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Radial temperature-gradient assay. (A) Radial temperature-gradient assay was performed using a 9-cm agar plate and a vial containing frozen acetic acid. Thermograph (TVS-610, Nippon Avionics) showed the surface temperature of the plate contacting a glass vial. The stable radial temperature-gradient ranging from 17° to 25° was established for at least 50 min on the agar surface. One or two animals were placed on the agar at 22°, indicated by a cross on the assay plate. (B) The assay plate was divided into three areas: (1) an area at a distance of 0–2 cm from the center of the assay plate (open area), (2) an area at a distance of 2–3 cm from the center of the assay plate (dark shading), and (3) an area at a distance of 3–4.5 cm from the center of the assay plate (light shading). (C) Animal tracks were categorized into four groups after thermotaxis assay. Typical tracks of each category are shown in a–e. (a) Animals that moved on the open area, possibly at 17°–20°, were classified as "17." (b) Animals that moved on the dark shaded area, possibly 20°, were classified as "20." (c) Animals that moved on the light shaded area, possibly at 20°–25°, were classified as "25." (d) Animals that moved back and forth between 17° and 25° were classified as "17/25." (e) Animals that moved almost randomly on the plate were also classified as "17/25," which stands for athermotactic phenotype.