C. elegans exhibits robust negative thermotaxis bias under varying conditions.
A) Diagram of assay conditions. Gradient steepness was maintained at 1.0°C/cm. Animals were placed in the middle of the gradient at the start of the assay (T_start). The temperature ranges of the gradient for varying cultivation temperatures (T_c) and T_start are shown. Conditions examined further in this study are boxed.
B–C) Thermotaxis bias (see text) exhibited by wild-type and tax-4(p678) animals for T_c=15°C (B) or T_c=20°C (C) with varying T_start. For each data point, n=105 animals; 7 independent assays. Error bars are the standard error of the mean (SEM). *, ** and *** - different from wild-type values at p<0.05, p<0.01 and p<0.001, respectively, by one-way ANOVA.

Different sensory neuron subsets are sufficient to restore negative thermotaxis bias to tax-4 mutants under different conditions.
A–C) Thermotaxis bias of transgenic tax-4(p678) mutants expressing wild-type tax-4 cDNA under the indicated cell-specific promoters (also see Materials and Methods). Conditions for each assay are indicated above each panel. Values shown are from one transgenic line for each construct; additional transgenic lines showed similar behaviors. For each data point, n=105 animals; 7 independent assays. *, ** and *** - different from tax-4 values at p<0.05, p<0.01 and p<0.001, respectively by one-way ANOVA with Bonferroni posthoc correction. Error bars are the SEM.

The AFD, AWC and ASI neurons are required in a degenerate manner to generate negative thermotaxis bias under different conditions.
A, B) Thermotaxis bias of wild-type and neuron-ablated strains (see Materials and Methods) under the indicated conditions. Wild-type data from Figure 1 are included for comparison. Data from wild-type, tax-4 and neuron-ablated strains shown in Figures 1B, 1C, 3A and 3B were obtained together on multiple days. For each data point, n=105 animals; 7 independent assays. *, ** and *** - different from wild-type values at p<0.05, p<0.01 and p<0.001, respectively by one-way ANOVA with Bonferroni posthoc correction. Conditions examined further are shaded.
C–E) Thermotaxis bias of indicated strains under the specified conditions. EC indicates that the promoter::caspase constructs were driven under different promoter combinations and present as extrachromosomal arrays (see Materials and Methods). n=105 animals; 7 independent assays. *, ** and *** - different from wild-type values at p<0.05, p<0.01 and p<0.001, respectively by one-way ANOVA with Bonferroni posthoc correction. Error bars are the SEM. Average speeds (mm/sec) of strains were: (T_c=15°C) wild-type − 0.17±0.008, AFD-ablated − 0.13±0.002, AWC-ablated − 0.17±0.007, ASI-ablated − 0.15±0.005; (T_c=20°C) wild-type − 0.18±0.005, AFD-ablated − 0.14±0.004, AWC-ablated − 0.20±0.003, ASI-ablated − 0.15±0.004.
F) Thermotaxis bias of wild-type or AFD-ablated strains expressing pkc-1(gf) under cell-specific promoters from extrachromosomal arrays. Numbers shown are from one transgenic line each. n=105 animals; 7 independent assays. * and *** - different at p<0.05 and p<0.001, respectively, between values indicated by brackets (one-way ANOVA with Bonferroni posthoc correction). Error bars are the SEM.

The ASI neurons exhibit intracellular changes in Ca²⁺ dynamics in response to temperature changes.
A) Representative example of a rising temperature stimulus (black) and Ca²⁺ events (green) in an ASI neuron expressing G-CaMP 2.2. T_c=15°C.
B–C) Heat maps and histograms showing responses of individual ASI neurons in wild-type or tax-4 mutants to a linear upward (B) or downward (C) temperature ramp at the indicated T_c. The slope of the ramps was 0.01°C/sec. Each line in the heat maps represents the responses of one neuron. Red dotted lines indicate T_c. Red bars indicate number of observed Ca²⁺ events in tax-4 mutants; responses are different at p<0.001 (Bi), and p<0.01 (Bii, Ci-ii) compared to wild-type values using a Kruskal-Wallis non-parametric test. Amplitudes of events in tax-4 mutants are not shown. A fluorescence change of >10% was considered a response. n=10 neurons each.

The operating range of ASI may be regulated by AFD-mediated neuromodulation.
A, C) Heat maps of responses of individual ASI neurons upon temperature shifts. T_c=15°C (A) or 20°C (C). T_h indicates the holding temperature at which responses were quantified.
B, D) Box-and-whisker plots of the frequency range of Ca²⁺ events upon temperature shifts to T_h in the indicated genetic backgrounds. For this quantification, a fluorescence change of >10% was considered a response. Boxes indicate the 25^th (lower boundary), 50th (median indicated by a line), and 75th percentiles (upper boundary). Whiskers show the minimum and maximum numbers of events. *, ** and *** indicate values that are different at p<0.05, 0.01 and 0.001, respectively from the corresponding wild-type values using the Kruskal-Wallis nonparametric test. n=10 neurons for each data point.
E) Average response frequencies and amplitudes of wild-type, unc-13(e51) and unc-31(e169) mutants at T_c=20°C. Averages are calculated from data shown in C.

Necessity and sufficiency of distinct subsets of thermosensory neurons under different conditions.
A) Shown are the inferred activity levels of AFD, AWC and ASI at different temperatures above T_c (Kimura et al., 2004; Chung et al., 2006; Biron et al., 2008; Kuhara et al., 2008; Ramot et al., 2008a). Filled and striped boxes indicate deterministic and stochastic Ca²⁺ events in response to temperature, respectively. All temperatures above T_c have not yet been systematically tested for AFD and AWC.
B) Neurons shown to be necessary and/or sufficient for negative thermotaxis bias are indicated below for each examined condition and gradient temperature range.