Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline in the mosquito, producing nitric oxide which is toxic to the parasite ( Clayton, Dong & Dimopoulos, 2014). Silencing of the TEP1, LRIM1 or APL1 gene abolishes parasite melanization and converts refractory or non-compatible Anopheles strain into a susceptible one. quadrinnulatus ( Habtewold et al., 2008). gambiae ( Blandin et al., 2004 Fraiture et al., 2009) and An. The precise mode of action of this TEP1-associated complex is not known but it has been implicated in the vector competence of An. In the hemolymph of the mosquito, TEP1, leucine-rich repeat immune protein 1 (LRIM1) and Anopheles- Plasmodium-responsive leucine-rich repeat 1 (APL1) protein combine to form a stable complex ( Fraiture et al., 2009 Povelones et al., 2011). Thioester-containing protein 1 (TEP1) has been shown to mediate anti- Plasmodium responses in various mosquito species ( Clayton, Dong & Dimopoulos, 2014 Jaramillo-Gutierrez et al., 2009). The qRT-PCR is used as a tool to validate expression data from microarray analyses and to detect changes in the expression levels of target genes. The immunological changes caused by Plasmodium infection at the transcriptome level can be captured in microarray gene expression and quantitative reverse transcription PCR (qRT-PCR) analyses ( Aguilar et al., 2005). Understanding of this immunity complex may lead to development of novel approaches for controlling malaria transmission, such as transgenic mosquitoes and inhibition of parasite development in the mosquito. berghei infection.Īdvances in molecular biology and gene expression techniques have enabled researchers to identify genes involved in the immunological response of Anopheles mosquitoes towards Plasmodium infection ( Chen, Mathur & James, 2008 Volohonsky et al., 2017).
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dirus were found to be significantly induced after P. Prior to that, the elongation factor 1-alpha ( EF1), actin 1 ( Act) and ribosomal protein S7 ( S7) genes were validated for their suitability as a set of reference genes. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. This may lead to erroneous quantification of expression levels. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Two anti- Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1) and nitric oxide synthase (NOS), play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. Quantitative reverse transcription PCR (qRT-PCR) has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion.