Identification and characterization of a new putative c-type lysozyme from malaria vector Anopheles stephensi

Lysozyme (E.C. 3.2.1.17) activity is reported from the malaria vector Anopheles stephensi. The activity was detected in the salivary gland and midgut using bacteriolytic radial diffusion assay. Spectrophotometric analysis indicated that higher level of lysozyme activity was maintained in both midgut and salivary gland tissues. The activity reached the highest level in 4-8 days old mosquitoes. Genomic PCR amplification revealed the presence of at least two putative lysozyme genes in the mosquito genome. Preliminary analysis of one of the 413 bp genomic fragments showed 56% identity to the lysozyme of mosquito A. gambiae. However, the nature and origin of the putative cloned lysozyme gene remains elusive.     

Molecular evidence for a kdr-like pyrethroid resistance mechanism in the malaria vector mosquito Anopheles stephensi

The mosquito Anopheles stephensi Liston (Diptera: Culicidae) is the urban vector of malaria in several countries of the Middle East and Indian subcontinent. Extensive use of residual insecticide spraying for malaria vector control has selected An. stephensi resistance to DDT, dieldrin, malathion and other organophosphates throughout much of its range and to pyrethroids in the Middle East. Metabolic resistance mechanisms and insensitivity to pyrethroids, so-called knockdown resistance (kdr), have previously been reported in An. stephensi. Here we provide molecular data supporting the hypothesis that a kdr-like pyrethroid-resistance mechanism is present in An. stephensi. We found that larvae of a pyrethroid-selected strain from Dubai (DUB-R) were 182-fold resistant to permethin, compared with a standard susceptible strain of An. stephensi. Activities of some enzymes likely to confer pyrethroid-resistance (i.e. esterases, monooxygenases and glutathione S-transferases) were significantly higher in the permethrin-resistant than in the susceptible strain, but the use of synergists--piperonyl butoxide (PBO) to inhibit monooxygenases and/or tribufos (DEF) to inhibit esterases--did not fully prevent resistance in larvae (permethrin LC50 reduced by only 51-68%), indicating the involvement of another mechanism. From both strains of An. stephensi, we obtained a 237-bp fragment of genomic DNA encoding segment 6 of domain II of the para type voltage-gated sodium channel, i.e. the putative kdr locus. By sequencing this 237 bp fragment, we identified one point mutation difference involving a single A-T base change encoding a leucine to phenylalanine amino acid substitution in the pyrethroid-resistant strain. This mutation appears to be homologous with those detected in An. gambiae and other insects with kdr-like resistance. A diagnostic polymerase chain reaction assay using nested primers was therefore designed to detect this mechanism in An. stephensi.     

Midgut specific immune response of vector mosquito Anopheles stephensi to malaria parasite Plasmodium

Innate immune-related polypeptides expression in midgut in the ageing vector mosquito A. stephensi following infection by malaria parasite, Plasmodium yoelii yoelii has been studied. Twenty polypeptides were induced by an infected blood meal during various stages of adult life. A 24 kDa polypeptide was induced generally in most of the stages. Maximum parasite induced polypeptides i.e. 22, 33, 111, 122, 127, 140, 143 and 146 kDa were found in 5 days of post blood feeding (PBF) which coincides with the presence of oocysts on the midgut. However, in addition, three polypeptides in 11 days PBF and 8 polypeptides in 20 days PBF were also induced due to parasite infection in aged mosquitoes. Quantitatively, the amount of soluble proteins in the midgut in oocyst-sporozoite-positive mosquitoes was always less as compared to their normal counterparts. The parasite evidently elicits defined immune responses by inducing specific polypeptides in the midgut of the mosquito.     

Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans

Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.     

Post-integration behavior of a Minos transposon in the malaria mosquito Anopheles stephensi

Transposable elements represent important tools to perform functional studies in insects. In Drosophila melanogaster, the remobilization properties of transposable elements have been utilized for enhancer-trapping and insertional mutagenesis experiments, which have considerably helped in the functional characterization of the fruitfly genome. In Anopheles mosquitoes, the sole vectors of human malaria, as well as in other mosquito vectors of disease, the use of transposons has also been advocated to achieve the spread of anti-parasitic genes throughout field populations. Here we report on the post-integration behavior of the Minos transposon in both the germ-line and somatic tissues of Anopheles mosquitoes. Transgenic An. stephensi lines developed using the piggyBac transposon and expressing the Minos transposase were tested for their ability to remobilize an X-linked Minos element. Germ-line remobilization events were not detected, while somatic excisions and transpositions were consistently recovered. The analysis of these events showed that Minos activity in Anopheles cells is characterized by unconventional functionality of the transposon. In the two cases analyzed, re-integration of the transposon occurred onto the same X chromosome, suggesting a tendency for local hopping of Minos in the mosquito genome. This is the first report of the post-integration behavior of a transposable element in a human malaria vector. Christina Scali and Tony Nolan contributed equally to the work.     

Exploring the salivary gland transcriptome and proteome of the Anopheles stephensi mosquito

Anopheles stephensi is the main urban mosquito vector of malaria in the Indian subcontinent, and belongs to the same subgenus as Anopheles gambiae, the main malaria vector in Africa. Recently the genome and proteome sets of An. gambiae have been described, as well as several protein sequences expressed in its salivary glands, some of which had their expression confirmed by amino terminal sequencing. In this paper, we randomly sequenced a full-length cDNA library of An. stephensi and performed Edman degradation of polyvinylidene difluoride (PVDF)-transferred protein bands from salivary homogenates. Twelve of 13 proteins found by aminoterminal degradation were found among the cDNA clusters of the library. Thirty-three full-length novel cDNA sequences are reported, including a novel secreted galectin; the homologue of anophelin, a thrombin inhibitor; a novel trypsin/chymotrypsin inhibitor; an apyrase; a lipase; and several new members of the D7 protein family. Most of the novel proteins have no known function. Comparison of the putatively secreted and putatively housekeeping proteins of An. stephensi with An. gambiae proteins indicated that the salivary gland proteins are at a faster evolutionary pace. The possible role of these proteins in blood and sugar feeding by the mosquito is discussed. The electronic tables and supplemental material are available at http://www.ncbi.nlm.nih.gov/projects/Mosquito/A_stephensi_sialome/ .     

DNA-binding proteins of the malaria vector Anopheles stephensi: purification and characterization of an endonuclease

DNA-binding proteins present in fourth instar larvae of Anopheles stephensi were isolated by affinity chromatography on native and denatured DNA cellulose columns and analyzed by electrophoresis on polyacrylamide gels. A denatured DNA-specific protein with an approximate molecular weight of 30 kDa was the predominant DNA binding protein of larvae. This protein was purified to electrophoretic homogeneity by ammonium sulfate fractionation followed by phosphocellulose chromatography. The purified 30 kDa binding protein showed an endonucleolytic activity capable of converting pBR 322 supercoiled DNA to the circular form. Maximum endonucleolytic activity was observed in the presence of 5 mM Mg(2+) at pH 7.4. Enzyme activity was completely inhibited by EDTA.     

Towards a species-selective acetylcholinesterase inhibitor to control the mosquito vector of malaria, Anopheles gambiae

Anopheles gambiae is the major mosquito vector of malaria in sub-Saharan Africa. At present, insecticide-treated nets (ITNs) impregnated with pyrethroid insecticides are widely used in malaria-endemic regions to reduce infection; however the emergence of pyrethroid-resistant mosquitoes has significantly reduced the effectiveness of the pyrethroid ITNs. An acetylcholinesterase (AChE) inhibitor that is potent for An. gambiae but weakly potent for the human enzyme could potentially be safely deployed on a new class of ITNs. In this paper we provide a preliminary pharmacological characterization of An. gambiae AChE, discuss structural features of An. gambiae and human AChE that could lead to selective inhibition, and describe compounds with 130-fold selectivity for inhibition of An. gambiae AChE relative to human AChE.     

Stable and heritable gene silencing in the malaria vector Anopheles stephensi

Heritable RNA interference (RNAi), triggered from stably expressed transgenes with an inverted repeat (IR) configuration, is an important tool for reverse genetic studies. Here we report on the development of stable RNAi in Anopheles stephensi mosquitoes, the major vector of human malaria in Asia. Trans genic mosquitoes stably expressing a RNAi transgene, designed to produce intron-spliced double-stranded RNA (dsRNA) targeting the green fluorescent protein EGFP gene, were crossed to an EGFP-expressing target line. EGFP expression was dramatically reduced at both the protein and RNA levels. The levels of gene silencing depended upon the RNAi gene copy number and its site of integration. These results demonstrate that specific RNAi-mediated knockdown of gene function can be achieved with high efficiency in Anopheles. This will be invaluable to systematically unravel the function of Anopheles genes determining the vectorial capacity of the malaria parasite.     

Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi.

Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.     

Detection and characterization of a mannan-binding lectin from the mosquito, Anopheles stephensi (Liston).

Two lectins from the serum of the mosquito, Anopheles stephensi (Liston), with distinct characteristics, were detected by agglutination of various animal erythrocytes. The lectins were developmental stage-specific and/or sex-related. One adult female-specific lectin was identified as mannan-specific, and named mosquito mannan-binding lectin (MBL). MBL cross-reacted immunologically with antibodies against a previously characterized cockroach lectin, Blaberus discoidalis lectin (BDL1), and its activity was almost completely blocked by the antibodies. Mosquito MBL agglutinated erythrocytes from human, sheep, goat and rabbit, but not chicken or mouse, and agglutination was inhibited by mannan and nitrophenol-modified sugar derivatives, but not by simple sugars. Using affinity chromatography with immobilized mannan on Sepharose 6B, the mosquito MBL was partially purified. Purified mosquito MBL shared biochemical properties with BDL1, containing two subunits of molecular mass of 28 and 30 kDa under reducing conditions in SDS/PAGE. Its activity is dependent on Ca(2+), and it is stable at pH 7-9 and at temperatures less than 30 degrees C.     

nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito,Anopheles stephensi

Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so.     

Isolation and characterization of polymorphic microsatellite markers from the mosquito Anopheles moucheti,malaria vector in Africa

Anopheles moucheti is a major human malaria vector in Equatorial Africa. The screening of an Anopheles moucheti genomic microsatellite library allowed us to select 36 sequences with AC/GT dinucleotide tandem repeats. Primer pairs were designed to amplify the loci and 25 out of 36 gave a repeatable and scorable amplification. In total, 17 loci were selected for their high degree of polymorphism (the number of alleles per locus ranged from four to 16, and observed heterozygosity from 0.43 to 0.87) and suspicion of absence of null alleles, using 30 wild females from South‐Cameroon. No linkage disequilibrium was found between the loci.     

Identification and characterization of the receptor for the Bacillus sphaericus binary toxin in the malaria vector mosquito, Anopheles gambiae

The binary toxin (Bin) from Bacillus sphaericus exhibits a highly insecticidal activity against Culex and Anopheles mosquitoes. The cytotoxicity of Bin requires an interaction with a specific receptor present on the membrane of midgut epithelial cells in larvae. A direct correlation exists between binding affinity and toxicity. The toxin binds with high affinity to its receptor in its primary target, Culex pipiens, and displays a lower affinity to the receptor in Anopheles gambiae, which is less sensitive to Bin. Although the Bin receptor has previously been identified and named Cpm1 in C. pipiens, its structure in Anopheles remains unknown. In this study, we hypothesize that the Anopheles Bin receptor is an ortholog of Cpm1. By screening the Anopheles genomic database, we identified a candidate gene (Agm3) which is expressed primarily on the surface of midgut cells in larvae and which functions as a receptor for Bin. A Cpm1-like gene is also present in the Bin-refractory species Aedes aegypti. Overall, our results indicate that the three mosquito genes examined share a very similar organization and are strongly conserved at the amino acid level, in particular in the NH(2)-terminus, a region believed to contain the ligand binding site, suggesting that relatively few amino acids residues are critical for high affinity binding of the toxin.