Fish Perception of Visual Stimuli

In both treatments, 10 focal fish were kept individually in separate tanks (45 cm × 28 cm × 30 cm) for at least 1 wk in visual isolation to other fish. On day nine, all fish were anesthetized with 1/5,000 solution of anesthetic (FA100, Tanabe Pharmacy Inc.) and photographed outside of the tank using a digital camera (Nikon D610, Nikon, Tokyo, Japan).
Using these photograph images and software GIMP 2.2, we created four types of photographic models: self-face and self-body (SS model), unfamiliar face and unfamiliar body (UU model), self-face and unfamiliar body (SU model), and unfamiliar face and self-body (US model; Fig. 2). Since fish had been observing their mirror image, we reversed the self-face photograph to replicate the perspective that focal fish had been observing in the mirror. All photographs were size matched to each subject fish and printed on high-quality photo paper and laminated.
Photograph models were presented to focal fish on the outside of the aquarium glass, 15 cm above the tank bottom. A white plastic sheet (45 cm × 28 cm) had been placed outside the tank glass before the laboratory lights came on, and the photograph model was shown between the glass and the white sheet. Hence, focal fish could view but not touch the photograph models. Video cameras were set 60 cm from the front of the tank and photograph models were made visible around noon for 5 min with focal fish behavior video recorded.