A continuous cell line of a nonhematophagous mosquito,Toxorhynchites amboinensis

Summary A continuous cell line of a nonhematophagous mosquito was obtained by using partially incapacitated but living larvae as the source The cells were predominantly epithelioid and diploid (2N=6). Syncytia were occasionally observed in the normal cell cultures.     

Uptake and persistence of ingested antibody in the mosquito Anopheles stephensi

A sensitive ELISA was developed to monitor the persistence of a specific antibody, rabbit anti-BSA, in the bloodmeal, haemolymph and tissues of the mosquito Anopheles stephensi Liston. Different concentrations of anti-BSA were fed to female mosquitoes in sheep blood, via a membrane-feeder, and it was found that antibody persisted in the gut as the bloodmeal was digested: concentrations present at 24 h were directly related to those fed. Homogenates of mosquito bodies, from which the intact guts had been removed, were always antibody-positive up to 9 days post-feeding, indicating that undigested antibody had passed through the gut wall into the haemocoele. Haemolymph was extracted from mosquitoes at different times post-feeding, using a microcapillary and manipulator, and antibody was detected in several of the assays. The level of antibodies in the haemolymph 24 h post-feeding was less than half of the level in mosquito heads, indicating removal of antibodies from the haemolymph, perhaps by binding onto haemocoelic tissues. The relevance of these results to the ingestion, survival and fate of antibody against malaria sporozoites is discussed.     

Larvicidal toxicity of Japanese Bacillus thuringiensis against the mosquito Anopheles stephensi

Japanese isolates of Bacillus thuringiensis were screened for larvicidal activity against the mosquito Anopheles stephensi , the urban malaria vector of the Indian subcontinent. Among more than 30 strains identified, larvicidal activity causing >80% mortality in 72 h was demonstrated for 41/1449 (2.8%) isolates. The majority of strains and isolates (97.2%) exhibited little or no larvicidal activity. Anopheles -active strains belonged to more than 12 H serotypes, especially H3ade (serovar fukuokaensis ) and H44 (serovar higo ). SDS-PAGE profiles of inclusion proteins showed 4 distinct types among 6 active strains examined. The most active Japanese isolates were H20 strain 89-T-34-14 (LC₅₀ 4.4 μg/ml) and H44 serovar higo strain 74-E-45-24 (LC₅₀ 7.6 μg/ml), respectively, 13-fold and 23-fold less active than the international standard H14 serovar israelensis (LC₅₀ 0.33 μg/ml).     

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/ .     

Induced immunity against the mosquito Anopheles stephensi: reactivity characteristics of immune sera

This study shows the progression of immune responses in mice during five sequential immunizations with Anopheles stephensi mosquito extracts, characterized by ELISA, Western blot and immunohistochemistry. When exposed repeatedly to mosquito bites, control mice developed antibodies which reached titres of 1:10(5), reacted weakly in Western blot analysis and were localized to the salivary glands. Mice immunized with mosquito head plus salivary glands, midgut, ovary, fat body, midgut microvilli (Mv) and midgut basolateral plasma membrane (Blm), showed increased titre with each successive boost. Epitopes were shared between sera or were specific to the immunizing or heterologous extract. Anti-Mv and Blm sera recognized proteins labelled by anti-midgut serum and gave specific reactions with the midgut and head. Cross-reactivity was confirmed immunohistochemically.     

Genetics of refractoriness to Plasmodium falciparum in the mosquito Anopheles stephensi

We previously selected a line of the malaria vector mosquito Anopheles stephensi refractory (resistant) to the human malaria parasite Plasmodium falciparum , using in vitro infections with P. falciparum gametocytes. This report presents data on the genetic background of refractoriness. The results of F₁-crosses and backcrosses show that refractoriness to P. falciparum in our A. stephensi line is autosomal and semi-dominant to susceptibility. The expression of refractoriness is apparently affected by a cytoplasmic factor. Interpretation of data from the crosses by quantitative trait locus analysis shows that one gene or two unlinked interacting autosomal genes, or groups of closely linked genes, are involved.     

Toxin-binding proteins from midgut epithelium membranes of Anopheles stephensi larvae

Proteins of 65 and 57 kD were isolated from the apical membranes of midgut epithelium of Anopheles stephensi larvae by affinity chromatography. These proteins can specifically bind endotoxin Cry11A and activate toxin Cry4B (Cry4B-tox) under conditions of ligand blotting, and both Cry proteins compete for this binding. At least in the case of Cry4B-tox, the binding with 65 and 57 kD proteins is reversible. The ability of the products of limited proteolysis of Cry11A and Cry4B to bind the 65 and 57 kD proteins correlates with their toxicity to A. stephensi larva. The N-terminal amino acid sequence of the 57 kD protein is unique and absent in the NCBI GenBank. The proteins of 65 and 57 kD share most of the properties studied with Aedes aegypti toxin-binding proteins. It is possible that they altogether represent a novel class (or classes) of delta-endotoxin receptors.     

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.     

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.     

Isolation and characterization of microsatellite loci in the mosquito Anopheles stephensi Liston (Diptera: Culicidae)

The mosquito Anopheles stephensi is an important malaria vector in India, Pakistan, Iran and Afghanistan. Differences in egg morphology and chromosomal characters have been described between urban and rural forms of this mosquito but the population genetic structure remains unclear. In India this species is mainly urban, rural populations are largely zoophilic and not thought to transmit malaria. In eastern Afghanistan and the Punjab and Northwest Frontier Province, Pakistan, it is the major malaria vector. We have developed primers for 16 microsatellite loci to assist in defining the population structure and epidemiological importance of this mosquito.     

Partial characterization of oligosaccharides expressed on midgut microvillar glycoproteins of the mosquito, Anopheles stephensi Liston

Midguts of the malaria-transmitting mosquito, Anopheles stephensi, were homogenized and microvillar membranes prepared by calcium precipitation and differential centrifugation. Oligosaccharides present on the microvillar glycoproteins were identified by lectin blotting before and after in vitro and in situ treatments with endo- and exo-glycosidases. Twenty-eight glycoproteins expressed a structurally restricted range of terminal sugars and oligosaccharide linkages. Twenty-three glycoproteins expressed oligomannose and/or hybrid N-linked oligosaccharides, some with alpha1-6 linked fucose as a core residue. Complex-type N-linked oligosaccharides on eight glycoproteins all possessed terminal N-acetylglucosamine, and alpha- and beta-linked N-acetylgalactosamine. Eight glycoproteins expressed O-linked oligosaccharides all containing N-acetylgalactosamine with or without further substitutions of fucose and/or galactose. Galactosebeta1-3/4/6N-acetylglucosamine-, sialic acidalpha2-3/6galactose-, fucosealpha1-2galactose- and galactosealpha1-3galactose- were not detected. Terminal alpha-linked N-acetylgalactosamine residues on N-linked oligosaccharides are described for the first time in insects. The nature and function of these midgut glycoproteins have yet to be identified, but the oligosaccharide side chains are candidate receptors for ookinete binding and candidate targets for transmission blocking strategies.     

Growth of Anopheles mosquito larvae on dietary microbiota in aquatic surface microlayers

Abstract. Hydrophobic organic matter accumulates under the surface film of water bodies to form the surface microlayers. Heterotrophic microorganisms use this organic matter for growth, and they, in turn, are fed upon by Anopheles mosquito larvae and other animals. From laboratory experiments we show that two species of mosquito larvae, Anopheles gambiae and An.quadrimaculatus , grew most rapidly where surface micro-layers were present and, especially, where labile dissolved organic matter was added to promote growth of microorganisms. The importance of microorganisms was confirmed by the addition of gentamicin antibiotic, which suppressed the microbiota and reduced the growth of larvae feeding on surface microlayers. Anopheles larvae grew well on a suspension of finely ground fish food to which the antibiotic had been added, showing that reduced growth was not due to gentamicin itself. Because sub-surface microorganisms are the components of the larval diet that most affect growth, we discuss their relevance to strategies for larval control of Anopheles mosquitoes.     

Enhanced protective potential of novel citronella essential oil microsponge hydrogel against Anopheles stephensi mosquito

Citronella oil has been frequently used as an insect repellant and antibacterial agent for management of vector borne diseases. In this study, the fabrication of citronella oil microsponge loaded hydrogel (HG-COMS) was conceptualized in order to provide future insight for developing delayed release formulation. The hydrogel was characterized for drug content, drug interaction studies, spreadability, texture analysis and in vitro occlusive behaviour and results were found satisfactory. Further, in vitro antimicrobial studies were carried out to compare the antimicrobial inhibitory potential of the HG-COMS against citronella oil loaded hydrogel (HG-CO). HG-COMS formulation showed better antimicrobial efficacy than HG-CO (zone of inhibition of E. coli, P. aeruginosa and S. aureus; with P value less than 0.01, 0.001 and 0.05, respectively). In addition, the safety (irritation potential) of the oil loaded hydrogel formulation was assessed by Hen’s Egg Test Chorioallantoic Membrane (HAT-CAM) method. Mosquito repellent activity against Anopheles stephensi (malaria vector mosquito) was also performed in a net cage having blood starved female mosquitoes. The repellent potential of prepared HG-COMS (34% repellency for 6 h) was found dependent on release of CO from the microsponges as well as from the gel matrix. HET-CAM test revealed that HG-COMS (irritation score: 6.43 ± 0.77) was found very promising in comparison to HG-CO (irritation score: 12.77 ± 0.36), and was thus, considered safer for dermal use. HG-COMS showed reduced frequency of application, no skin irritation and potential for controlling A. stephensi for longer time periods. Hence, HG-COMS is found as a promising eco-friendly protective option, to minimize the burden of mosquito-transmitted diseases, especially malaria in future.     

Laboratory evaluation of Toxorhynchites splendens (Diptera: Culicidae) for predation of Aedes albopictus mosquito larvae

Biology of the mosquito Toxorhynchites splendens (Wiedemann) was studied in the laboratory to provide baseline data for using the predatory larvae of this species against those of Aedes albopictus (Skuse) in a biological control programme. The mean incubation time of Tx.splendens eggs was 43.8 h and the time required for newly-hatched larvae to initiate predation was 2.5 h. Mean numbers of prey larvae consumed and killed by each Tx.splendens larva totalled 389 and 345 respectively. The larval period of Tx.splendens was not significantly different for rearing individually or in groups of nine, with equal prey density, and duration of larval development was proportional to prey density. In mass rearing, larval cannibalism was usually observed during days 1-3 post-eclosion. The incidence of cannibalism decreased sharply on the fourth day after hatching when some larvae became fourth-instar. Adult female Tx.splendens usually commenced oviposition on day 4 after emergence. The number of eggs laid daily increased on day 7 and the peak oviposition of 6.3 eggs/female/day occurred on day 11. When oviposition containers were provided only intermittently, gravid females of Tx.splendens scattered most of their eggs on the dry floor of the cage. Viability of eggs laid by females aged 4-14 days was high (60-90%) but decreased to less than 40% as the females aged.     

Plasmodium berghei: plasmodium perforin-like protein 5 is required for mosquito midgut invasion in Anopheles stephensi

During its life cycle the malarial parasite Plasmodium forms three invasive stages which have to invade different and specific cells for replication to ensue. Invasion is vital to parasite survival and consequently proteins responsible for invasion are considered to be candidate vaccine/drug targets. Plasmodium perforin-like proteins (PPLPs) have been implicated in invasion because they contain a predicted pore-forming domain. Ookinetes express three PPLPs, and one of them (PPLP3) has previously been shown to be essential for mosquito midgut invasion. In this study we show through phenotypic analysis of loss-of-function mutants that PPLP5 is equally essential for mosquito infection. Deltapplp5 ookinetes cannot invade midgut epithelial cells, but subsequent parasite development is rescued if the midgut is bypassed by injection of ookinetes into the hemocoel. The indistinguishable phenotypes of Deltapplp5 and Deltapplp3 ookinetes strongly suggest that these two proteins contribute to a common process.