Garrity_SupplementaryFig.1 A

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1 Garrity_SupplementaryFig.1 dtrp1 dtrp1 ins otential protein roducts of dtrp1ins Supplementary Figure 1 : The dtrp ins mutant. a, dtrp1 ins was created via a sitedirected insertional disruption. This loop-in created a partial duplication of exons 2 through 14 of the dtrp1 gene (duplicated region shown in gray) and the insertion of a white minigene flanked by an FRT site (arrowhead) between duplicated gene segments. In addition, a frame-shift mutation was introduced in the downstream copy of the dtrp1 locus at amino acid 183. The upstream copy of dtrp1 lacks sequences encoding the sixth transmembrane domain and C-terminus of, while the downstream copy lacks promoter sequences and the normal start codon and also contains the frame-shift mutation that is predicted to truncate the protein within the third ankyrin repeat. b, dtrp1 mutant animals lack normal protein expression. 1

2 Fraction within Interval Fraction within Interval Fraction within Interval Fraction within Interval Garrity_Supplementary Figure 2 C dtrp1 ins / + (n=9) Df4415/ + (n=8) Cha(7.4)-Gal4/ US-dTrp1 RNi (n=8) Cha(7.4)-Gal4/ + (n=5) US-dTrp1 RNi / + (n=9) appl-gal4/ US-dTrp1 RNi (n=5) appl-gal4/ + (n=4) US-dTrp1 RNi / + (n=9) D Cha(1.2)-Gal4/ US-dTrp1 RNi (n=5) Cha(1.2)-Gal4/ + (n=4) US-dTrp1 RNi / + (n=9) Supplementary Figure 2: Effects of partial loss of dtrp1 function and dtrp1 RNi knockdown on thermal preference. a-d, Distribution of animals of indicated genotype thermal gradient. 2

3 Fraction within Interval Garrity_Supplementary Figure WTab-antennae (n=8) WTab-antennae & proboscis (n=5) Supplementary Figure 3 : The proboscis contributes to cold avoidance. Distribution of animals on thermal gradient after bilateral removal of third antennal segment and aristae (Wtab-antennae) or after bilateral removal of third antennal segment and aristae as well as the proboscis (Wtab-antennae&proboscis). Removal of the proboscis increases the cold avoidance defect of antennal ablation. n= number assays. Data are mean +/- SEM. 3

4 Garrity_Supplementary Figure 4 KCl F/F=25% 2S ' Supplementary Figure 4: C neurons respond to KCl in dtrp1 mutant. Representative trace of C neuron in ins ;dtrp1 SH -Gal4;US-G-CaMP animal ( ins ). after 1µl of 3M KCl was added to the perfusion chamber. The trace and images are of the same cell shown in Figure 2j and were obtained immediately after the temperature ramp shown. a and a are grayscale and psuedocolor images of neuron prior to KCl addition, b is psuedo-color image after KCl addition. Fluorescence increased within ~3 sec of KCl addition. Maximum F/F was 1% in this cell. 4

5 Garrity_Supplementary fig.5 a c155-gal4 (control) US- (control) -6 mv Temperature Course 23 C 26 C 29 C 26 C 23 C resting potential decreases at 29 C c155-gal4; US- ( misexpression) denote ~12 sec intervals b c155-gal4 (n=7) Resting Membrane Potentials (mv) 23 C 26 C 29 C 26 C 23 C -62 +/ / / / /- 3 US- (n=8) -57 +/ / / / /- 3 c155-gal4; US-(n=13) -57 +/- 2 ND ND -51 +/ /- 2 Supplementary Figure 5: a, Representative recordings of temperature-responsive activity at the neuromuscular junctions of control and mis-expressing flies. The overall temperature courses were as in Figure 4, but the time intervals depicted in these panels are much shorter, permitting the resolution of individual Excitatory Junction Potentials (EJPs). Note that warming to 29 C slightly decreased the resting membrane potential of control muscles. b, Resting membrane potentials (in mv). Mean +/- SEM. ND= not determined; the high frequency of warmth-activated EJPs in these animals prevented accurate determination of muscle resting membrane potential. 5

6 Garrity_Supplementary fig.6 a b c d -expressing oocyte (no PT) -expressing oocyte (+ PT) control oocyte (no PT) control oocyte (+ PT) µ 1s µ 1s µ 1s µ 1s e (H48R) mutant-expressing oocyte (no PT) Supplementary Figure 6: Heat-activated currents in -expressing oocytes are -dependent and are not blocked by treatment with the calcium chelator PT. In contrast, an endogenous cold-activated current is present in all oocytes and is efficiently blocked by PT. a, Current from -expressing oocyte exposed to cooling and warming. b, Current from -expressing oocyte injected with 5 ml of 2 mm PT 3 min prior to recording, yielding an approximate final concentration of 1 mm within the oocyte. c, Current from control oocyte that was not injected with RN. d, Current from control oocyte injected with 5 ml of 2 mm PT 3 min prior to recording. : 5 micromolar Ruthenium Red. e, Current from oocyte expressing the H48R mutant channel. The paper originally reporting warmth activation of (ref. 12) unknowingly used this H48R mutant channel rather than a wild-type channel. Unlike the wild-type, the H48R mutant channel rapidly inactivates and does not respond to repeated warming. 6

7 a Garrity_Supp Fig7 % amino acid identity Drosophila melanogaster TRP1 () nopheles gambiae TRP1 () 68 edes aegypti TRP1 () Culex pipiens TRP1a () Culex pipiens TRP1b () Pediculus humanis corporis TRP1 () Tribolium castaneum TRP1 () Homo sapiens TRP1 () b NK1 NK2 NK3 NK4 Supplementary Figure 7: Highly related orthologs are present in insects that act as disease vectors and agricultural pests. mino acid identity matrix (a) and sequence alignment (b) of TRP1 proteins.,, cp TRP1b, and predicted based on genomic sequence. Culex pipiens contains a tandem array of TRP1 orthologs. Human TRP1 () is included for comparison. NK=ankyrin repeat, TM= transmembrane region, P-loop=pore region. Location of H48 in is noted with asterisk in red. NK6 NK5 NK7 NK8 H48 * NK9 NK1 NK11 NK13 NK12 NK15 NK14 NK17 NK16 TM1 TM3 TM2 TM4 TM5 P-loop TM6 7