosophila melanogaster (in the following, Drosophila) and the malaria mosquito Anopheles gambiae (in the following, Anopheles) are both highly adapted, successful dipteran species that diverged about 250 [...]

We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster. Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire., Malaria transmission requires survival and development of the Plasmodium parasite in two invaded organisms: the human host and the mosquito vector. Interactions between the immune system of either organism with [...]

The complex gene expression responses of Anopheles gambiae to microbial and malaria challenges, injury, and oxidative stress (in the mosquito and/or a cultured cell line) were surveyed by using cDNA microarrays constructed from an EST-clone collection. The expression profiles were broadly subdivided into induced and down-regulated gene clusters. [Gram.sup.+] and [Gram.sup.-] bacteria and microbial elicitors up-regulated a diverse set of genes, many belonging to the immunity class, and the response to malaria partially overlapped with this response. Oxidative stress activated a distinctive set of genes, mainly implicated in oxidoreductive processes. Injury up- and down-regulated gene clusters also were distinctive, prominently implicating glycolysis-related genes and citric acid cycle/oxidative phosphorylation/redox-mitochondrial functions, respectively. Cross-comparison of in vivo and in vitro responses indicated the existence of tightly coregulated gene groups that may correspond to gene pathways.

Two classes of genomic clones coding for genes expressed after a blood meal in the mosquito, Aedes aegypti, were isolated. One of the clones hybridized to a 6.5 kb message that was shown to code for vitellogenin. The other clone, the 15a gene, hybridized to a 0.65 kb message, which was examined in the study. It was shown that the transcription of the 15a gene occurs in the follicle cells of the ovary under the control of 20-hydroxyecdysone. Moreover, analysis revealed that the two genomic clones encode related proteins that are similar to the vitelline membrane proteins of Drosophila melanogaster.

Mosquito midgut invasion by ookinetes of the malaria parasite Plasmodium disrupts the barriers that normally prevent the gut microbiota from coming in direct contact with epithelial cells. This triggers a long-lived response characterized by increased abundance of granulocytes, a subpopulation of hemocytes that circulates in the insect's hemocoel, and enhanced immunity to bacteria that indirectly reduces survival of Plasmodium parasites upon reinfection. In mosquitoes, differentiation of hemocytes was necessary and sufficient to confer innate immune memory. 10.1126/science.1190689

A study was conducted on rat outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) using gene synthesis and bacterial expression with Escherichia coli and nuclear magnetic resonance spectroscopy. Results showed that the rhombic perturbation of the cytochrome's ferric center is similar to that of microsomal beef, rabbit, chicken and rat cytochromes b5. Further observations also determined the OM cytochrome b5 heme orientation ratio to be 1.0 plus or minus 0.1.