Ecological Archives E096-168-A1
Kunio Takatsu and Osamu Kishida. 2015. Predator cannibalism can intensify negative impacts on heterospecific prey. Ecology 96:1597–1608. http://dx.doi.org/10.1890/14-1616.1
Appendix A. Methods of collection, keeping, and manipulation of experimental animals.
A1: Collection and keeping of eggs of Hynobius retardatus salamanders and Rana pirica frogs
Ninety egg clusters of H. retardatus salamanders and 10 egg masses of R. pirica frogs were collected from several ponds in the Teshio Experimental Forest of Hokkaido University, Hokkaido, Japan, in mid-May 2012. Each of the 10 frog egg masses was kept in a separate 22 L semi-transparent polypropylene tank (51.3 cm × 37.2 cm × 16.6 cm high) filled with 5 L of aged tap water, and the tanks were placed in our experimental room, which was maintained at 17 °C with a natural light–dark (14h/10h) regime. The eggs had started to hatch by late May. After the frog tadpoles hatched, we put eight pieces of rabbit chow (dry weight: 1.6 g) into each tank as food for the frog tadpoles every 2 days, and we also exchanged the water every 2 days. We cultured the hatchling frog tadpoles under the conditions described above two weeks (i.e., until the start of the experiment). Each of the 90 salamander egg clusters was placed separately in a colander (9 cm × 6.5 cm × 5 cm high), and the colanders were placed in 13 L semi-transparent polypropylene tanks (43.6 cm × 28.4 cm × 14.1 cm high; 20 colanders per tank) filled with 10 L of aged tap water. Then the tanks were placed in a refrigerator and maintained at 3 °C under natural light–dark (14h/10h) conditions.
A2: Method to manipulate timing of hatching of salamanders
We obtained the early and late salamander hatchlings (hatch time difference, 1 week) by manually controlling the water temperature experienced by the embryos in a single egg as described below (i.e., full or half sibs). This method allowed us to preclude possible confounding genetic effects on cannibalism.
To obtain both early and late hatchlings from a single egg cluster for the large-variation treatment, each egg cluster was split in half before the larvae hatched. Then one half of the cluster was kept in an experimental room maintained at 17 °C to accelerate hatching, and the other half was kept in a refrigerator maintained at 3 °C to delay hatching by 1 week relative to the early-hatch group. The early-hatch group hatched two weeks after the frog tadpoles had hatched.