Toll signaling in Anopheline mosquitoes

The Toll pathway is a central regulator of immunity in insects and acts to induce transcription of genes that limit infection.  We aim to identify immune-functioning Toll-like receptors (TLRs), initiators of this signaling cascade, in Anopheles gambiae.  Phylogenetic analysis highlighted putative orthologs of immune-functioning Drosophila melanogaster genes, Toll and Tehao, in A. gambiae.  These genes have undergone duplication events in A. gambiae resulting in four distinct genes: Toll1A, Toll1B, Toll5A, and Toll5B.  We present here our efforts to utilize the fungus Beauveria bassiana I93-825 to activate the Toll pathway in A. gambiae to analyze immune-functioning TLRs.  The cuticle of 2-4 day old females was exposed by direct contact to an oil suspension of spores [1.24 x 10⁹ spores/mL] applied to filter paper substrate.  The expression of key regulators REL1 (transcription factor) and Cactus (inhibitor) were knocked down by RNAi (injection of 69nL dsRNA [3µg/µL]).  Overall, we found infection with B. bassiana decreased median survival of A. gambiae by 10.17 ± 1.364 days compared to controls (T-test P=0.0033).  Knockdown of cactus increased survival after infection, while knockdown of REL1 decreased survival. RT-qPCR analysis revealed 2-fold transcriptional upregulation of Cactus 4 days post exposure (2-Way ANOVA, P=0.0258).  The data show that B. bassiana can successfully probe the Toll pathway in A. gambiae, as knockdown of critical pathway components alters survivorship to fungal challenge.  Future studies aim to utilize this assay to search for putative immune-acting TLRs in A. gambiae.  Partial funding provided by the National Institutes of Health through R01-AI095842.