Phylogenetic characterization and prevalence of Spirobacillus cienkowskii : a red - pigmented , 1 spiral - shaped bacterial pathogen of the freshwater Cladoceran , Daphnia 2 3 Running title : Phylogeny and

1 2 Microscopic examination of the hemolymph from diseased daphniids in 17 lakes in southwestern 3 Michigan and 5 rock pools in southern Finland revealed the presence of tightly coiled bacteria that 4 bore striking similarities to the drawings of a morphologically unique pathogen, Spirobacillus 5 cienkowskii, first described by Elya Metchnikoff more than 100 years ago. The uncultivated 6 microbe was identified as a deeply branching member of the delta-Proteobacteria through 7 phylogenetic analyses of two conserved genes: the 16S rRNA-encoding gene (rrs) and the b-8 subunit of topoisomerase (gyrB). Fluorescence in situ hybridization confirmed that the rRNA 9 gene sequence originated from bacteria with the tightly coiled morphology. Microscopy and PCR 10 amplification with pathogen-specific primers confirmed infections by this bacterium in four 11 species of Daphnia: D. dentifera, D. magna, D. pulicaria, and D. retrocurva. Extensive field 12 surveys reveal that this bacterium is widespread geographically and able to infect many different 13 Cladoceran species. In a survey of populations of D. dentifera in lakes in Michigan, USA, we 14 found the bacterium in 17 of 18 populations studied. In these populations, 0 to 12% of the 15 individuals were infected, with an average of 3% during midsummer and early autumn. 16 Infections were less common in rock pool populations of D. magna in southern Finland, where the 17 pathogen was found in 5 of 137 populations. The broad geographic distribution, wide host range, 18 and high virulence of S. cienkowskii suggest it plays an important role in the ecology and 19 evolution of daphniids.