Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area led to i mportant findings regarding changes in vectors' physiology upon blood feedi ng and parasite infection. Mechanisms for interfering with the vectorial ca pacity of insects responsible for the transmission of diseases such as mala ria, Chagas disease and dengue fever are being devised with the ultimate go al of developing transgenic insects. A primary necessity for this goal is i nformation on gene expression and control in the target insect. Our group i s investigating molecular aspects of the interaction between Leishmania par asites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made fr om head/thorax and abdomen of sugar fed L. longipalpis for the identificati on of expressed sequence tags (EST). We applied differential display revers e transcriptase-PCR and randomly amplified polymorphic DNA-PCR to character ize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identifi ed 37 cDNAs that have shown homology to known sequences from GeneBank. Of t hese, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Thre e are putative differentially expressed cDNAs from blood fed and Leishmania -infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two se quences are homologous to Drosophila melanogaster gene products recently di scovered through the Drosophila genome initiative.