Transcriptoma parcial de Triatoma brasiliensis: estudo do intestino médio anterior através da geração de etiquetas de sequências expressas

Abstract

Triatoma brasiliensis is a hematophagous arthropod and the main vector responsible for the transmission of the protozoa Trypanosoma cruzi in the semiarid areas of the Northeast region of Brazil. Since T. cruzi life cycle is influenced by the insect digestive tract, is of utmost importance to understand the factors that influence the insect feeding performance. In this work we report the characterization of the anterior midgut transcriptome of T. brasiliensis using Expressed Sequence Tags (ESTs). We have isolated the mRNA from the anterior midgut of 101 fifth-instar nymphs and generated a cDNA library. A total of 768 clones were randomly selected and sequenced with M13 forward and reverse primers. ESTs were edited in silico to remove low quality regions and sequences of vector, adapters and poli(A) tail using DNA Baser and SeqClean software. Sequences were also clustered using the CAP3 software and submitted to similarity searches, functional annotation and classification with blastn, blastx and blast2GO. We have obtained 311 uniques (138 contigs e 173 singletons). Some transcripts were considered as contaminants and further removed from analysis. From the remaining transcripts, 180 uniques did not present similarity with sequences deposited in databases and are, possibly, T. brasiliensis specific genes. The library exhibited a high expression level of secreted transcripts and transcripts related to energetic metabolism, protein synthesis and modification machinery, immunity and viral transcripts. A considerable variety of transcripts was isolated and these are consistent with their tissue of expression (anterior midgut) and organism. For instance, we observed transcripts coding for defensin, lysozyme, brasiliensin and digestive enzymes. Five transcripts were submitted to RT-qPCR analysis. The expression of three transcripts tends to be influenced by fasting and the other two, by blood feding. Three hours proved to be a very short time for gene expression analysis and a larger sample size and other time points after blood feding are desirable for further analysis. The characterization of the T. brasiliensis midgut transcriptome will help us to understand the feeding evolution of arthropod vectors, as well as to provide useful information to develop new strategies for vector control and to understand the processes involved in vector vertebrate host T. cruzi complex interaction.