![A greater variation in avoidance temperatures compared with CTmax among anuran species. (A) The experimental setup for the two-chamber choice assay. Thermal probes were placed on the wall at each end of the chamber (arrows, see supplementary movie 1, Supplementary Material online). (B) Representative heat maps showing the distribution of a single Buergeria japonica tadpole during the entire assay period (480 s). A temperature difference was formed between the two chambers in the test assay. The time spent in each area during the 480-s period is shown in the representative examples (29 °C vs. 40 °C, see supplementary movie 1, Supplementary Material online). (C) A plot of the swimming speed of a B. japonica tadpole versus time in the representative assay shown in B. The background colors represent the location of tadpoles in each area (left: blue, middle: white, right: green). (D) Graphs summarizing the avoidance indexes of tadpoles for five anuran species under various temperature conditions (n = 5–15, 5–8, 6–10, 5–9, and 10–12 for B. japonica, Buergeria buergeri, Gandirana rugosa, Rhacophorus schlegelii, and Rana japonica, respectively). Avoidance indexes of tadpoles acclimated at 26 °C and 35 °C are indicated by the circles and triangles, respectively. Data points marked with asterisks indicate the temperature conditions at which the time spent in the test chamber was significantly shorter than the time spent in the control chamber (Welch’s t-test, P < 0.05). (E) The effect of warm acclimation on thermal selection in the two-chamber choice assay. Tadpoles were acclimated (Acc.) at 35 °C for 1 day before use in the assay (see supplementary movies 2 and 3, Supplementary Material online). Tadpoles reared at 26 °C were used as controls. Each data point represents the avoidance index of an individual tadpole. P-values are indicated for cases in which the avoidance index was significantly smaller for warm-acclimated tadpoles than those of control tadpoles. Temperatures in the test chamber are shown below the x-axis. (F) The effect of warm acclimation on the CTmax of tadpoles of the five anuran species. The average CTmax values of tadpoles reared at lower (basal) and higher (Acc.) temperatures are shown. Note that the acclimation temperatures of Rh. schlegelii and Ra. japonica were different from those of the other species considering their lower avoidance temperatures shown in D. The number of observations is shown in each bar. Welch’s t-test was used for statistical comparisons. (G) A significant (Spearman correlation coefficient 0.929, P = 0.022) positive correlation between CTmax and avoidance temperature in the anuran species. Note that the avoidance temperature of B. japonica tadpoles was conservatively determined as 43 °C since tadpoles showed weak avoidance at 42 °C in supplementary fig. S7B (Supplementary material online), although they did not show significant avoidance in the same test temperature in D and E. (H) Typical traces of the [Ca2+]i response to heat in DRG neurons from B. japonica. The upper and lower traces show changes in [Ca2+]i and temperature, respectively. (I, J) Distributions (I) and average thermal activation thresholds (J) of heat-responsive DRG neurons from B. japonica, B. buergeri, and Ra. japonica. P-values <0.05 are indicated (one-way ANOVA followed by post-hoc Tukey’s HSD test). Among all DRG neurons observed, 12 of 47 (four individuals), 6 of 26 (four individuals), and 39 of 83 (three individuals) were heat-responsive in B. japonica, B. buergeri, and Ra. japonica, respectively. The average thermal activation thresholds of heat-responsive DRG neurons in B. japonica, B. buergeri, and Ra. japonica were 43.3 °C ± 1.0 (n = 12), 41.7 °C ± 1.7 (n = 6), and 36.9 °C ± 0.6 (n = 39), respectively.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/mbe/39/9/10.1093_molbev_msac180/2/msac180f2.jpeg?Expires=1750677290&Signature=B~pfP6p0EfACxxV4TjlxktDlfzfNmd1cIk55WAiF7YlMr5UdqaHoDN6B~~kiCyHfsg2F5rvSZxainZlzK94JDD0mBYs73i5SeZOYvtV4-3RKcPD~e7NAOeXURiLWVTA-3c9RwSbaQlJyjHs8bG58RNpsT-ahSkmOWS9eHCd15YK0xgCf5lyPzXM~wS6uo4a~TlPNe4aTIav5QwFs9Km~pin0aSfDtzKQlqbnumviD2xRDsVC6pPp1Kk9Ns2IA66iyDSNghvQ4Qnb4mXuyprEvsoeLWGK2-evjoENW-QEN8~uIpRAXIi5a~skm6WXj-MdWc~R5ilOeoSIPAea7Ft2Pg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
A greater variation in avoidance temperatures compared with CTmax among anuran species. (A) The experimental setup for the two-chamber choice assay. Thermal probes were placed on the wall at each end of the chamber (arrows, see supplementary movie 1, Supplementary Material online). (B) Representative heat maps showing the distribution of a single Buergeria japonica tadpole during the entire assay period (480 s). A temperature difference was formed between the two chambers in the test assay. The time spent in each area during the 480-s period is shown in the representative examples (29 °C vs. 40 °C, see supplementary movie 1, Supplementary Material online). (C) A plot of the swimming speed of a B. japonica tadpole versus time in the representative assay shown in B. The background colors represent the location of tadpoles in each area (left: blue, middle: white, right: green). (D) Graphs summarizing the avoidance indexes of tadpoles for five anuran species under various temperature conditions (n = 5–15, 5–8, 6–10, 5–9, and 10–12 for B. japonica, Buergeria buergeri, Gandirana rugosa, Rhacophorus schlegelii, and Rana japonica, respectively). Avoidance indexes of tadpoles acclimated at 26 °C and 35 °C are indicated by the circles and triangles, respectively. Data points marked with asterisks indicate the temperature conditions at which the time spent in the test chamber was significantly shorter than the time spent in the control chamber (Welch’s t-test, P < 0.05). (E) The effect of warm acclimation on thermal selection in the two-chamber choice assay. Tadpoles were acclimated (Acc.) at 35 °C for 1 day before use in the assay (see supplementary movies 2 and 3, Supplementary Material online). Tadpoles reared at 26 °C were used as controls. Each data point represents the avoidance index of an individual tadpole. P-values are indicated for cases in which the avoidance index was significantly smaller for warm-acclimated tadpoles than those of control tadpoles. Temperatures in the test chamber are shown below the x-axis. (F) The effect of warm acclimation on the CTmax of tadpoles of the five anuran species. The average CTmax values of tadpoles reared at lower (basal) and higher (Acc.) temperatures are shown. Note that the acclimation temperatures of Rh. schlegelii and Ra. japonica were different from those of the other species considering their lower avoidance temperatures shown in D. The number of observations is shown in each bar. Welch’s t-test was used for statistical comparisons. (G) A significant (Spearman correlation coefficient 0.929, P = 0.022) positive correlation between CTmax and avoidance temperature in the anuran species. Note that the avoidance temperature of B. japonica tadpoles was conservatively determined as 43 °C since tadpoles showed weak avoidance at 42 °C in supplementary fig. S7B (Supplementary material online), although they did not show significant avoidance in the same test temperature in D and E. (H) Typical traces of the [Ca2+]i response to heat in DRG neurons from B. japonica. The upper and lower traces show changes in [Ca2+]i and temperature, respectively. (I, J) Distributions (I) and average thermal activation thresholds (J) of heat-responsive DRG neurons from B. japonica, B. buergeri, and Ra. japonica. P-values <0.05 are indicated (one-way ANOVA followed by post-hoc Tukey’s HSD test). Among all DRG neurons observed, 12 of 47 (four individuals), 6 of 26 (four individuals), and 39 of 83 (three individuals) were heat-responsive in B. japonica, B. buergeri, and Ra. japonica, respectively. The average thermal activation thresholds of heat-responsive DRG neurons in B. japonica, B. buergeri, and Ra. japonica were 43.3 °C ± 1.0 (n = 12), 41.7 °C ± 1.7 (n = 6), and 36.9 °C ± 0.6 (n = 39), respectively.
