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.     

ACE inhibitors reduce fecundity in the mosquito,Anopheles stephensi

Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase, which cleaves dipeptides and, in some instances, dipeptide or tripeptide amides from the C-terminus of regulatory peptides (e.g. angiotensin I, bradykinin and substance P). The expression of ACE is highly regulated in insects, where it is thought to have a role in the metabolism of peptide hormones involved in regulating reproduction. After a blood meal, ACE activity in the female mosquito Anopheles stephensi, increases four-fold with much of the enzyme finally accumulating in the ovary. In the present study, we have studied the effect on reproduction of adding two selective inhibitors of ACE, captopril and lisinopril, to the blood meal. Both ACE inhibitors reduced the size of the batch of eggs laid by females in a dose-dependent manner, with no observable effects on the behaviour of the adult insect. The almost total failure to lay eggs after feeding on either 1 mM captopril or 1 mM lisinopril, did not result from interference with the development of the primary follicle, but was due to the inhibition of egg-laying. Since very similar effects on the size of the egg-batch were observed with two selective ACE inhibitors, belonging to different chemical classes, we suggest that these effects are mediated by the selective inhibition of the induced mosquito ACE, a peptidase probably involved in the activation/inactivation of a peptide regulating egg-laying activity in A. stephensi.     

Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae)

Rabbits were immunized three times with extracts of Anopheles stephensi midgut. Immunized rabbits showed a high titer of antibodies when characterized by ELISA. We investigated the effect of anti-mosquito midgut antibodies on mosquito fecundity, longevity, mortality, engorgement, and the development of the malaria parasite in mosquitoes. Fecundity was reduced significantly (38%) and similarly hatchability by about 43.5%. There was no statistically significant effect on mortality, longevity, and engorgement. When the mosquito blood meal contained anti-midgut antibodies, fewer oocysts of Plasmodium vivax developed in the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. We also found that the midgut antibodies inhibit the development and/or translocation of the sporozoites. Antisera raised against midgut of A. stephensi recognized eight polypeptides (110, 92, 70, 45, 38, 29, 15, 13 kDa) by Western blotting. Cross-reactive antigens/epitopes present in other tissues of A. stephensi were also examined both by Western blotting and in vivo ELISA. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission blocking vaccine and/or anti-mosquito vaccine.     

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.     

Partial genomic organization of ribosomal protein S7 gene from malaria vector Anopheles stephensi

In this study, we describe the partial genomic organization of ribosomal protein S7 gene isolated from the mosquito Anopheles stephensi. Initially a 558 bp partial cDNA sequence was amplified as precursor mRNA sequence containing 223 bp long intron. 5＇ and 3＇ end sequences were recovered using end specific rapid amplification of cDNA ends （RACE） polymerase chain reaction. The full-length cDNA sequence was 914 nucleotide long with an open reading frame capable of encoding 192 amino acid long protein with calculated molecular mass of 22174 Da and a pI point of 9.94. Protein homology search revealed 〉75% identity to other insect＇s S7 ribosomal proteins. Analysis of sequence alignment revealed several highly conserved domains, one of which is related to nuclear localization signal （NLS） region of human rpS7. Interestingly, intron nucleotide sequence comparison with A. gambiae showed a lesser degree of conservation as compared to coding and untranslated regions. Like this, early studies on the genomic organization and cDNA/ Expressed sequence tag analysis （EST） could help in genome annotation ofA. stephensi, and would be likely to be sequenced in the future.     

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.     

Effect of adult nutrition on the melanization immune response of the malaria vector Anopheles stephensi

Two dietary resources - blood and sugar - were assessed for effects on the melanization immune response of the mosquito Anopheles stephensi Liston (Diptera: Culicidae) towards inoculated Sephadex beads (negatively charged C-25). This melanization is conferred by genetic factors capable of making the mosquito refractory to malaria parasites. If An. stephensi females had obtained a bloodmeal one day before inoculation with a bead, the efficacy of their immune response increased with the concentration of sugar ingested. At the highest sugar concentration (6%) tested, 38% of the mosquitoes completely melanized their bead, whereas at the lowest sugar concentration (2%), none of the mosquitoes were able to melanize their bead completely. Among mosquitoes not having a bloodmeal, the immuno-competence was low (c. 9% of the mosquitoes completely melanized their bead) and independent of sugar concentration. The observed interaction between these two resources indicates that both resources are required for the Anopheles female to develop an effective melanization immune response.     

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.     

Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi

Some genetic strategies for controlling transmission of mosquito-borne diseases call for the introgression of antipathogen effector genes into vector populations. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the expression of the effector molecules to maximize their efficacy. Vitellogenin (Vg)-encoding gene control sequences are candidates for driving tissue-, stage- and sex-specific expression of exogenous genes. One of the Anopheles stephensi Vg genes, AsVg1, was cloned and a full-length cDNA, as well as 850 base pairs adjacent to the 5'-end, were sequenced and characterized. Expression of AsVg1 is restricted to the fat body tissues of blood-fed females, and the amino acid sequence of the conceptual translation product is >85% identical to those of other anopheline Vgs. These characteristics support the conclusion that AsVg1 is a Vg-encoding gene. Functional analyses of the AsVg1 putative cis-regulatory sequences were performed using transgenic mosquitoes. The results showed that DNA fragments encompassing the 850 base pairs immediately adjacent to the 5'-end of the gene and the 3'-end untranslated region are sufficient to direct sex-, stage- and tissue-specific expression of a reporter gene. These data indicate that the AsVg1 promoter is a good candidate for controlling the expression of anti-pathogen effector molecules in this malaria vector mosquito.     

Comparative susceptibility of three important malaria vectors Anopheles stephensi, Anopheles fluviatilis, and Anopheles sundaicus to Plasmodium vivax

The 3 laboratory-colonized malaria vectors, i.e., Anopheles stephensi, An. sundaicus, and An. fluviatilis, were studied for their comparative susceptibility to Plasmodium vivax sporogony. There was no significant difference in oocyst and sporozoite recruitment by these 3 species, whereas the geometric mean (GM) of the oocyst number per midgut was significantly lower in An. fluviatilis as compared with that in the other 2 species. There was no difference in the GM of oocyst between An. stephensi and An. sundaicus. Adaptability to laboratory conditions and susceptibility to plasmodial infection suggest that An. fluviatilis and An. sundaicus can also be used as a vector model for vector-parasite interaction studies.     

Heat shock response during development of the malaria vector Anopheles stephensi (Culicidae: Diptera)

The pattern of synthesis of heat shock proteins (HSP) and thermotolerance to elevated temperatures during the development of the malaria vector Anopheles stephensi normally reared at 28 +/- 2 degrees C was studied using SDS-PAGE. In total twelve heat shock proteins (i.e. 31, 33, 38, 43, 44, 51, 57, 62, 69, 71, 113 and 121 kD were induced by heat shock during various stages of development. Eight polypeptides (HSP during one or other of the instars) appeared during normal development of the adult, which showed very little response towards heat shock. Only two polypeptides (57 and 69 kD) were induced while the 22.5 kD protein disappeared during adult life. The HSP 62 and 71 kD induced during the larval stages showed a sharp decline in quantity in male and female adults upon heat shock. Three HSP (31, 43 and 44 kD) were induced in pupae due to heat shock. The synthesis of HSP in A. stephensi was correlated with the various morphological and physiological events occurring during development.     

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.     

Physiological aspects of multiple blood feeding in the malaria vector Anopheles tessellatus

The malaria vector Anopheles tessellatus is able to take several blood meals in a gonotrophic cycle. The fecundity is largely dependent on the first blood meal and is not generally increased by subsequent blood meals during a gonotrophic cycle. Larval rearing densities influenced adult body size. There is an inverse relationship between wing length and larval rearing densities. Smaller mosquitoes produced from larvae reared at higher densities had reduced body reserves of protein, lipid and carbohydrates. At emergence, ovarian development in An. tessellatus is in the previtellogenic stage and it remained at this stage until the intake of a blood meal. The number of ovarian follicles is related to wing length and, irrespective of adult body size, An. tessellatus developed oocytes to maturity with a single blood meal. This is attributed to the availability of metabolic reserves above the threshold level required for further development of oocytes. Mosquitoes that took more than one blood meal had largely digested their previous blood meal and had ongoing vitellogenesis. Blood meals subsequent to the first one apparently contribute mainly to increasing metabolic reserves. The stimulus for a second and third blood meal in An. tessellatus appears to be completion of the digestion of the previous blood meal. There was no evidence that multiple blood meals taken in the first gonotrophic cycle influenced fecundity significantly in the second cycle.     

Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection

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.