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Science 334 (6056): 670-674

Copyright © 2011 by the American Association for the Advancement of Science

Drosophila Microbiome Modulates Host Developmental and Metabolic Homeostasis via Insulin Signaling

Seung Chul Shin1,3,*,{dagger}, Sung-Hee Kim1,{dagger}, Hyejin You1,2, Boram Kim1,2, Aeri C. Kim1,2, Kyung-Ah Lee1, Joo-Heon Yoon3, Ji-Hwan Ryu3, and Won-Jae Lee1,{ddagger}

1 School of Biological Science, Seoul National University and National Creative Research Initiative Center for Symbiosystem, Seoul 151-742, South Korea.
2 Department of Bioinspired Science and Division of Life and Pharmaceutical Science, Ewha Woman’s University, Seoul 120-750, South Korea.
3 Research Center for Human Natural Defense System, Yonsei University College of Medicine, CPO Box 8044 Seoul, South Korea.


Figure 1
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Fig. 1. Genome-wide screening of the Acetobacter genes essential for host growth. (A) The time to reach puparium formation of conventionally reared w1118 larvae and germ-free w1118 larvae fed a standard laboratory diet containing different yeast concentrations (2, 1, 0.5, 0.25, or 0.1%) or the casamino acid diet (casamino acid). The sizes of larvae at the 120th hour of development after egg laying are shown. Data were analyzed using an analysis of variance (ANOVA) followed by Tamhane’s T2 post hoc test; values represent mean ± SEM (***P < 0.001). (B) A. pomorum is sufficient for full larval development. Germ-free embryos were associated with each of the five species of commensal bacteria or all five commensal bacteria (+A. pomorum/C. intestini/G. morbifer/L. plantarum/L. brevis). Germ-free embryos and conventionally reared embryos were also used as control. The time to reach puparium formation was measured in the casamino acid diet. The sizes of larvae at the 120th hour of development after egg laying are shown. Data were analyzed using an ANOVA followed by Tamhane’s T2 post hoc test; values represent mean ± SEM (***P < 0.001). (C) All 14 screened mutant strains were defective in promoting host development. Germ-free embryos were colonized with each of the 14 mutant A. pomorum strains and the fly larvae fed the casamino acid diet. Mutated gene names and clone identification numbers are shown. Germ-free embryos associated with WT A. pomorum (wild-type) were used as the control. The time to reach puparium formation of WT A. pomorum–monoassociated embryos was arbitrarily designated as 1, and the results are shown as relative time to puparium formation. Data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (**P < 0.01, ***P < 0.001). In box-plot diagrams, black lines and boxes represent the median and first and third quartiles of the values; whiskers extend to minimum and maximum values. In an independent experiment, the time to reach puparium formation of each group of monoassociated larvae (n = ~20) was measured.

 

Figure 2
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Fig. 2. Commensal PQQ-ADH activity is required for diverse ranges of host homeostatic programs controlling developmental rate, body size, and metabolism. In all experiments, germ-free embryos monoassociated with the WT A. pomorum (+wild-type) and P3G5 mutant strain (+P3G5) maintained in the casamino acid media were compared. (A) Time to puparium formation and sizes of larvae 120 hours after egg laying. A standard cornmeal-agar medium containing 0.1% yeast was also used. Data were analyzed using the Mann-Whitney U test (***P < 0.001). (B) Adult body sizes. Body weights of adult flies (5 days old) were measured. Data were analyzed using the Mann-Whitney U test (***P < 0.001). (C) Wing area, cell number, and size. Female adult flies (5 days old) were used. Data were analyzed using the Mann-Whitney U test (***P < 0.001). (D) Sugar and lipid levels. The early third instar larvae (10 to 15 larvae per each experiment) were used. Data were analyzed using the Mann-Whitney U test (*P < 0.05 and **P < 0.01).

 

Figure 3
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Fig. 3. Deregulation of developmental and metabolic homeostasis in P3G5-monoassociated animals is rescued by enhancing host IIS. In all experiments, animals maintained in the casamino acid media were compared. The animals used in this study were: wild-type + Hs-GAL4, WT A. pomorum-monoassociated control flies carrying Hs-GAL4 alone; P3G5 + Hs-GAL4, P3G5-monoassociated control flies carrying Hs-GAL4 alone; and P3G5 + Hs-GAL4>UAS-DILP2, P3G5-monoassociated flies carrying Hs-GAL4>UAS-DILP2. (A) P3G5-induced dFOXO nuclear localization is abolished upon ectopic DILP2 expression leading to cytoplasmic retention of dFOXO. Fat body tissues of the early third instar larvae were used. (B) Time to puparium formation and the sizes of the larvae at the 120th hour of development after egg laying. Data are analyzed using an ANOVA followed by Tamhane’s T2 post hoc test; values represent mean ± SEM (***P < 0.001). (C) Adult body size. Body weights of female adults (5 days old) were measured. Data are analyzed using an ANOVA followed by Tamhane’s T2 post hoc test; values represent mean ± SEM (***P < 0.001). (D) Wing area, cell number, and size. Wings of female adult flies (5 days old) were used in this study. Data were analyzed using Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (*P < 0.05, **P < 0.01, ***P < 0.001). (E) Sugar and lipid levels. The early third instar larvae (10 to 15 larvae per each experiment) were used. Data were analyzed using Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (**P < 0.01).

 

Figure 4
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Fig. 4. Deregulation of developmental and metabolic homeostasis in P3G5-monoassociated animals is rescued by acetic acid supplementation. In all experiments, animals maintained in the casamino acid media were compared. The animals used in this study were: wild-type, WT A. pomorum-monoassociated control flies; P3G5, P3G5-monoassociated control flies; P3G5 + acetic acid, P3G5-monoassociated flies in the presence of 0.2% acetic acid; and Germ-free + acetic acid, germ-free control flies in the presence of 0.2% acetic acid. (A) Acetic acid supplementation in casamino acid media induces IIS activation in P3G5-monoassociated larvae. PI3K activation state was evaluated by examining membrane targeting of pleckstrin homology–green fluorescent protein (PH-GFP), and localization of dFOXO was examined by immunostaining with an antibody to dFOXO. Fat body tissues of the early third instar larvae were used. DAPI, 4',6-diamidino-2-phenylindole. (B) Time to puparium formation, and sizes of larvae at the 120th hour of development after egg laying. Data were analyzed using an ANOVA followed by Tamhane’s T2 post hoc test. Values represent mean ± SEM (***P < 0.001). (C) Adult body size. Body weights of adult flies (5 days old) were measured. Data were analyzed using an ANOVA followed by Tamhane’s T2 post hoc test; values represent mean ± SEM (***P < 0.001). (D) Wing area, cell number, and size. Wings of female adult flies (5 days old) were used in this study. Data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (***P < 0.001). (E) Sugar and lipid levels. The early third instar larvae (10 to 15 larvae per each experiment) were used. Data were analyzed using the Kruskal-Wallis test followed by the Mann-Whitney U test using Bonferroni correction to adjust the probability. Bonferroni-adjusted P values were used (*P < 0.05, **P < 0.01).

 


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