Exploring the loss of blood-feeding within the Sabethini tribe by analyzing evolutionary relationships

Perhaps the most important behavior exhibited by mosquitoes  is blood-feeding. However, more than 150 species in three genera are unable to blood-fed. Due to the lack of genetic data available for most mosquitoes, whether this represents a single event in the evolutionary history of mosquitoes, or multiple independent events, is unknown. Advances in sequencing methods have enabled extensive sampling of non-model mosquito species, but the best way to analyze such data has been untested. To simultaneously address the times blood-feeding has been lost, and the best methods with which to analyze orthologous sequence data for non-model organisms, we evaluated two bioinformatic pipelines and sequence data from 59 mosquito species. We hypothesize that the loss of blood-feeding was a single evolutionary event within the common ancestor of the three genera that do not blood-feed, Malaya, Topomyia, and Toxorynchites. To test this hypothesis, we generated alignments of orthologous sequences from two bioinformatic pipelines, Orthograph and HybPiper. From resulting sequence alignments, we will generate phylogenetic trees to evaluate how many times blood-feeding has been lost in mosquitoes. We compared these pipelines in the number of orthologous sequences each recovered, as well as support values from phylogenetic analyses of sequence alignments. Using the outputs of the pipelines, we will discuss support for a single loss of blood-feeding in mosquitoes, which gave rise to non-biting lineages.