Protein Biochemistry and Structural Biology Laboratory, National Institute of Malaria Research (ICMR), Dwarka, New Delhi, India.
PLoS One. 2011 Apr 4;6(4):e18400. doi: 10.1371/journal.pone.0018400.
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.
致倦库蚊是印度农村地区人类疟疾的主要传播媒介，它是一个由五个亲缘种组成的复杂种系。尽管亲缘关系密切，但在这个亲缘种系复杂种中，自然选择的亚组 B 种被发现是疟疾的低效传播媒介。我们试图从转录激活的一氧化氮合酶（AcNOS）生理学方面理解媒介和非媒介致倦库蚊之间的差异，以阐明这种抗性的机制。鉴定可能赋予抗性表型的基因和基因产物之间的差异，可以促进新的疟疾传播阻断策略的发展。
方法/主要发现：我们对亲缘关系密切的敏感（种 A）和抗性（种 B）致倦库蚊亲缘种进行了研究，以描述 AcNOS 基因和基因元件的生化和分子差异及其抑制卵囊生长的能力。我们证明，在种 B 中，与种 A 相比，在间日疟原虫入侵中肠并在吸血过程中，种 B 的中肠和血淋巴中的 AcNOS 比活和亚硝酸盐/硝酸盐更高。AcNOS 的半定量 RT-PCR 和实时 PCR 数据表明，该基因在种 B 的中肠中比在种 A 中表达更为丰富，并且在血餐之后转录上调。与血餐一起用 L-NAME 进行饮食喂养可显著抑制中肠 AcNOS 活性，导致种 B 致倦库蚊卵囊产量增加。
结论/意义：我们假设，由于对间日疟原虫感染的抗性，蚊子先天细胞毒性的 NOS 上调可能有助于致倦库蚊种群的自然抗性。这种抗性蚊的先天能力可能代表了蚊子免疫系统对寄生虫的原始功能，并且可以用于理解规划有效媒介控制策略时抗性的分子基础。
