of 15
Letter
https://doi.org/10.1038/s41586-018-0634-9
A gut microbial factor modulates locomotor
behaviour in
Drosophila
Catherine
e. Schretter
1
*
, Jost Vielmetter
2
, Imre Bartos
3
, Zsuzsa Marka
3
, Szabolcs Marka
3
, Sulabha Argade
4
& Sarkis K. Mazmanian
1
*
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
2
Protein Expression Center, Beckman Institute, California Institute of Technology, Pasadena,
CA, USA.
3
Department of Physics, Columbia University, New York, NY, USA.
4
GlycoAnalytics Core, University of California, San Diego, CA, USA.
*
e-mail:
cschrett@caltech.edu
;
sarkis@caltech.edu
NATURE|
www.nature.com/nature
SUPPLEMENTARY INFORMATION
https://doi.org/10.1038/s41586-018-0634-9
In the format provided by the authors and unedited.
© 2018 Springer Nature Limited. All rights reserved.
Sample Size and Statistics
Figure 1b
i
Analysis
(
1b,
1i): Average speed
calculated over the 10 min. testing period
.
(1d): Average bout length
calculated
over the 10 min. testing period
.
(1e): Average pause length
calculated over the 10 min. testing
period
.
(1f): Number
of bouts
over 10 min. testing period
.
(1g): Daily crossings
sampled ever
y
min.
per day.
(1h): Stance linearity index
,
see ref. 64 for calculations
All are biological replicates
assessed with a two
-
tailed test
and d
ata are representat
ive of at least 3 independent trials
for each experiment
.
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
1b
Conv
1
36
Kruskal
-
Wallis test
(P < 0.0001)
Kruskal
-
Wallis test
(
P =
0.0026
)
Kruskal
-
Wallis test
(
P < 0.0001
)
Kruskal
-
Wallis test
(
P = 0.6685
)
Kruskal
-
Wallis test
(
P = 0.0042
)
Kruskal
-
Wallis test
(
P = 0.0057
)
1 v 2
0.0070
Ax
2
36
1 v 3
0.0024
L.p
3
35
1 v 4
0.9999
L.b
4
36
2 v 3
0.9999
2 v 4
0.0112
1d
1e
1f
1g
1h
Conv
Ax
L.p
L.b
Conv
Ax
L.p
L.b
Conv
Ax
L.p
L.b
Conv
Ax
L.p
L.b
Conv
Ax
L.p
L.b
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
1
2
3
4
32
36
22
20
32
36
22
20
32
36
22
20
8
8
8
8
6
7
5
5
3 v 4
1 v 2
1 v 3
1 v 4
2 v 3
2 v 4
3 v 4
1 v 2
1 v 3
1 v 4
2
v 3
2 v 4
3 v 4
1 v 2
1 v 3
1 v 4
2 v 3
2 v 4
3 v 4
1 v 2
1 v 3
1 v 4
2 v 3
2 v 4
3 v 4
0.0040
0.0014
0.2111
0.9999
0.9999
0.2814
0.9999
< 0.0001
0.0034
0.9999
0.9999
0.0335
0.3321
0.0100
0.1980
0.9999
0.9999
0.0335
0.4716
0.0063
0.8248
0.9999
0.6907
0.0508
0.9999
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
1i
Conv
1
25
Kruskal
-
Wallis test
(
P = 0.0016
)
1 v 2
0.2846
Ax
2
29
1 v 3
0.0470
L.p
3
24
1 v 4
0.9999
L.b
4
35
2 v 3
0.9999
2 v 4
3 v 4
0.0453
0.0047
Figure 2a
f
Analysis
(2a
d, 2f): Average speed
.
(2e):
Carbohydrate analysis of whole fly homogenates measured using
HPAEC
-
PAD. E
ach
sample contains
5 flies
.
All are biological replicates assessed with a two
-
tailed test and data
are
representative of at least 2
independent trials
for each experiment
, except for Figure 2e in which one trial was performed
.
Figure
Condition
Measurement
#
Sample
Size
Statistical Test
Comparison
P
-
value
2a
Ax
1
57
Kruskal
-
Wallis
test
(P = 0.0004
)
Kruskal
-
Wallis
test
(
P < 0.0001
)
Kruskal
-
Wallis
test
(
P = 0.0014
)
Kruskal
-
Wallis
test
(
P = 0.0003
)
Mann
-
Whitney
test
Mann
-
Whitney
test
Mann
-
Whitney
test
Mann
-
Whitney
test
Mann
-
Whitney
test
1 v 2
0.0004
L.b
2
42
1 v 3
0.0294
L.b
CFS
3
36
1 v 4
0.9999
L.b
HK
4
24
2 v 3
0.9999
2 v 4
0.1188
2b
2c
2d
2e
Conv
Ax
Xi*
Ax
L.b
CFS
Xi*
Ai*
Ax
L.b
L.b
xylA
Ax
Xi*
Ax
Xi*
Ax
Xi*
Ax
Xi*
Ax
Xi*
Total
Gluc
Fruc
Ribo
Treh
1
2
3
1
2
3
4
1
2
3
17
45
29
31
12
28
13
28
29
18
5
5
5
5
5
5
5
5
5
5
3 v 4
1 v 2
1 v 3
2 v 3
1 v 2
1 v 3
1 v 4
2 v 3
2 v 4
3 v 4
1 v 2
1 v 3
2 v 3
0.8799
< 0.0001
0.9999
< 0.0001
0.0253
0.0048
0.9999
0.9999
0.2720
0.2177
0.0002
0.9056
0.0424
0.0079
0.5000
0.2222
0.0079
0.0079
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
2f
Ax
1
16
Kruskal
-
Wallis test
(
P = 0.0057
)
1 v 2
0.0465
Xi*
2
18
1 v 3
0.8352
Ax+Treh
3
16
1 v 4
0.9999
Xi*+Treh
4
17
2 v 3
0.9999
2 v 4
3 v 4
0.0090
0.3024
Figure
3a
g
Anal
ysis
(3a): Difference
in average speed between flies calculated for each GAL4 line crossed with
UAS
-
dTRPA1
tested at 27 ̊C.
E
ach point represents an independent trial
,
containing between 10
-
15
flies per group
.
Tdc2/Ddc/Th/ChAT, n = 5; Tβh, n
= 6; Gad1, n = 3.
F
or further details and graphs of each GAL4 line, see Extended
Data Fig. 8
.
(3b
g):
Average speed
.
For 3b and c, statistical significance was found for both variables
(microbial
status x
genotype)
calculated
by
Two
-
way ANOVA
.
In 3c,
there is
a significant
interaction between microbial status
and genotype
(P = 0.0437)
.
When examined
separately, t
he Kruskal
-
Wallis test on
only Conv flies across genotypes
was significant
(P = 0.0030) and
the Kruskal
-
Wallis test
on
only ABX flies
was not significant (P =
0.
8426).
All are biological replicates assessed with a two
-
tailed test and data
are representative of at least 2
independent trials
for each experiment
.
Figure
Condition
Genotype
Sample
Size
Statistical
Test
P
-
value
3b
ABX
UAS
-
TrpA1
15
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
0.0139
Xi*
12
ABX
Tdc2
-
GAL4
23
0.0177
Xi*
ABX
GAL4>UAS
23
14
0.6308
Xi*
(27°C)
12
3c
ABX
Xi*
Conv
ABX
Conv
ABX
Conv
ABX
Conv
ABX
GAL4>UAS
(20°C)
UAS
-
TrpA1
Tdc2
-
GAL4
GAL4>UAS
(27°C)
GAL4>UAS
(20°C)
12
15
45
30
48
39
49
39
22
31
0.0238
0.0033
0.0263
0.4929
0.0143
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
3d
Conv
1
13
Kruskal
-
Wallis test
(
P < 0.0001
)
1 v 2
0.0008
Ax
2
33
1 v 3
0.9999
Xi*
3
21
2 v 3
0.0015
Conv+OA
4
29
4 v 5
0.9999
Ax+OA
Xi*+OA
5
6
27
32
4 v 6
5 v 6
0.9999
0.9999
Figure
Condition
#
Sample
Size
Statistical
Test
Comparison
P
-
value
3e
Ax
1
58
Kruskal
-
Wallis
test
(
P = 0.0008
)
1 v 2
0.9999
Ax
+OA
2
13
1 v 3
0.9999
Ax+TA
3
10
1 v 4
0.0047
Xi*
4
54
1
v 5
0.9999
Xi*
+OA
Xi*+T
A
5
6
46
27
1
v 6
4 v 5
4 v 6
5 v 6
0.0351
0.0980
0.9999
0.2695
Figure
Condition
Genotype
Sample
Size
Statistical
Test
P
-
value
3f
ABX
Wt (w+)
12
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
0.0002
Xi*
17
ABX
Tdc
R023
19
0.4517
3g
Xi*
ABX
Wt (CS)
17
15
0.0128
Xi*
15
ABX
Xi*
Tβh
M18
11
12
0.5254
Extended Data Figure 1
a
h
Analysis
(
1a): Colony forming units
.
(1b
e, h
): Average speed
.
(
1
f): Average speed
calculated only
within
walking bout
s
.
(
1
g): Tripod index: see ref. 64 for calculations
.
All are biological replicates
assessed with a two
-
tailed test
.
Figure
Condition
Sample
Size
Statistical
Test
P
-
value
1a
L.p
15
Unpaired
Student’s T
-
test
0.6800
L.b
18
Figure
Condition
Sex
#
Sample
Size
Statistical Test
Comparison
P
-
value
1b
Conv
F
1
90
Kruskal
-
Wallis
test
(
P
< 0.0001
)
Kruskal
-
Wallis
test
(P = 0.0302)
1 v 2
< 0.0001
Ax
F
2
92
1 v 3
0.0251
L.b
F
3
89
2 v 3
< 0.0001
Conv
M
4
100
4 v 5
0.0669
Ax
L.b
M
M
5
6
100
95
4 v 6
5 v 5
0.9999
0.0664
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
1c
Ax
1
58
Kruskal
-
Wallis
test
(P < 0.0001)
Kruskal
-
Wallis
test
(P < 0.0001)
Mann
-
Whitney
test
Mann
-
Whitney
test
Mann
-
Whitney
test
Kruskal
-
Wallis
test
(P = 0.1731)
Kruskal
-
Wallis
test
(P = 0.1358)
Kruskal
-
Wallis
test
(P < 0.0001)
1 v 2
0.0008
L.b
EW
2
57
1 v 3
< 0.0001
L.b
Bb14
3
57
2 v 3
0.9999
1d
Ax
1
45
1 v 2
0.0463
L.b
EW
2
28
1 v 3
< 0.0001
1e
1f
1g
1h
L.b
P
-
2
Diet 1 Ax
Diet 1
L.b
Diet 2 Ax
Diet 2
L.b
Diet 3 Ax
Diet 3
L.b
Conv
Ax
L.p
L.b
Conv
Ax
L.p
L.b
Ax
L.p
L.b
L.p
+
L.b
3
1
2
3
4
5
6
1
2
3
4
1
2
3
4
1
2
3
4
42
20
21
18
16
6
6
23
35
22
22
6
7
5
5
18
24
24
24
2 v 3
1 v 2
3 v 4
5 v 6
1 v 4
1 v 4
1 v 2
1 v 3
1 v 4
2 v 3
2 v 4
4 v 5
0.0348
0.0006
0.0004
0.0411
0.9999
0.9999
0.9999
0.0090
< 0.0001
0.2590
0.0037
0.9631
Extended Data Figure 2b
l
Analysis
(
2b, e
g, i
):
Average speed
.
(2c, j): Average bout length
.
(
2d, k
): Average speed within walking bout
s
.
(
2l
): D
aily crossings.
All are biological replicates assessed with
a two
-
tailed test
.
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
2b
Ax
L.b
0d
1
2
46
47
Kruskal
-
Wallis
test
(P = 0.0015)
Kruskal
-
Wallis
test
(P = 0.0041
)
Kruskal
-
Wallis
test
(P = 0.0004
)
1 v 2
1 v 3
0.0032
0.0096
L.b
3
-
5d
3
43
2
v 3
0.9999
2c
Ax
L.b
0d
L.b
3
-
5d
1
2
3
18
18
6
1 v 2
1 v 3
2 v 3
0.0295
0.0126
0.8985
2d
Ax
L.b
0d
L.b
3
-
5d
1
2
3
36
36
12
1 v 2
1 v 3
2 v 3
0.0063
0.0019
0.6410
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
2e
Conv
Ax
1
2
11
53
Kruskal
-
Wallis
test
(P = 0.0004
)
Kruskal
-
Wallis
test
(P = 0.0038)
Mann
-
Whitney
test
Mann
-
Whitney
test
Kruskal
-
Wallis
test
(P = 0.0011
)
Kruskal
-
Wallis
test
(P = 0.0203
)
Kruskal
-
Wallis
test
(P = 0.0079
)
Kruskal
-
Wallis
test
(P = 0.0109
)
1 v
2
1 v 3
0.0127
0.9999
L.b
0d
3
53
1 v 4
0.9999
L.b
3
-
5d
4
52
2 v 3
2 v 4
0.0065
0.0056
3 v 4
0.9999
2f
Conv
1
32
1 v 2
0.0122
2g
2i
2j
2k
2l
Ax
ABX
Conv (OR)
ABX (OR)
Conv (CS)
ABX (CS)
ABX
L.p
L.b
ABX
L.p
L.b
ABX
L.p
L.b
ABX
L.p
L.b
2
3
1
2
3
4
1
2
3
1
2
3
1
2
3
1
2
3
36
36
20
22
12
17
29
24
35
36
30
35
42
30
35
6
6
6
1 v 3
2 v 3
1 v 2
3 v 4
1 v 2
1 v 3
2 v 3
1 v 2
1 v 3
2 v 3
1 v 2
1 v 3
2 v 3
1 v 2
1 v 3
2 v 3
0.0091
0.9999
0.0265
0.0093
0.9999
0.0203
0.0019
0.9999
0.0698
0.0350
0.9999
0.0607
0.0094
0.8385
0.0148
0.2507
Extended Data Figure 3a
f
Analysis
(
3
a
,
b
, f
):
Average speed
.
(3c):
Average bout length
.
(3d
): Average speed within walking bout
s
.
(
3
e
): Daily
crossings.
All are biological replicates
assessed with a two
-
tailed test.
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
3
a
Ax
L.p
1
2
45
17
Kruskal
-
Wallis
test
(P = 0.01
)
Kruskal
-
Wallis
test
(P < 0.0001)
1 v 2
1 v 3
0.9999
0.3301
L.b
3
42
1 v 4
0.9999
3b
L.p
CFS
L.b
CFS
Ax
4
5
1
17
16
23
1 v 5
3 v 5
4 v 5
1 v 2
0.0445
0.9999
0.0906
0.1287
L.p
CFS
L.b
CFS
2
3
20
20
1 v 3
2 v 3
< 0.0001
0.0025
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
3c
Ax
1
23
Kruskal
-
Wallis
test
(P = 0.0
0
1
6
)
Kruskal
-
Wallis
test
(P < 0.0001
)
Kruskal
-
Wallis
test
(P = 0.0469)
Kruskal
-
Wallis
test
(P = 0.0009)
1 v 2
0.5833
3d
L.p
CFS
L.b
CFS
Ax
L.p
CFS
2
3
1
2
20
17
22
21
1 v
3
2 v 3
1 v 2
1
v 3
0.0010
0.0696
0.0231
< 0.0001
3e
3f
L.b
CFS
Ax
L.p
CFS
L.b
CFS
Ax
L.b
0.1 Uracil
10 Uracil
3
1
2
3
1
2
3
4
17
8
8
4
96
88
41
18
2 v 3
1 v 2
1 v 3
2 v 3
1 v 2
1 v 3
1 v 4
0.0493
0.9999
0.2535
0.0472
0.0004
0.9999
0.4509
Extended Data Figure 4a
e
Analysis
(
4a, b
):
Average speed
.
(4c): Expression
of gene transcripts
, samples from whole body homogenates
.
(4d
):
Amount ingested
.
(
4e
):
Gut content
.
All are biological replicates assessed with a two
-
tailed test.
Figure
Condition
Measurement
#
Sample
Size
Statistical
Test
Comparison
P
-
value
4a
Wt Conv
1
16
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
Mann
-
Whitney test
One
-
way
ANOVA
test
(P = 0.8386)
One
-
way
ANOVA
test
(P =
0.9441
)
One
-
way
ANOVA
test
(P =
0.5100
)
One
-
way
ANOVA
test
(P =
0.4687
)
One
-
way
ANOVA
test
(P =
0.6952
)
1 v 2
0.0032
4b
Wt ABX
IMD
Conv
IMD
ABX
Wt Conv
Wt ABX
Ti
Conv
Ti
ABX
2
3
4
1
2
3
4
17
24
25
15
17
10
11
3 v 4
1 v 2
3 v 4
0.0168
0.0299
< 0.0001
4c
Ax
L.p
CFS
L.b
CFS
Ax
L.p
CFS
L.b
CFS
Ax
L.p
CFS
L.b
CFS
Ax
L.p
CFS
L.b
CFS
Ax
L.p
CFS
L.b
CFS
Dpt
Drs
Cec
AttA
Duox
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
8
10
10
10
10
10
8
10
10
5
5
5
3
5
5
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
4d
Ax
L.p
CFS
1
2
6
5
Kruskal
-
Wallis
test
(P = 0.8626
)
Kruskal
-
Wallis
test
(P =
0.4236
)
4e
L.b
CFS
Conv
Ax
L.p
CFS
L.b
CFS
3
1
2
3
4
5
7
13
7
10
Extended Data Figure 5a
j
Analysis
(
5a
f, h
):
Average speed
.
(5g):
Daily crossings.
(5i
):
Percent survival
. Each sample consists of 15
25
flies
.
(
5j
):
Percentage of
apoptotic cells
.
All are biological replicates assessed with a two
-
tailed test.
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
5a
Ax
L.b
CFS
1
2
18
18
Kruskal
-
Wallis
test
(P = 0.0008
)
Kruskal
-
Wallis
test
(P = 0.0037
)
Kruskal
-
Wallis
test
(P = 0.0102
)
Kruskal
-
Wallis
test
(P = 0.0016
)
Kruskal
-
Wallis
test
(P < 0.0001
)
Kruskal
-
Wallis
test
(P < 0.0001)
1 v 2
1 v 3
0.0566
0.9999
5b
5c
5d
5e
5f
+ Typ
-
Typ
Ax
L.b
CFS
+PK
-
PK
Ax
L.b
CFS
+100°C
Ax
+amyl
-
amyl
Ax
L.b
L.p
A.p
E.c
Ax
E.c
tyrA
trpC
manX
treA
xylA
3
4
1
2
3
4
1
2
3
1
2
3
1
2
3
4
5
1
2
3
4
5
6
7
17
17
23
18
23
23
18
18
18
30
17
30
30
30
29
30
18
65
52
18
17
45
46
20
1 v 4
3 v 4
1 v 2
1 v 3
1 v 4
3 v 4
1 v 2
1 v 3
2 v 3
1 v 2
1 v 3
2 v 3
1 v 2
1 v 3
1
v 4
1 v 5
2 v 3
1 v 2
1 v 3
1 v 4
1 v 5
1 v 6
1 v 7
0.0021
0.0181
0.0081
0.5168
0.0139
0.9999
0.0081
0.1867
0.7709
0.0170
0.0036
0.9999
< 0.0001
0.2185
0.9140
< 0.0001
0.0074
0.0183
0.0003
0.0300
< 0.0001
< 0.0001
0.9999
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
5g
Conv
Ax
1
2
16
24
Kruskal
-
Wallis
test
(P = 0.0027
)
Kruskal
-
Wallis
test
(P = 0.0167
)
Kruskal
-
Wallis
test
(P =
0.4117
)
Kruskal
-
Wallis
test
(P = 0.0383
)
1 v 2
2 v 3
0.0432
0.0640
5h
5i
5j
L.b
CFS
xylA
CFS
Xi
Ax
L.b
CFS
10 Xi
100 Xi
Ax
L.p
CFS
L.b
CFS
Xi
Conv
Ax
L.p
CFS
L.b
CFS
Xi
3
4
5
1
2
3
4
1
2
3
4
1
2
3
4
5
19
20
8
16
11
12
14
4
5
5
4
7
5
4
6
6
2 v 4
2 v 5
1 v 2
1 v 3
1 v 4
1 v 2
2
v 3
2
v 4
2
v 5
0.9999
0.0134
0.0274
0.9999
0.0263
0.9999
0.9999
0.0343
0.9999
Extended Data Figure 6a
b
Analysis
(
6
a, b
):
Sleep analysis
.
All are biological replicates assessed with a two
-
tailed test.
Figure
Condition
#
Sample
Size
Statistical Test
Comparison
P
-
value
6a
Light
Conv
Ax
1
2
8
8
Kruskal
-
Wallis
test
(P =
0.0036
)
Kruskal
-
Wallis
test
(P =
0.0585
)
Kruskal
-
Wallis
test
(P =
0.0314
)
Kruskal
-
Wallis
test
(P =
0.1477
)
1 v 2
2 v 3
0.0193
0.9999
Phase
6a
Dark
Phase
6b
Light
Phase
6b
Dark
Phase
L.p
L.b
Conv
Ax
L.p
L.b
Conv
Ax
L.p
CFS
L.b
CFS
Xi
Conv
Ax
L.p
CFS
L.b
CFS
Xi
3
4
1
2
3
4
1
2
3
4
5
1
2
3
4
5
8
8
8
8
8
8
17
25
19
21
8
17
25
19
21
8
2 v 4
1 v 2
2 v 3
2 v 4
2 v 5
0.0193
0.0911
0.4850
0.9999
0.0932