The ecophysiology of parasites may play a crucial role in driving epidemics, yet the ecology of parasites is rarely considered, particularly in the case of parasites with non-human hosts. Here, we focus on Spirobacillus cienkowskii, a bacterium in the delta-Proteobacteria subphylum that infects Daphnia and other freshwater Cladocera.  S. cienkowskii has received recent attention due to its virulent effects on Daphnia populations. However, the role of the ecophysiology of this parasite in driving infection patterns has been ignored, largely because it has proven difficult to culture in the laboratory. While S. cienkowskii has distinct coloration and unique spiral morphology, it demonstrates morphological plasticity, making it difficult to definitively identify microscopically. In this study, we have combined cultivation efforts with molecular techniques targeting the 16S rRNA gene to characterize the ecophysiology of S. cienkowskii. Liquid media were selected based on their osmotic similarity to Daphnia hemolymph, and their previous use for culturing parasites of other freshwater crustaceans (e.g., Aeromonas hydrophila). Additionally, oxygen partial pressures were varied in consideration of the possible decrease in oxygen within the organism. PCR analyses were used to evaluate the success of the various culturing techniques without depending on cell morphology. Further, analysis of environmental samples was used to elucidate the seasonal cycles of S. cienkowskii physiology. 

Results/Conclusions

PCR analysis of preliminary culturing attempts suggests that S. cienkowskii demonstrated the most growth on tryptic soy broth (TSB) incubated at 4°C. Surprisingly, cultures incubated at higher temperatures demonstrated very limited growth. Environmental samples analyzed with previously developed PCR primers suggest that S. cienkowskii are present as planktonic cells as well as within the Daphnia hosts. In fact, DNA samples derived from the water surrounding infected Daphnia apparently contain as many or more S. cienkowskii 16S rRNA gene sequences than DNA derived from infected organisms.  Other environmental samples included in the analysis include lake sediment, Daphnia resting eggs, and fish fecal pellets. Molecular analyses targeting this fastidious bacterial parasite enhanced culturing attempts by utilizing morphology-independent assessment of culture success. In addition, environmental sample analyses contributed insight to the epidemiology of the Spirobacillus-Daphnia interaction by elucidating the non-infectious aspects of S. cienkowskii ecology. The data presented here regarding low optimal growth temperatures have led to re-examination of the role of temperature in dictating S. cienkowskii epidemics. Overall, this study demonstrates the importance of considering the ecophysiology of the parasite to understanding natural host-parasite interactions.