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Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development.

ABSTRACT: A cDNA encoding a lysozyme was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) from females of the malaria vector Anopheles dirus A (Diptera: Culicidae). The 623 bp lysozyme (AdLys c-1) cDNA encodes the 120 amino acid mature protein with a predicted molecular mass of 13.4 kDa and theoretical pI of 8.45. Six cysteine residues and a potential calcium binding motif that are present in AdLys c-1 are highly conserved relative to those of c-type lysozymes found in other insects. RT-PCR analysis of the AdLys c-1 transcript revealed its presence at high levels in the salivary glands both in larval and adult stages and in the larval caecum. dsRNA mediated gene knockdown experiments were conducted to examine the potential role of this lysozyme during Plasmodium berghei infection. Silencing of AdLys c-1 resulted in a significant reduction in the number of oocysts as compared to control dsGFP injected mosquitoes.

SUBMITTER: Lapcharoen P

PROVIDER: S-EPMC3200504 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development.

Lapcharoen Parichat P Komalamisra Narumon N Rongsriyam Yupha Y Wangsuphachart Voranuch V Dekumyoy Paron P Prachumsri Jetsumon J Kajla Mayur K MK Paskewitz Susan M SM

Developmental and comparative immunology 20110628 1

A cDNA encoding a lysozyme was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) from females of the malaria vector Anopheles dirus A (Diptera: Culicidae). The 623 bp lysozyme (AdLys c-1) cDNA encodes the 120 amino acid mature protein with a predicted molecular mass of 13.4 kDa and theoretical pI of 8.45. Six cysteine residues and a potential calcium binding motif that are present in AdLys c-1 are highly conserved relative to those of c-type lysozymes found in oth ...[more]

PMID: 21741400

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Project description:BACKGROUND: Anopheles culicifacies, the main vector of human malaria in rural India, is a complex of five sibling species. Despite being phylogenetically related, a naturally selected subgroup species B of this sibling species complex is found to be a poor vector of malaria. We have attempted to understand the differences between vector and non-vector Anopheles culicifacies mosquitoes in terms of transcriptionally activated nitric oxide synthase (AcNOS) physiologies to elucidate the mechanism of refractoriness. Identification of the differences between genes and gene products that may impart refractory phenotype can facilitate development of novel malaria transmission blocking strategies. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a study on phylogenetically related susceptible (species A) and refractory (species B) sibling species of An. culicifacies mosquitoes to characterize biochemical and molecular differences in AcNOS gene and gene elements and their ability to inhibit oocyst growth. We demonstrate that in species B, AcNOS specific activity and nitrite/nitrates in mid-guts and haemolymph were higher as compared to species A after invasion of the mid-gut by P. vivax at the beginning and during the course of blood feeding. Semiquantitative RT-PCR and real time PCR data of AcNOS concluded that this gene is more abundantly expressed in midgut of species B than in species A and is transcriptionally upregulated post blood meals. Dietary feeding of L-NAME along with blood meals significantly inhibited midgut AcNOS activity leading to an increase in oocyst production in An. culicifacies species B. CONCLUSIONS/SIGNIFICANCE: We hypothesize that upregulation of mosquito innate cytotoxicity due to NOS in refractory strain to Plasmodium vivax infection may contribute to natural refractoriness in An. culicifacies mosquito population. This innate capacity of refractory mosquitoes could represent the ancestral function of the mosquito immune system against the parasite and could be utilized to understand the molecular basis of refractoriness in planning effective vector control strategies.

Project description:BackgroundThe malaria vector Anopheles funestus is increasingly recognized as a dominant vector of residual transmission in many African settings. Efforts to better understand its biology and control are significantly impeded by the difficulties of colonizing it under laboratory conditions. To identify key bottlenecks in colonization, this study compared the development and fitness characteristics of wild An. funestus from Tanzania (FUTAZ) and their F1 offspring during colonization attempts. The demography and reproductive success of wild FUTAZ offspring were compared to that of individuals from one of the only An. funestus strains that has been successfully colonized (FUMOZ, from Mozambique) under similar laboratory conditions.MethodsWild An. funestus (FUTAZ) were collected from three Tanzanian villages and maintained inside an insectary at 70-85% RH, 25-27 °C and 12 h:12 h photoperiod. Eggs from these females were used to establish three replicate F1 laboratory generations. Larval development, survival, fecundity, mating success, percentage pupation and wing length were measured in the F1 -FUTAZ offspring and compared with wild FUTAZ and FUMOZ mosquitoes.ResultsWild FUTAZ laid fewer eggs (64.1; 95% CI [63.2, 65.0]) than FUMOZ females (76.1; 95% CI [73.3, 79.1]). Survival of F1-FUTAZ larvae under laboratory conditions was low, with an egg-to-pupae conversion rate of only 5.9% compared to 27.4% in FUMOZ. The median lifespan of F1-FUTAZ females (32 days) and males (33 days) was lower than FUMOZ (52 and 49 for females and males respectively). The proportion of female F1-FUTAZ inseminated under laboratory conditions (9%) was considerably lower than either FUMOZ (72%) or wild-caught FUTAZ females (92%). This resulted in nearly zero viable F2-FUTAZ eggs produced. Wild FUTAZ wings appear to be larger compared to the lab reared F1-FUTAZ and FUMOZ.ConclusionsThis study indicates that poor larval survival, mating success, low fecundity and shorter survival under laboratory conditions all contribute to difficulties in colonizing of An. funestus. Future studies should focus on enhancing these aspects of An. funestus fitness in the laboratory, with the biggest barrier likely to be poor mating.

Dataset Information

Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development.

Publications

Investigations on the role of a lysozyme from the malaria vector Anopheles dirus during malaria parasite development.

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