Expressão diferencial de genes relacionados à resposta imune do mosquito Anopheles (Nyssorhynchus) darlingi

Abstract

The Anopheles (Nyssorhynchus) darlingi Root, 1926 is considered the primary ma-laria vector in South America, with the highest records in the Amazon region. In recent decades, studies have shown that immune responses in insects such as Anopheles gambiae in, vector of malaria are largely regulated by pathways Toll and Imd (immunodeficiency), through the NF-kappa β transcription factors and Rel1 Rel2 which are controlled by negative regulators Cactus and Cas-par. Alternatives for detection and quantification of gene expression at the genomic level have been carried out through analysis of cDNA fragments amplified by the method of Polymerase Chain Reaction in Real Time (qRT-PCR). The present study examined for the first time, quantitative mRNA expression of Rel1, Rel2, Caspar and Cactus genes related to the immune system in fed adult females and females not fed Anopheles darlingi, using GAPDH and β-actin markers, as refer-ence genes. Anopheles darlingi larvae were collected in the vicinity of Manaus, Amazonas, which were identified after being kept in the laboratory until adult-hood. Adult females were fed with blood meal, and after 4, 8, 14 and 24 hours were frozen at negative 80 oC. The extraction and purification of RNA were ob-tained with the Qiagen® kit and the complementary strand of mRNA (cDNA) with a Promega® kit. Primers of Rel1, Rel2, Caspar and Cactus genes were designed with the "Gene Runer" Program. The protocol for qRT-PCR was according to SYBR ® Green (Applied Biosystems ®) and the amplifications carried out in the ABI-7500 thermocycler ™ Real-Time PCR System, Applied Biosystems ®. The analyzes showed that the expression levels of Rel1, Rel2, Caspar and Cactus genes varied widely among these genes during 4, 8, 14 and 24 hours after blood feeding compared to adult females control not fed (0 h). The Rel1 gene, compared to the controls, showed higher expression level compared to Rel2, Caspar and Cactus genes after the blood meal, in the intervals of 8 and 14 hours. This differ-ence in the differential gene expression of these samples may be related to their immune status. These findings are important for understanding the response of the immune system of A. darlingi, and useful to respond other questions and perspec-tives about mosquito control.