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
te
mp
era
ture
(°C
)
24
25
26
9
6
98
Pre
ferr
ed
te
mp
era
ture
(°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
ferr
ed
te
mp
era
ture
(°C
)
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.
Pre
ferr
ed
te
mp
era
ture
(°C
)
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
S1. Kaneko, H., Head, L.M., Ling, J., Tang, X., Liu, Y., Hardin, P.E., Emery, P., and
Hamada, F.N. (2012). Circadian Rhythm of Temperature Preference and Its Neural Control in Drosophila. Current biology : CB 22, 1851-1857.
S2. Rajan, A., and Perrimon, N. (2012). Drosophila cytokine unpaired 2 regulates
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,
a Drosophila JAK/STAT pathway ligand. Developmental biology 288, 420-433. S4. Isabel, G., Martin, J.R., Chidami, S., Veenstra, J.A., and Rosay, P. (2005). AKH-
producing neuroendocrine cell ablation decreases trehalose and induces behavioral changes in Drosophila. Am J Physiol Regul Integr Comp Physiol 288, R531-538.
S5. Lee, G., and Park, J.H. (2004). Hemolymph sugar homeostasis and starvation-induced
hyperactivity affected by genetic manipulations of the adipokinetic hormone-encoding gene in Drosophila melanogaster. Genetics 167, 311-323.
S6. Kim, S.K., and Rulifson, E.J. (2004). Conserved mechanisms of glucose sensing and
regulation by Drosophila corpora cardiaca cells. Nature 431, 316-320. S7. Baines, R.A., Uhler, J.P., Thompson, A., Sweeney, S.T., and Bate, M. (2001). Altered
electrical properties in Drosophila neurons developing without synaptic transmission. The Journal of neuroscience : the official journal of the Society for Neuroscience 21, 1523-1531.