Annotation of the Toll and IMD Pathways in Anopheles stephensi

The Toll and Immune Deficiency (IMD) pathways are central regulators of immunity in arthropods, and in mosquitoes limit infection by viruses, bacteria, fungi, and eukaryotes. Identification of the molecular make-up of these signaling pathways is thus a critical first step in the molecular analysis of vector competence in any arthropod vector species. We describe here these intracellular signaling cascades in the recently sequenced genome of the Indo-Pakistan malaria vector, Anopheles stephensi Indian strain. An extensive literature search was used to compile a list of 19 Toll and 21 IMD pathway components in D. melanogaster, and their coding sequences were retrieved from FlyBase. These sequences were used to identify 1:1 orthologs using best reciprocal blast hits with tblastn against An. stephensi scaffolds and putative transcripts available from VectorBase. Automatic gene annotations were refined manually using Artemis software, based on amino acid alignments produced with ClustalW. Seventeen of the 19 Toll pathway components and 20 of the 21 molecules in the IMD pathway were identified in An. stephensi. Overall, we observed high sequence conservation between orthologs in the two anopheline species, with percent identities varying between 51-91% at cDNA and 47-100% at protein levels. Of note, the inhibitor Pirk was not identified in the anopheline genomes, suggesting differences in IMD pathway inhibition in mosquitoes. Taken together, this study has greatly improved the annotation of the Toll and IMD pathway components in mosquitoes (updated gene lists available), and will enable future immunological studies in An. stephensi.