ALBERS, Melissa A*; BRADLEY, Timothy J; University of California, Irvine; University of California, Irvine: A Phylogenetic Analysis of the Evolution of Saline-Tolerance in Mosquito Larvae

Saline-tolerance has evolved in mosquito larvae several times in various genera. The Ochlerotatus genus is of interest because there are both freshwater and saline-tolerant forms within this group and both forms are osmoregulators. The physiological ability to cope with saline water versus freshwater in the Ochlerotatus mosquitoes is associated with a morphological difference in the rectum, an organ that serves an osmoregulatory function in these insects. The saline-tolerant forms have a two-part rectum while the freshwater obligates have a one-part rectum. Of the many species of this genus whose saline-tolerance has not been described, we have collected several in their larval stages. Our study includes 16 Ochlerotatus species collected in North America and one closely related and saline-tolerant New Zealand Opifex species. For each species we performed a morphological analysis of the larval recta. To determine saline-tolerance, mosquito larvae were gradually taken from dilute water to water adjusted up to 1000 mOsm using InstantOcean. Those with high survival to pupation in solutions up to 1000 mOsm were determined to be saline-tolerant. We then looked at the evolutionary patterns of saline-tolerance in this genus by mapping this character onto a phylogenetic tree. We constructed this tree by PCR amplifying and direct sequencing of the 18s rDNA gene, the D2 region of the 28s rDNA gene and two mitochondrial genes, COI and COII for each species. We then used PAUP 4.0 to construct and analyze a phylogenetic tree. Our tree does not suggest a single point when the saline-tolerant and freshwater forms split. Instead, it provides evidence that saline-tolerance evolved once from a freshwater ancestor and was subsequently lost or arose multiple times.