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.     

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.     

Larvicidal efficacy of Aloe barbadensis and Cannabis sativa against the malaria vector Anopheles stephensi (Diptera: Culicidae)

Anopheles stephensi is the primary vector of malaria, an endemic disease in India. An effort to control An. stephensi larvae by leaf extracts of Aloe barbadensis (Liliaceae) and Cannabis sativa (Moraceae) was made under laboratory conditions. A carbon tetrachloride extract of A. barbadensis was the most effective of all the extracts tested for larvicidal activity against the anopheline larvae, with LC₅₀ 15.58 and 8.04 p.p.m. after 24 and 48 h of exposure, respectively. Thus, the leaf extract of A. barbadensis has active components that could be useful as a larvicide of ecocongenial nature against malaria vectors.     

Dendritic cells subsets mediated immune response during Plasmodium berghei ANKA and Plasmodium yoelii infection

The roles of dendritic cells (DCs) in mediating immunity against Plasmodium infection have been extensively investigated, but immune response during pathogenesis of malaria is still poorly understood. In the present study, we compared the splenic DCs phenotype and function during P. berghei ANKA (PbA) or P. yoelii (P. yoelii) infection in Swiss mice. We observed that PbA-infected mice developed more myeloid and mature DCs capable of secreting IL-12, while P. yoelii-infected mice had more plasmacytoid and immature DCs secreting higher levels of IL-10. Expression of FoxP3, IL-17, TGF-β and IL-6 were also different between these two infections. Thus, these results suggest that the phenotypic and functional subsets of splenic DCs are key factors for regulating immune responses to PbA and P. yoelii infections.     

Glycosylphosphatidyl-inositols in murine malaria: Plasmodium yoelii yoelii

Glycosylphosphatidyl-inositols (GPIs) are vital major glycoconjugates in intraerythrocytic stages of Plasmodium. Here, we report on the biosynthesis and the characterization of GPIs synthesized by the murine malarial parasite P. yoelii yoelii YM. Parasitized erythrocytes were labeled in vivo and in vitro with either radioactive nucleotide sugar precursors, ethanolamine or glucosamine. The pathway leading to the formation of GPI precursors was found to resemble that described for P. falciparum; however, in P. yoelii, the formation of an additional hydrophilic precursor containing an acid-labile modification was detected. The data suggest that this modification is linked to the fourth mannose attached to the trimannosyl backbone in an alpha1-2 linkage. The modification was susceptible to hydrofluoric acid (HF), but not to nitrous acid (HNO(2)). Data obtained from size-exclusion chromatography on Bio-Gel P4, and Mono Q analysis of the fragments generated by HNO(2) deamination suggest that the modification is due to the presence of an additional ethanolamine linked to the fourth mannose via a phosphodiester bond.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

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.     

Role of Anopheles (Kerteszia) bellator as malaria vector in southeastern Brazil (Diptera: Culicidae)

New research concerning Anopheles bellator in the southeast of the State of S?o Paulo, Brazil, are reported. Adult females of this mosquito showed remarkable endophily and endophagy which was even greater than An. cruzii. The epidemiological role of this anopheline as a malaria vector is discussed.     

Allozyme variation in Anopheles stephensi Liston from Pakistan (Diptera: Culicidae)

Allozyme variability was analyzed at 16 loci in 11 lines of Anopheles stephensi Liston from Pakistan. Six lines were considered as samples from natural populations. For these lines the mean number of alleles was 1.31-1.63, the degree of polymorphism was 0.188-0.375, the observed heterozygosity was 0.065-0.086, and the genetic distance ranged from 0.001 to 0.016. No population-specific alleles were found. Interbreeding was considerable (mean Fit = 0.183). Differences in allele frequencies were due considerable (mean Fit = 0.183). Differences in allele frequencies were due primarily to local inbreeding (Fis greater than Fst at most loci). The Lahore line, reared for more than 20 generations, had more homozygotes than the other lines. A line refractory to Plasmodium falciparum and a genetic sexing line exhibited decreased allozyme variability. The latter line showed reduced staining intensity at 10 loci. Linkage studies are recommended for the following loci with rare alleles: Acp, Gapdh, Icd-1, Icd-2, Mpi, and Pgd.     

Immune haemolymph proteins in response to bacterial infection and identification of a putative bacteria binding protein in malaria vector Anopheles stephensi

Induction of haemolymph proteins in mosquito A. stephensi due to wounding or bacterial infection (E. coli) was analyzed using SDS-PAGE. Wounding response of pupa revealed subsequent induction of two polypeptides (21 and 74 kDa). Two other polypeptides (44 and 57 kDa) were induced commonly in both pupa and adult female haemolymph upon bacterial infection. In vitro binding assay revealed identification of 44 kDa, a putative bacterial binding protein, a more relevant protein for further elucidation of molecular mechanism involved in host parasite interactions.     

Plasmodium yoelii: semiquantitative analyses of circumsporozoite protein gene expression during the sporogonic development of P. y. yoelii and P. y. nigeriensis in the mosquito vector Anopheles stephensi

Malaria infection in the mosquito vector can be modulated by the vertebrate host, mosquito factors, and interactions between different parasite populations. Modulation of parasite development can be assessed through the study of gene expression. The present report describes a specific, sensitive, and nonradioactive method that permits assessment of parasite load and quantification of circumsporozoite protein gene expression during the sporogonic stages of Plasmodium yoelii yoelii and P. y. nigeriensis. A decrease in parasite load was observed when comparing DNA of oocysts on day 7 postinfection with that of oocysts and sporozoites on day 19. On day 7, parasites (oocysts) showed a marked increase of circumsporozoite protein expression when compared with that (sporozoites and oocysts) on day 19. The method developed in this work can be a valuable tool to understand parasite interaction mechanisms that are involved in mosquito malaria infections.     

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.     

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.     

Immunogold localization of circumsporozoite protein of the malaria parasite Plasmodium falciparum during sporogony in Anopheles stephensi midguts

The occurrence of the circumsporozoite (CS) proteins of Plasmodium falciparum sporozoites was monitored during sporogonic development in Anopheles stephensi mosquitoes. Using a monoclonal anti-CS protein antibody (3Sp2) and immunogold labeling on ultrathin cryosections it was found that CS protein is synthesized in immature oocysts from day 6 onwards when there are not yet signs of sporozoite formation. The CS protein is rapidly incorporated in the oocyst plasmalemma, which subsequently invaginates into the parasite. In the oocyst only the external sporozoite membrane contains CS protein. The inner pellicle membranes, rhoptries and micronemes do not react with monoclonal antibody (MoAb) 3Sp2.     

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.     

The effect of Plasmodium yoelii nigeriensis infection on the feeding persistence of Anopheles stephensi Liston throughout the sporogonic cycle.

Vector-borne parasites such as malaria have been shown to modify the feeding behaviour of their invertebrate hosts so as to increase the probability of transmission. However, evolutionary consideration of developmental changes in malaria within Anopheles mosquitoes suggests that the nature of altered feeding by mosquitoes should differ depending on the developmental stage of the parasite. We present laboratory evidence that the feeding persistence of female Anopheles stephensi towards a human host is decreased in the presence of Plasmodium yoelii nigeriensis oocysts (which cannot be transmitted), but increased when the malaria has developed into transmissible sporozoites in the salivary glands. In ten-minute trials, 33% of uninfected mosquitoes gave up their feeding attempt before the test period had ended, 53% of those harbouring oocysts had given up, but only 20% of those infected with sporozoites gave up by this time. We conclude that changes in feeding behaviour of mosquitoes mediated by parasite infection are sensitive to the developmental stage of the parasite and that these changes have important implications for malaria epidemiology.