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icroarray Analysis of Anopheles gambiae Gene Expression in Response to Chloroquine Treatment

P. Abrantes1, G. Dimopoulos2, V.E. do Rosário1 and H. Silveira1
Centro de Malária e Outras Doenças Tropicais/ UEI de Malária, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa, Portugal
2W. Harry Feinstone Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, USA

Although knowledge of the mosquito immune response and mosquito-host interaction mechanisms has recently improved, very little is known about the impact of the antimalarial chloroquine on mosquito immunity. In order to further characterize that impact and to help clarify the poorly characterized mechanism of action of chloroquine, cDNA microarrays were used to analyze global gene expression.

Anopheles gambiae spotted 20K EST microarrays comprising 20,000 EST clone inserts from various developmental and tissue specific EST libraries were used to examine mosquito midgut and cell line gene expression in response to a blood meal containing chloroquine. Microarray slides were scanned on a TECAN LS 300 SCANNER and images processed by means of the Chipskipper software version 1.1. Intensities of hybridized targets and associated background were estimated. For the within-array normalization, spots with bad quality were removed and background was subtracted. Normalized ratio was log2 transformed and LOESS (locally weighted polynomial regression) regression was applied. For the between-array normalization, a centering-scale step was used. Final data set was processed with GEPAS tools.

Approximately 4 % of the total ESTs analyzed was expressed at least two-fold above or below the controls. Affected genes represent a variety of functional classes that include immunity, oxidative stress, transporters, cytoskeleton, replication, transcription and translation, as well as a number of unknown genes. Five immunity-related genes (two serine proteases, two serpins and one prophenoloxidase) and three oxidative stress-related genes (two glutathione-s-transferases and one thioredoxin) were analyzed by quantitative real-time PCR in order to validate microarray results.

This work was supported by POCTI/MGI/44905/2002. P. Abrantes was supported by F.C.T grant SFRH/BD/6346/2001.