Asexual Planarian Clonal Line Maintenance and Microbial Analysis

The S. mediterranea asexual clonal line ClW4^{24 (link)} was maintained at 18 °C in water. The water was first filtered through charcoal and ceramics with pores of 0.2 µm (manufactured by Fairey Industrial Ceramics Limited) and through a membrane of 0.2 µm (Thermo Scientific Nalgene Filtration Products) for 10 years. Microbiological analysis of the filtered water was performed by inoculation of 5% sheep blood-enriched Columbia agar plates (bioMérieux, Marcy l’étoile, France) with 25, 50, or 100 µL of filtered water. Inoculated plates were then incubated at 19, 28, or 37 °C for 4 days. Any bacteria were detected.S. mediterranea were fed once per week with homogenized calf liver (batch 20118-5814, origin: Saprimex (a local supermarket), 13310 St Martin de Crau) and then starved 2 weeks prior to experiments. In some experiments, worms were starved for 1 or 4 weeks. The bacterial constituents of homogenized calf liver were defined by inoculation of 5% sheep blood-enriched Columbia agar plates (bioMérieux, Marcy l’étoile, France) with 25, 50, or 100 µL of homogenized calf liver. Inoculated plates were then incubated at 19, 28, or 37 °C for 1, 2, 3, and 4 days.