Cloning and blood‐meal dependent induction pattern of apolipophorin‐III from Anopheles sinensis

Apolipophorin‐III is known to play a role in transporting lipids in insects, and much attention has been paid to lepidopteran insects' apolipophorin. Thus, we were interested in examining the effects of blood‐meal on the expression pattern of apolipophorin‐III in mosquitoes. This led us to clone and partially characterize the full‐length cDNA of apoLp‐III (AnsiApoLp‐III) from Anopheles sinensis. Analysis of AnsiApoLp‐III cDNA shows that the 728‐bp sequence has a 582‐bp protein‐coding region with 94 bp of putative 5′ untranslated region and 152 bp of 3′ untranslated region. The deduced amino acid sequence begins with a methionine codon at position 95 and extends to position 674, encompassing a polypeptide of 193 amino acids. AnsiApoLp‐III has the highest identity (63%) to Culex quinquefasciatus apoLp‐III. Temporal expression pattern analysis shows that although AnsiApoLp‐III was expressed at all developmental stages, it was highly detected at egg and adult stages in the female mosquitoes. In addition, we found out that AnsiApoLp‐III was induced in An. sinensis adult females after uptaking a blood‐meal. Spatial expression patterns of AnsiApoLp‐III shows that AnsiApoLp‐III mRNA was strongly induced at day 1 and gradually decreased from day 1 to day 4 in the ovaries. Most interestingly, AnsiApoLp‐III mRNA in the Malpighian tubule was strongly induced at day 1, decreased during days 1–3, and then became elevated again at day 4. These data suggest that blood‐meal influences AnsiApoLp‐III mRNA induction in ovaries and Malpighian tubules. It remains to further elucidate the biological roles of AnsiApoLp‐III in these organs.     

Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.     

Wolbachia strain wAlbB enhances infection by the rodent malaria parasite Plasmodium berghei in Anopheles gambiae mosquitoes

Wolbachia, a common bacterial endosymbiont of insects, has been shown to protect its hosts against a wide range of pathogens. However, not all strains exert a protective effect on their host. Here we assess the effects of two divergent Wolbachia strains, wAlbB from Aedes albopictus and wMelPop from Drosophila melanogaster, on the vector competence of Anopheles gambiae challenged with Plasmodium berghei. We show that the wAlbB strain significantly increases P. berghei oocyst levels in the mosquito midgut while wMelPop modestly suppresses oocyst levels. The wAlbB strain is avirulent to mosquitoes while wMelPop is moderately virulent to mosquitoes pre-blood meal and highly virulent after mosquitoes have fed on mice. These various effects on P. berghei levels suggest that Wolbachia strains differ in their interactions with the host and/or pathogen, and these differences could be used to dissect the molecular mechanisms that cause interference of pathogen development in mosquitoes.     

Susceptibility of Anopheles campestris-like and Anopheles barbirostris species complexes to Plasmodium falciparum and Plasmodium vivax in Thailand

Nine colonies of five sibling species members of Anopheles barbirostris complexes were experimentally infected with Plasmodium falciparum and Plasmodium vivax. They were then dissected eight and 14 days after feeding for oocyst and sporozoite rates, respectively, and compared with Anopheles cracens. The results revealed that Anopheles campestris-like Forms E (Chiang Mai) and F (Udon Thani) as well as An. barbirostris species A3 and A4 were non-potential vectors for P. falciparum because 0% oocyst rates were obtained, in comparison to the 86.67-100% oocyst rates recovered from An. cracens. Likewise, An. campestris-like Forms E (Sa Kaeo) and F (Ayuttaya), as well as An. barbirostris species A4, were non-potential vectors for P. vivax because 0% sporozoite rates were obtained, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. barbirostris species A1, A2 and A3 were low potential vectors for P. vivax because 9.09%, 6.67% and 11.76% sporozoite rates were obtained, respectively, in comparison to the 85.71-92.31% sporozoite rates recovered from An. cracens. An. campestris-like Forms B and E (Chiang Mai) were high-potential vectors for P. vivax because 66.67% and 64.29% sporozoite rates were obtained, respectively, in comparison to 90% sporozoite rates recovered from An. cracens.     

Effect of anti-mosquito antibodies on the infectivity of the rodent malaria parasite Plasmodium berghei to Anopheles farauti

The effect of mouse anti-mosquito antibodies, present in the bloodmeal, on the infectivity of Plasmodium berghei Vincke to Anopheles farauti Laveran was investigated. Significantly fewer oocysts developed in mosquitoes feeding on mice immunized with sugar-fed mosquito midgut antigens than in mosquitoes feeding on control mice. Mosquitoes feeding on mice immunized with the midgut antigens derived from sugar-fed mosquitoes also showed reduced mortality and had lower infection rates than those fed on unimmunized mice. Blood-fed midgut antigen was less effective in producing these effects than sugar-fed midgut antigen.     

Accelerated clearance of uninfected red cells from Plasmodium berghei-infected mouse blood in normal mice

51Cr-labelled uninfected cells, separated from Plasmodium berghei-infected mouse blood using the fluorescence-activated cell sorter, were cleared more rapidly than normal mouse erythrocytes after intravenous injection into normal mice.     

CLIP proteases and Plasmodium melanization in Anopheles gambiae

Melanization is a potent immune response mediated by phenoloxidase (PO). Multiple Clip-domain serine proteases (CLIP) regulate PO activation as part of a complex cascade of proteases that are cleaved sequentially. The role of several CLIP as key activators or suppressors of the melanization responses of Anopheles gambiae to Plasmodium berghei (murine malaria) has been established recently using a genome-wide reverse genetics approach. Important differences in regulation of PO activation between An. gambiae strains were also identified. This review summarizes these findings and discusses our current understanding of the An. gambiae melanization responses to Plasmodium.     

Plasmodium vivax: impaired escape of Vk210 phenotype ookinetes from the midgut blood bolus of Anopheles pseudopunctipennis

The site in the midguts of Anopheles pseudopunctipennis where the development of Plasmodium vivax circumsporozoite protein Vk210 phenotype is blocked was investigated, and compared to its development in An. albimanus. Ookinete development was similar in time and numbers within the blood meal bolus of both mosquito species. But, compared to An. pseudopunctipennis, a higher proportion of An. albimanus were infected (P=0.0001) with higher ookinete (P=0.0001) and oocyst numbers (P=0.0001) on their internal and external midgut surfaces, respectively. Ookinetes were located in the peritrophic matrix (PM), but neither inside epithelial cells nor on the haemocoelic midgut surface by transmission electron microscopy in 24h p.i.-An. pseudopunctipennis mosquito samples. In contrast, no parasites were detected in the PM of An. albimanus at this time point. These results suggest that P. vivax Vk210 ookinetes cannot escape from and are destroyed within the midgut lumen of An. pseudopunctipennis.     

A Plasmodium falciparum host-targeting motif functions in export during blood stage infection of the rodent malarial parasite Plasmodium berghei

Plasmodium falciparum (P. falciparum) secretes hundreds of proteins--including major virulence proteins--into the host erythrocyte. In order to reach the host cytoplasm, most P. falciparum proteins contain an N terminal host-targeting (HT) motif composed of 11 amino acids. In silico analyses have suggested that the HT motif is conserved throughout the Plasmodium species but experimental evidence only exists for P. falciparum. Here, we show that in the rodent malaria parasite Plasmodium berghei (P. berghei) a reporter-like green fluorescent protein expressed by the parasite can be exported to the erythrocyte cytoplasm in a HT-specific manner. This provides the first experimental proof that the HT motif can function as a signal for protein delivery to the erythrocyte across Plasmodium species. Further, it suggests that P. berghei may serve as a model for validation of P. falciparum secretome proteins. We also show that tubovesicular membranes extend from the vacuolar parasite into the erythrocyte cytoplasm and speculate that these structures may facilitate protein export to the erythrocyte.     

Silencing an Anopheles gambiae catalase and sulfhydryl oxidase increases mosquito mortality after a blood meal

Catalase is a potent antioxidant, likely involved in post-blood meal homeostasis in mosquitoes. This enzyme breaks down H2O2, preventing the formation of the hydroxyl radical (HO*). Quiescins are newly classified sulfhydryl oxidases that bear a thioredoxin motif at the N-terminal and an ERV1-like portion at the C-terminal. These proteins have a major role in generating disulfides in intra- or extracellular environments, and thus participate in redox reactions. In the search for molecules to serve as targets for novel anti-mosquito strategies, we have silenced a catalase and a putative quiescin/sulfhydryl oxidase (QSOX), from the African malaria vector Anopheles gambiae, through RNA interference (RNAi) experiments. We observed that the survival of catalase- and QSOX-silenced insects was reduced over controls following blood digestion, most likely due to the compromised ability of mosquitoes to scavenge and/or prevent damage caused by blood meal-derived oxidative stress. The higher mortality effect was more accentuated in catalase-silenced mosquitoes, where catalase activity was reduced to low levels. Lipid peroxidation was higher in QSOX-silenced mosquitoes suggesting the involvement of this protein in redox homeostasis following a blood meal. This study points to the potential of molecules involved in antioxidant response and redox metabolism to serve as targets of novel anti-mosquito strategies and offers a screening methodology for finding targetable mosquito molecules.     

Anopheles and Plasmodium: from laboratory models to natural systems in the field

Parasites that cause malaria must complete a complex life cycle in Anopheles vector mosquitoes in order to be transmitted from human to human. Previous gene-silencing studies have shown the influence of mosquito immunity in controlling the development of Plasmodium. Thus, parasite survival to the oocyst stage increased when the parasite antagonist gene LRIM1 (leucine-rich repeat immune protein 1) of the mosquito was silenced, but decreased when the C-type lectin agonist gene CTL4 or CTLMA2 (CTL mannose binding 2) was silenced. However, such effects were shown for infections of the human mosquito vector Anopheles gambiae with the rodent parasite Plasmodium berghei. Here, we report the first results of A. gambiae gene silencing on infection by sympatric field isolates of the principal human pathogen P. falciparum. In contrast with the results obtained with the rodent parasite, silencing of the same three genes had no effect on human parasite development. These results highlight the importance of following up discoveries in laboratory model systems with studies on natural parasite-mosquito interactions.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Plasmodium yoelii: blood oxygen and brain function in the infected mouse

The carriage of oxygen by the blood and the in vivo response of the brain were investigated in mice infected with a lethal strain of Plasmodium yoelii. All mice with parasitaemia exceeding 70% were severely anaemic (Hb 3.5 +/- 1.8 g/dl; mean +/- 1 SD), acidotic (blood pH 7.04 +/- 0.06) and hypoglycaemic (blood glucose 0.6 +/- 0.76 mumol/ml). The oxyhaemoglobin dissociation curve (ODC) of blood from heavily infected mice was shifted right as compared to controls, but the increase in p50 was less than expected from the accompanying acidosis. The reduced shift right was due to a decrease in the 2,3-DPG/Hb ratio in infected animals (0.72 +/- 0.12, n = 17 vs 1.10 +/- 0.09, n = 12 in controls). Despite the severity of terminal infection, the cerebral pH and the relative steady-state concentrations of PCr, ATP and Pi measured in vivo by nuclear magnetic resonance (31P NMR) were normal. Alterations in brain energy status and pH cannot account for cerebral signs or death in this proposed mouse model of cerebral malaria.