Supplementary figure legends
For statistical analysis of all figures, see Supplementary Methods.
Supplementary Figure 1 Slow killing of wild type C. elegans by the pathogenic
bacterium Serratia marcescens ATCC 13880 (PDF file, 159kB). Wild type animals
were assayed in two independent trials (≥ 25 animals in each trial). Slow-killing
experiments were performed as described1.
Supplementary Figure 2 Two-choice learning assays on OP50 and PA14 (PDF file,
170kB). adp-1(ky20), egl-4(ly95) and tol-1(nr2033) animals learn as well as wild type
animals (n ≥ 4 assays). Error bars represent standard error of the mean (SEM).
Supplementary Figure 3 Two-choice assays for olfactory preferences (PDF file,
170kB). Animals were grown either on OP50 on control growth plates or trained with
PA14 with a small amount of OP50 on training growth plates. Choice indices for animals
of each genotype were calculated as described in Fig. 1 (* P ≤ 0.05, n ≥ 4 assays). Error
bars, SEM.
Supplementary Figure 4 Pathogenic bacteria and starvation elicit different changes
in olfactory preference (PDF file; 170kB). Olfactory preferences of wild type animals
that were either grown on OP50, or grown on OP50 until adults and then on PA14 for
four hours, or grown on OP50 until adults and then starved for four hours, analyzed in the
four-choice maze assays. Four bacteria strains were used as food choices: E. coli OP50;
P. aeruginosa PA14; P. fluorescens; S. marcescens ATCC 13880. Compared to animals
grown on OP50, animals trained on PA14 for four hours acquired aversion to PA14.
Animals starved for four hours before the assay did not show decreased attraction
towards PA14 compared to animals grown on OP50. Therefore, olfactory learning on
pathogens is distinct from starvation. Neither starved animals nor animals trained with
PA14 for four hours acquired increased preferences towards OP50 (* P ≤ 0.05, n ≥ 14
assays). Error bars, SEM.
Supplementary Figure 5 Food-leaving behavior is normal in tph-1 mutants (PDF
file; 166kB). Wild type animals stay on a lawn of E. coli OP50 or the non-virulent S.
marcescens strain Db1140, but leave a lawn of the pathogenic S. marcescens strain Db11
(food-leaving behavior). tol-1(nr2033) animals are defective in food-leaving, whereas
tph-1(mg280) mutants are normal. The weaker defect of tol-1 mutants compared to the
results in Pujol et al.2 is due to different bacteria culture conditions (J. Ewbank, personal
communications). Each genotype was assayed in at least 3 independent trials (* P ≤ 0.05,
n ≥ 15 animals for each genotype in each trial). Error bars, SEM.
Supplementary Figure 6 Slow killing kinetics of serotonin mutants by PA14 (PDF
file; 170kB). tph-1(mg280) mutants, mod-1(ok103) mutants and wild type animals
showed identical killing kinetics by PA14 under slow-killing conditions. Each genotype
was assayed in two independent trials (≥ 30 animals for each genotype in each trial).
Slow-killing experiments were performed as described1.
Supplementary Figure 7 Serotonin from NSM neurons but not ADF neurons is
sufficient for enhanced slowing (PDF file; 170kB). The locomotory speed of well-fed
C. elegans decreases when the animal encounters a bacteria lawn, and food-deprived
animals slow down even more. The enhanced slowing response of food-deprived
animals is mostly dependent on NSM serotonergic neurons3. Expression of tph-1 in
NSM in tph-1(mg280) animals partially rescued the slowing response to food after
starvation, but expression of tph-1 in ADF did not rescue (** P ≤ 0.01; n ≥ 19 animals
each). Error bars, SEM.
Supplementary Figure 8 (PDF file; 430kB) Anti-serotonin immunostaining in wild
type animals fed on (a) OP50 and nonpathogenic P. aeruginosa 12A1, (b) OP50 and
nonpathogenic P. aeruginosa 50E12 (c) OP50 and nonpathogenic P. aeruginosa
gacA::kan. Compared to wild type animals fed on OP50 (Figure 5a), no increase was
observed in either ADF (arrowhead) or NSM (arrow) neurons. Quantification of the
straining is shown in Figure 5i.
Supplementary Figure 9 Expression of tph-1 in ADF neurons rescues aversive
olfactory learning on PA14 (PDF file; 198kB). Four-choice maze assay using two nonpathogenic bacteria strains, E. coli OP50 (OP) and P. fluorescens (PF), two pathogenic
strains, P. aeruginosa PA14 (PA) and S. marcescens ATCC 13880 (SM), as food choices.
A, B, tph-1 animals are defective in both attractive and aversive components of the
experience-dependent olfactory learning when trained either (A) with OP50 and PA14 or
(B) with P. fluorescens and S. marcescens ATCC 13880 (n ≥ 7 assays). C, Expression of
tph-1 cDNA in ADF, NSM, I3, M4, and M3 neurons rescued the defects of tph-1 mutants
in both attractive and aversive components of the olfactory learning when animals are
trained with OP50 and PA14 (* P ≤ 0.05, n ≥ 6 assays). D, E, Expression of a tph-1
cDNA in ADF neurons in tph-1(mg280) animals restored aversive learning on PA14
when animals are trained with OP50 and PA14, but did not rescue attractive learning
towards OP50 (* P ≤ 0.05, n ≥ 5 assays). D and E present the data from two stably
transmitted transgenic lines generated independently. Error bars, SEM.
Supplementary Figure 10 The serotonin-gated chloride channel MOD-1 regulates
aversive learning in interneurons (PDF file; 212kB). Four-choice maze assay using
two non-pathogenic bacteria strains, E. coli OP50 (OP) and P. fluorescens (PF), two
pathogenic strains, P. aeruginosa PA14 (PA) and S. marcescens ATCC 13880 (SM), as
food choices. A, B, mod-1(ok103) mutants are defective in aversive learning on training
pathogenic bacterium when trained with (A) OP50 and PA14 (B) P. fluorescens and S.
marcescens (*** P ≤ 0.001, n ≥ 7 assays). C, D, E, F, G, H, Four-choice maze assays
for aversive learning on PA14 for worms trained with OP50 and PA14. (C), (D),
Expression of mod-1 cDNA using mod-1 promoter completely rescues aversive learning
(** P ≤ 0.01, * P ≤ 0.05, n ≥ 5 assays, C and D present the results from two stably
transmitted transgenic lines generated independently). (E), (F), Expression of a mod-1
cDNA using ttx-3 promoter rescues aversive learning (** P ≤ 0.01, * P ≤ 0.05, n ≥ 5
assays, E and F present the results from two stably transmitted transgenic lines generated
independently). (G), (H), Expression of a mod-1 cDNA using odr-2(2b) promoter
rescues aversive learning (** P ≤ 0.01, n ≥ 12 assays, G and H present the results from
two stably transmitted transgenic lines generated independently). Error bars, SEM.
1.
2.
3.
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