An asexual clonal line of Schmidtea mediterranea (CIW4) was maintained at 18°C in the dark. All planarians were kept in worm water; worm water consists of Instant Ocean salts (0.5 g/liter) in ultrapure water of Type 1. Animals were fed no more than once a week with “natural” (no antibiotics or hormones) liver paste made from whole calf liver (Creekstone Farms). Liver was frozen and thawed only once before feeding animals. Worms 5 to 7 mm in length were starved at least 1 week before experimentation. S. mediterranea were amputated into trunk fragments via scalpel cuts made just above and below the pharynx. Amputations were done under a dissecting microscope on a custom-made cooling Peltier plate, as previously described (34). Untreated control animals were allowed to regenerate in standard biological oxygen demand incubators (VWR) at 18°C without light, which is the normal method for the planarian field.

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