1. An Intestine-Derived Neuropeptide Controls Avoidance Behavior in Caenorhabditis elegans 
Kiho Lee, Eleftherios Mylonakis 
Cell Reports 
Volume 20, Issue 10, Pages (September 2017)
DOI: /j.celrep
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2. Cell Reports 2017 20, 2501-2512DOI: (10.1016/j.celrep.2017.08.053)
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3. Figure 1 PA14 Virulence Factor Affects Aversive Learning Behavior
(A) Animated two-choice preference assay. Worms were put on OP50 or PA14 or non-virulent PA14 mutant strains and then washed and put on the testing plates. After a 1-hr incubation, we counted and calculated the choice index and learning index as depicted. The worms shown on testing represent approximate numbers, and the testing results represented by the dotted line are not depicted. Worms trained with PA14 or virulence-compromised PA14 were tested with OP50 and PA14 or virulence-compromised PA14.
(B) Choice indexes of worms exposed to OP50 (naive) were not significantly different. Instead, worms that were trained with PA14 had significantly different choice indexes than worms trained with virulence-compromised PA14. p values are for each condition compared to OP50-PA14 (naive) or PA14-PA14 (trained) (Dunnett’s test).
(C) Learning indexes of worms trained on PA14 had a learning index on the test plate similar to that of worms on virulence-compromised PA14 (PA14-PA14, PA14-gacA or PA14-lasR). Learning indexes of worms trained on virulence-compromised PA14 showed no aversive learning behavior upon the test plate with PA14 or virulence-compromised PA14 (gacA-PA14, gac-A-gacA, lasR-PA14, and lasR-lasR) (unpaired two-tailed t test).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < ; n.s, non-significant. Results are presented as mean ± SEM from 6 to 13 independent experiments performed in triplicate assays. More than 40 worms were used for each assay.
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4. Figure 2 Intestinal ins-11 Controls Aversive Learning Behavior
(A) Choice index and learning index of animals after intestine-restricted knockdown of potential intestine-neuronal signal transmitters are shown. Each RNAi represents a gene for RNAi control(L4440), neuropeptide (egl-3), serotonin (tph-1), GABA (unc-47), tyramine and octopamine (tbc-1), dopamine and serotonin (cat-1), acetylcholine (cha-1), and choline transport (cho-1) (Dunnett’s test).
(B) Choice index and learning index of animals after intestine-restricted knockdown of neuropeptide-processing enzymes and dense core vesicle components (Dunnett’s test).
(C) Disruption of ins-11 by deletion, knockdown in whole tissue or intestine-restricted RNAi resulted in increased learning index. Paired two-tailed t test.
Results are presented as mean ± SEM from 3 to 12 independent experiments performed in triplicate assays. More than 40 worms were used for each assay. ∗p <0.05 and ∗∗p < See also Figure S1 and Table S1.
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5. Figure 3
Expression of ins-11 Depends on the Traits of the Stress Source
(A and B) In vivo transcriptional expression of ins-11 after exposure to OP50, P. aeruginosa, P. aeruginosa gacA, exotoxin A (1 mg/mL), and G418 (50 mg/mL). Transcriptional change was measured by qPCR (n = 4, Mann-Whitney test, p = ) (A) and GFP (B) in vivo. One data point in PA14 was not depicted in the graph in (B), as the value was too high (1, ). Results are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < ; n.s., non-significant (Tukey’s test). White dotted lines indicate the body shape of C. elegans.
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6. Figure 4
Expression of ins-11 Depends on hlh-30 and the p38 MAPK Pathway
(A and B) GFP fused to the promoter of ins-11 is increased upon PA14 (B) when compared to upon OP50 (A) in C. elegans.
(C and D) RNAi of hlh-30 diminished both basal (C) and elevated expression of ins-11 (D).
(E–J) Knockdown of p38 MAPK pathway, nsy-1 (E), sek-1 (G) and pmk-1 (I) did not affect basal expression upon OP50, but knockdown of each nsy-1 (F), sek-1 (H), and pmk-1 (J) diminished the elevated expression of ins-11 upon PA14.
Results are presented as mean ± SEM. We used an unpaired two-tailed t test to compare GFP intensity values on E. coli to those on PA14 in each knockdown condition. ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < ; n.s., non-significant (Dunnett’s test). White dotted lines indicate the body shape of C. elegans. See also Figures S2 and S3.
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7. Figure 5 ins-11 Changes Signal Pathways in Neuronal Cells
(A and B) Transcriptional expression of ins-6 in ASI neuron in the control RNAi was not changed on both on OP50 (A) and PA14 (B).
(C and D) With ins-11 RNAi, expression of ins-6 in ASI was not changed on OP50 (C) but was abnormally increased only when worms met PA14 (D).
(E, H, and G) (E) shows GFP intensity measurement of ins-6 promoter in ASI neuron. Transcriptional expression of tph-1 in ADF neuron was elevated only when worms are exposed to PA14 (H) compared to OP50 (G).
(I and J) With ins-11 RNAi, expression of tph-1 was abnormally increased even on OP50 (I) similar to on PA14 (J).
(F, K, and L) (K) shows GFP intensity measurement of tph-1 promoter in ASI neuron. Increased learning index by knockdown of ins-11 was suppressed in ins-6 (F) or tph-1 (L) mutant background.
Tukey’s multiple comparisons test was used for (E) and (K), and an unpaired two-tailed t test was used for (F) and (L). Results are presented as mean ± SEM from 3 to 12 experiments with triplicate assays. More than 40 worms were used in each assay. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < ; n.s., non-significant. The dotted line represents the shape of neuronal cells. See also Figure S4.
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8. Figure 6 Increased avoidance by ins-11 Changed the Survival Rate
(A) The choice indexes of trained wild-type N2 and ins-11 mutant animals were significantly changed by exposure to OP50. After an additional 2-hr incubation on OP50, the learning indexes of both animals were decreased, but ins-11 mutant animals still showed significant avoidance to PA14. Results are presented as mean ± SEM from more than 8 experiments with triplicate assays. More than 40 worms were used in each assay. ∗p < 0.05; ∗∗∗p < 0.001; n.s., non-significant.
(B) Lifespan of wild-type N2 and ins-11 mutant animals was tested on big lawns, which were covered by PA14, or small lawns, which were made using a single drop (10 μL) of PA14. Results are from 2 independent experiments.
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9. Figure 7
Increased Avoidance by ins-11 Is Associated with Fatty Acid Content and the Number of Hatched Eggs
(A) After a 12-hr incubation on PA14 small lawns, wild-type N2 and ins-11 mutant animals were stained with oil red O, which stains fatty acid content in worms. Because a monochromatic camera sensor was used, the black region of the worm’s body (red dots) represents the stained fatty acid.
(B) Light intensity is inversely proportional to fat content. ins-11 mutant animals contain more fat than wild-type N2 worms on a normal plate with OP50. The fat content of both wild-type N2 and ins-11 mutant animals decreased at a similar rate on plates with small PA14 lawns (Tukey’s multiple comparisons test).
(C) When we compared the reduced amount of fat on OP50 to PA14, fat content decreased faster in ins-11 mutant animals than in wild-type N2 worms (two-tailed unpaired t test).
(D) After a 12-hr incubation on PA14 small lawns, ins-11 mutant animals laid fewer hatched eggs than wild-type N2 worms. Results are from 3 independent experiments using more than 6 parents in each group (unpaired two-tailed t test).
(E) Working model based on our findings. ins-11 secreted from the intestine negatively controls aversive learning behavior by ASI and ADF neurons. Transcription factors, hlh-30, and the p38 MAPK pathway regulate the expression of ins-11 in the intestine. In addition to negative regulation by ins-11, we speculate that there could be positive regulation that activates aversive behavior from the intestine.
Results are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p <
Cell Reports  , DOI: ( /j.celrep )
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