Current Biology, Volume 28

Supplemental Information

Feeding-State-Dependent Modulation of Temperature

Preference Requires Insulin Signaling

in Drosophila Warm-Sensing Neurons

Yujiro Umezaki, Sean E. Hayley, Michelle L. Chu, Hanna W. Seo, PrasunShah, and Fumika N. Hamada

Figure S1: Starvation-induced change in temperature preference behavior in

various controls, related to Figure 1, Table S1 and S2.

(A) Comparison of Tp between fed (F: fed, white box) and starved overnight (S:

starved O/N, gray box) conditions using w1118 and yw flies. The same w1118 data

employed in Figure 1B are used. The plotting pattern and the statistical analysis are

the same as in Figure 2A. **p<0.01. ****p<0.0001.

(B) Temperature preference rhythms (TPR) in w1118 during the daytime (ZT1-12)

under fed (blue line) and starved (orange line) conditions. The flies were starved for

24 hr, and their temperature preference behavior was tested at ZT 1-3, 4-6, 7-9 and

10-12 (during the daytime). As we previously reported [S1], in the normally fed w1118

flies, Tp was significantly higher at ZT 7-9 and 10-12 than at ZT 1-3, suggesting that

Tp increased during the daytime (blue line). Similarly, in the starved w1118 flies, Tp

was significantly higher at ZT 10-12 than at ZT 4-6, suggesting that Tp increased

during the daytime (orange line) (Table S1). As noted, Tp appeared to be lowest at ZT

4-6 but was not significantly different from that at ZT 1-3 (p>0.05, Dunn’s test).

Because the starved flies sustained a rhythmic Tp, the data indicate that starvation

does not eliminate the rhythmic Tp. One-way ANOVA and the Tukey-Kramer post

hoc test (fed condition) or the Kruskal-Wallis test and the Dunn’s post hoc test

(starved condition) was used (Table S1). The Tukey-Kramer test or the Dunn’s test

was used for multiple comparison tests to compare to ZT1-3 (fed conditions) and

ZT4-6 (starved conditions), respectively. Each data shows an average with sem.

*p<0.05. **p<0.01. ***p<0.001.

Pre

ferr

ed

te

mp

era

ture

(°C

)

21

25

23

27****

w1118 yw

10 14 7 7

**

22

23

24

25

1-3 4-6 7-9 10-12

ZT

12

20

15 23

169

14

12P

refe

rre

d t

em

pe

ratu

re (°C

)Fed

Starved

w1118

*****

*

F S F S

A B

w1118 Ilp1-/- Ilp2-/- Ilp3-/- Ilp4-/- Ilp5-/- Ilp7-/- Ilp2-3,5-/-

5 5 5 8 7 5 5 6 5 5 5 5 10 710 14

20

22

24

26

18

** *** *** ** ** ****28

**** *

Pre

ferr

ed

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mp

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(°C

)

24

25

26

9

6

98

Pre

ferr

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(°C

)

ilp6 LOF

1-3 4-6 7-9 10-12

ZT

A

B

******

F S F S F S F S F S F S F S F S

Figure S2: ilp1, ilp2, ilp3, ilp4, ilp5 and ilp7 are not necessary for the starvation-

induced reduction in Tp, related to Figure 2, Table S1 and S2.

(A) Comparison of Tp between fed (F: fed, white box) and starved (S: starved O/N, gray

box) conditions using w1118, ilp1-/-, ilp2-/-, ilp3-/-, ilp4-/-, ilp5-/-, ilp7-/- and ilp2-3,5-/- flies.

The same w1118 data employed in Figure 1B were used. The plotting pattern and statistical

analysis are the same as in Fig. 2A. *p<0.05. **p<0.01. ***p<0.001. ****p<0.0001.

(B) TPR in ilp6 LOF flies during the daytime (ZT1-12) under fed conditions. The Kruskal-

Wallis test and the post hoc test (Dunn’s test) was used for multiple comparison testing for

comparisons to ZT1-3 (Table S1). Each data shows an average with sem. ***p<0.001. The

ilp6 LOF flies still exhibited a normal TPR during the daytime, in which Tp was higher at

ZT 10-12 than at ZT 1-3. The data indicate that Ilp6 is required for the starvation-induced

reduction in Tp, but not TPR.

Pre

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)

25

23

26

24

22upd2Δ

A upd2Δ

*F S

6 6

B AkhGal4>uas-Kir

25

23

26

24

22AkhG4

>Kir

Kir/+

6 5 5 5

***F S F S

*

Figure S3: Upd2 and AKH are not required for the starvation-induced

reduction in Tp, related to Figure 2, Table S1 and S2.

Comparison of Tp between fed (F: fed, white box) and starved overnight (S:

starved O/N, gray box) conditions using upd2Δ (A), Kir/+ and AkhG4>Kir

(Akh-Gal4/+; uas-Kir2.1/+) (B) flies. The same Kir/+ data employed in

Figure 3A were used. The plotting pattern and statistical analysis are the

same as in Figure 2A. *p<0.05. ***p<0.001.

The leptin ortholog Unpaired 2 (Upd2) is secreted from the fat body and is

involved in the regulation of growth and energy metabolism [S2]. We found

that a upd2 deletion mutant (upd2Δ) [S3] still exhibited a higher Tp in fed

conditions than in starved conditions (Figure S3A), suggesting that Upd2 is

not necessary for the starvation-induced reduction in Tp. Furthermore,

adipokinetic hormone (AKH) is a functional homolog of glucagon in

mammals and is secreted from another peripheral tissue, the corpus

cardiacum [S4, S5]. Because AKH influences the energy reserves in the fat

body as well [S6], we also tested whether the corpus cardiacum is involved in

the starvation-induced reduction in Tp. Although Akh-Gal4-expressing cells

in the corpus cardiacum were inhibited by Kir2.1 (AkhG4>Kir) [S7], the flies

still preferred a higher temperature in fed conditions than in starved

conditions (Figure S3B), suggesting that the corpus cardiacum is not required

for the starvation-induced reduction in Tp. Therefore, our data suggest that

neither Upd2 nor AKH is involved in the starvation-induced reduction in Tp.

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)

22

26

24

28

30

32

610

Rescue

in ACs

uas-

TrpA1/+;

TrpA1ins

TrpA1-

G4/+;

TrpA1ins

6

********

21

25

23

27

19

Kir/+ R11F02>

Kir

CA

22

26

24

28**

Kir/+ R11F02>

Kir

6 58

R11F02/+

***NS

B **

*******

F S F S

****

6 5 5 5

Figure S4: Effects of modulating ACs (A) and R11F02 cold-sensing neurons (B and C) on

Tp phenotypes, related to Figure 3, Table S1 and S2.

(A) TrpA1 in ACs is sufficient for the observed temperature preference behavior. Comparison of

Tp between TrpA1ins mutants and the corresponding rescued flies. TrpA1 was expressed in ACs

in the TrpA1ins mutant background (TrpA1SH-Gal4/uas-TrpA1; TrpA1ins) and the Tp of the

rescued flies was found to be significantly different from that of the TrpA1ins mutants

(TrpA1G4/+; TrpA1ins (TrpA1SH-Gal4/+; TrpA1ins), uas-TrpA1/+; TrpA1ins (uas-TrpA1/+;

TrpA1ins)).

(B) The flies in which R11F02 cold-sensing neurons were inhibited preferred a lower

temperature than the controls. Flies in which R11F02 neurons were inhibited: R11F02>Kir

(R11F02-Gal4/+; uas-Kir2.1/+) flies, Controls: (Kir/+ and R11F02/+). The same Kir/+ data

employed in Figure 3A were used. We found that the inhibition of R11F02-Gal4 neurons caused

defects in the cold avoidance phenotype in adults, suggesting that R11F02-Gal4 cold-sensing

neurons are also required for the cold avoidance phenotype in adults.

(C) Comparison of the Tp of flies with inhibited cold- sensing neurons between fed (F: fed,

white box) and starved (S: starved O/N, gray box) conditions. The same Kir/+ and R11F02>Kir

data employed in Figure S4B (fed condition) were used. One-way ANOVA and the post hoc test

(Tukey-Kramer test) were performed to compare Tp in each genotype with fed Kir/+ flies and

flies in which cold-sensing neurons were inhibited. **p<0.01. ***p<0.001. ****p<0.0001.

Reference

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physiological homeostasis by remotely controlling insulin secretion. Cell 151, 123-137. S3. Hombria, J.C., Brown, S., Hader, S., and Zeidler, M.P. (2005). Characterisation of Upd2,

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