T. Adak

Comparative susceptibility of different members of the Anopheles culicifacies complex to Plasmodium vivax

Three colonized species of the Anopheles culicifacies complex (species A, B and C) were compared with Anopheles stephensi (control) for susceptibility to Plasmodium vivax. In feeding experiments involving over 400 paired comparisons, the mean number of oocysts, oocyst rate and sporozoite rate were found to be significantly different. Of the test groups, species A had the highest percentage of mosquitoes with oocysts (> 60%) and sporozoites (> 50%). An. culicifacies species B were least susceptible, less than 5% had oocysts and less than 2% had sporozoites in the salivary glands. The results are discussed in the light of the vectorial potential of the members of the An. culicifacies complex observed in field studies.

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.

Genetic structure of Plasmodium vivax isolates in India

Variations in the allelic composition of glucose phosphate isomerase (GPI), NADP-dependent glutamate dehydrogenase (GDH) and adenosine deaminase (ADA) enzyme systems of Plasmodium vivax were observed in isolates of Indian origin in 1985-1993. No significant difference was observed in allelic frequencies in different years. The data indicated random distribution of GPI, GDH and ADA alleles among the isolates, suggesting that loci for these enzymes were not linked. A high proportion of the isolates comprised at least 2 genetically distinct clones, the mean number of clones per isolate being 1.4. There was no significant difference in the number of oocysts in Anopheles stephensi fed on uniclonal and multiclonal isolates. No difference was observed in the proportions of uniclonal and multiclonal isolates during low and high transmission periods.

Susceptibility of species A, B, and C of Anopheles culicifacies complex to Plasmodium yoelii yoelii and Plasmodium vinckei petteri infections.

The comparative susceptibilities of colonized species A, B, and C of Anopheles culicifacies complex and Anopheles stephensi were determined for 2 rodent malaria parasites Plasmodium vinckei petteri and Plasmodium yoelii yoelii. All the 3 members of the complex were found to support complete sporogony with varying success. Controls, A. stephensi, become readily infected, with >70% developing oocysts. Of the test groups, species A had the highest percentage of mosquitoes with oocysts (>25%) and sporozoites (>15%). Anopheles culicifacies species B were least susceptible; less than 10% had oocysts and sporozoites in the salivary glands. The results demonstrate that A. culicifacies species A is most susceptible and species B is least susceptible to infections with both the parasites.

House-scale Evaluation of Bifenthrin Indoor Residual Spraying for Malaria Vector Control in India

Abstract In an area of India where the main rural malaria vector, Anopheles culicifacies Giles, has developed triple resistance to DDT, HCH, and malathion sprayed indoors in antimalaria program, bifenthrin (10% wettable powder) was evaluated in a randomized house-scale trial between July 1999 and March 2000. Entomological impact of four serial doses of bifenthrin (25, 50, 100, and 200 mg/m2) sprayed in rooms in five villages was compared with malathion (2 g/m2) and unsprayed control. An. culicifacies was 100% susceptible to bifenthrin (0.1%), but only 57% to malathion (5%) test papers. Contact bioassays were carried out on sprayed surfaces for 24 wk, and 24 h mortality in An. culicifacies was recorded. Bifenthrin 100- and 200-mg doses caused ≥80% mortality until 24 wk. The 50-mg dose caused ≥80% mortality on tin, wood, and mud surfaces for 24 wk, and on brick walls for 16 wk. Bifenthrin 25-mg dose produced ≥80% mortality for 24 wk on tin, 20 wk on mud walls, 16 wk on brick walls, and 8 wk on wood surfaces. Persistence of ≥80% mortality did not differ for 25- and 50-mg doses on any surface except on wood (P < 0.05). Malathion sprayed in three rounds of 6 wk apart caused ≥80% mortality for 16 wk on the brick and mud walls, and for 20 wk on the tin and wood surfaces. Bifenthrin 25- and 50-mg doses produced a similar impact on the densities of An. culicifacies and other mosquitoes but a superior one to malathion or control. Bifenthrin 25-mg dose caused least excito-repellency. Overall, efficacy of bifenthrin was superior to malathion. Considering the duration of the persistence of significant insecticidal action of bifenthrin on the most common surfaces (mud and brick walls), least excito-repellency and a relative impact on the mosquito densities, the 25-mg dose was the most superior among all the four doses evaluated.

Gut microbes influence fitness and malaria transmission potential of Asian malaria vector Anopheles stephensi

The midgut of parasite transmitting vector, Anopheles stephensi is a physiologically dynamic ecological niche of resident microbes. The gut resident microbes of anisomorphic and physiologically variable male and female A. stephensi mosquitoes were different (Rani et al., 2009). To understand the possible interaction of gut microbes and mosquito host, we examined the contribution of the microbe community on the fitness of the adult mosquitoes and their ability to permit development of the malaria parasite. A. stephensi mosquitoes were fed with antibiotic to sterilize their gut to study longevity, blood meal digestion, egg laying and maturation capacity, and consequently ability to support malaria parasite development. The sterilization of gut imparted reduction in longevity by a median of 5 days in male and 2 days in female mosquitoes. Similarly, the sterilization also diminished the reproductive potential probably due to increased rate of the resorption of follicles in ovaries coupled with abated blood meal digestion in gut-sterilized females. Additionally, gut sterilization also led to increased susceptibility to oocyst development upon feeding on malaria infected blood. The susceptibility to malaria parasite introduced upon gut sterilization of A. stephensi was restored completely upon re-colonization of gut by native microbes. The information provided in the study provides insights into the role of the gut-resident microbial community in various life events of the mosquito that may be used to develop alternate malaria control strategies, such as paratransgenesis.

EFFECTIVENESS OF MOSQUITO NETS TREATED WITH A TABLET FORMULATION OF DELTAMETHRIN FOR MALARIA CONTROL IN A HYPERENDEMIC TRIBAL AREA OF SUNDARGARH DISTRICT, ORISSA, INDIA

ABSTRACT A village-scale trial on the efficacy of mosquito nets treated with a tablet formulation of deltamethrin (K-OTAB®) against malaria in comparison to untreated nets or no net was conducted in Sundargarh District of Orissa, India, which is characterized by perennial transmission with Plasmodium falciparum accounting for more than 80% of malaria cases. Three villages with similar topographical and epidemiological situations were selected and randomly assigned to 3 arms of the study: treated net, untreated net, and no net. Distribution of nets, based on a sleeping pattern survey, was carried out to cover 100% of the population in treated-net and untreated-net villages. Longitudinal and cross-sectional surveys were conducted to measure malaria incidence, prevalence, and splenomegaly. Malaria incidence was reduced by 64.3% in the village with treated nets, 45.2% in the village with plain nets, and 21.4% in the control village without nets. Comparison of malaria incidence data after 1 year of intervention showed significant difference between villages with treated net vs. untreated net (P < 0.05) and treated net vs. no net (P < 0.005). The incidence of clinical attack rate due to P. falciparum was significantly lower in the population using treated nets than in those using untreated nets and no nets. However, no age-specific protective efficacy of treated nets or untreated nets was observed. A significant reduction occurred in spleen rate and parasite rate in children aged 2–9 years using treated nets or untreated nets. An overall significant reduction was found in parasite rate in the total population using treated and untreated nets as compared to nonusers.

Salivary gland transcriptome analysis in response to sugar feeding in malaria vector Anopheles stephensi

In this study, we analyzed a small scale transcriptome of salivary glands in sugar fed female mosquitoes. Thirty five percent of the transcripts could not be assigned a function. Some of them may code for salivary gland specific products involved in sugar feeding. We identified and characterized two new putative cDNAs encoding a sugar transporter and a cAMP generating DAPIT (Diabetes-Associated proteins in insulin sensitive tissues). Down regulation of these two cDNAs in response to blood feeding suggest that both AsST and AsDAPIT salivary genes may specifically be involved in the facilitation of sugar metabolism and energy production. The inability to absorb or digest sugar may cause organ failure, improper functioning of nervous system, behavioral disorder and death. Further functional characterization of theses putative transcripts is under investigation to examine their role in the mosquito salivary glands.

Determination of nitric oxide metabolites, nitrate and nitrite, in Anopheles culicifacies mosquito midgut and haemolymph by anion exchange high-performance liquid chromatography: plausible mechanism of refractoriness

BackgroundThe diverse physiological and pathological role of nitric oxide in innate immune defenses against many intra and extracellular pathogens, have led to the development of various methods for determining nitric oxide (NO) synthesis. NO metabolites, nitrite (NO2-) and nitrate (NO3-) are produced by the action of an inducible Anopheles culicifacies NO synthase (AcNOS) in mosquito mid-guts and may be central to anti-parasitic arsenal of these mosquitoes.MethodWhile exploring a plausible mechanism of refractoriness based on nitric oxide synthase physiology among the sibling species of An. culicifacies, a sensitive, specific and cost effective high performance liquid chromatography (HPLC) method was developed, which is not influenced by the presence of biogenic amines, for the determination of NO2- and NO3- from mosquito mid-guts and haemolymph.ResultsThis method is based on extraction, efficiency, assay reproducibility and contaminant minimization. It entails de-proteinization by centrifugal ultra filtration through ultracel 3 K filter and analysis by high performance anion exchange liquid chromatography (Sphereclone, 5 μ SAX column) with UV detection at 214 nm. The lower detection limit of the assay procedure is 50 pmoles in all midgut and haemolymph samples. Retention times for NO2- and NO3- in standards and in mid-gut samples were 3.42 and 4.53 min. respectively. Assay linearity for standards ranged between 50 nM and 1 mM. Recoveries of NO2- and NO3- from spiked samples (1–100 μM) and from the extracted standards (1–100 μM) were calculated to be 100%. Intra-assay and inter assay variations and relative standard deviations (RSDs) for NO2- and NO3- in spiked and un-spiked midgut samples were 5.7% or less. Increased levels NO2- and NO3- in midguts and haemolymph of An. culicifacies sibling species B in comparison to species A reflect towards a mechanism of refractoriness based on AcNOS physiology.ConclusionHPLC is a sensitive and accurate technique for identification and quantifying pmole levels of NO metabolites in mosquito midguts and haemolymph samples that can be useful for clinical investigations of NO biochemistry, physiology and pharmacology in various biological samples.

Expression profile of prophenoloxidase-encoding (acppo6) gene of Plasmodium vivax-refractory strain of Anopheles culicifacies.

ABSTRACT Anopheles culicifacies is the main vector for transmission of Plasmodium vivax malaria in the Indian subcontinent. A strain of An. culicifacies isolated from its natural niche displayed complete refractoriness to P. vivax by melanotic encapsulation of ookinetes. Prophenoloxidases are key components of the phenoloxidase cascade that leads to recognition and melanization of invading organisms. We isolated and cloned prophenoloxidase-encoding acppo6 gene of An. culicifacies and analyzed its expression profile under various regimens of immune challenge. The acppo6 was differentially expressed during various stages of larval development. The acppo6 transcription was also up-regulated in response to bacteria and Plasmodium vinckei petteri challenge. The transcript levels of the acppo6 gene were higher in naive adult refractory female mosquitoes as compared with female susceptible mosquitoes. Furthermore, the induction of acppo6 in the susceptible strain upon Plasmodium infection was negligible as compared with that of the refractory strain. The observation is suggestive of the role of acppo6 in effectuating a melanotic response in Plasmodium-incompetent naturally occurring refractory An. culicifacies strain.