<i>amn</i> mutants show defects in egg-laying decisions.

<p>(A) In sucrose/caffeine chamber, <i>amn</i>^<i>1</i>, <i>amn</i>^<i>28A</i>, <i>amn</i>^<i>c651</i>, and <i>amn</i>^<i>X8</i> flies showed significant difference in egg-laying decisions compared to wild-type flies. <i>amn</i>^<i>X8</i> showed significant difference compared to the other <i>amn</i> mutants. Each value represents mean ± SEM (n = 34–35, ***P < 0.001). (B) In sucrose/plain chamber, <i>amn</i>^<i>1</i>, <i>amn</i>^<i>28A</i>, <i>amn</i>^<i>c651</i>, and <i>amn</i>^<i>X8</i> flies showed significant difference in egg-laying decisions compared to wild-type flies. <i>amn</i>^<i>X8</i> showed significant difference compared to the other <i>amn</i> mutants. Each value represents mean ± SEM (n = 20, ***P < 0.001, **P < 0.01). (C) In caffeine/plain chamber, <i>amn</i>^<i>1</i>, <i>amn</i>^<i>28A</i>, <i>amn</i>^<i>c651</i>, and <i>amn</i>^<i>X8</i> flies showed significant difference in egg-laying decisions compared to wild-type flies. Each value represents mean ± SEM (n = 23–27, ***P < 0.001).</p

Aging does not alter <i>Drosophila</i> egg-laying decisions.

<p>(A) Schematic representation of the chamber for egg-laying decisions assays. Two 1% agarose media, containing either 100 mM sucrose (orange) or 100 mM caffeine (blue), were loaded above the 3% hard agarose medium (grey). A gap (0.1 cm) was made on the 3% agarose medium in the middle of the chamber. (B) Egg-laying decisions of wild-type flies at different ages (5-day and 21-day). Each value represents mean ± SEM (N = 36–42, n.s., not statistically significant).</p

Neural activity in mushroom body αβ neurons is required for normal egg-laying decisions.

<p>(A) Preferential expression of <i>R16A06-GAL4</i> in mushroom body γ neurons (green). The brain was immunostained with DLG antibody (red). The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; R16A06-GAL4/UAS-mCD8</i>::<i>GFP</i>. (B) Preferential expression of <i>VT30604-GAL4</i> in mushroom body α´β´ neurons (green). The brain was immunostained with DLG antibody (red). The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; VT30604-GAL4/UAS-mCD8</i>::<i>GFP</i>. (C) Preferential expression of <i>VT49246-GAL4</i> in mushroom body αβ neurons (green). The brain was immunostained with DLG antibody (red). The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; VT49246-GAL4/UAS-mCD8</i>::<i>GFP</i>. (D) Effects of acute silencing of neuronal activity in different mushroom body neuron subsets on egg-laying decisions. The temperature shift protocols are shown schematically above each graph. Each value represents mean ± SEM (n = 10–25, ***P < 0.001. n.s., not statistically significant). Genotypes were as follows: (1) <i>tubP-GAL80</i>^<i>ts</i><i>/+; +/UAS-Kir2</i>.<i>1</i>, (2) <i>+/+; R16A06-GAL4/+</i>, (3) <i>+/+; VT30604-GAL4/+</i>, (4) <i>+/+; VT49246-GAL4/+</i>, (5) <i>tubP-GAL80</i>^<i>ts</i><i>/+;R16A06-GAL4/UAS-Kir2</i>.<i>1</i>, (6) <i>tubP-GAL80</i>^<i>ts</i><i>/+;VT30604-GAL4/UAS-Kir2</i>.<i>1</i>, and (7) <i>tubP-GAL80</i>^<i>ts</i><i>/+;VT49246-GAL4/UAS-Kir2</i>.<i>1</i>.</p

Expression of <i>amn</i> transgene in DPM neurons reverses the defects of egg-laying decisions in <i>amn</i> mutants.

<p>(A) The expression pattern of <i>C316-GAL4</i> (green). The brain was immunostained with DLG antibody (red). Arrowheads indicate the somata of DPM neurons. The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; C316-GAL4/UAS-mCD8</i>::<i>GFP</i>. (B) The expression pattern of <i>VT6412-GAL4</i> (green). The brain was immunostained with DLG antibody (red). Arrowheads indicate the somata of DPM neurons. The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; VT64246-GAL4/UAS-mCD8</i>::<i>GFP</i>. (C) The expression pattern of <i>VT64246-GAL4</i> (green). The brain was immunostained with DLG antibody (red). Arrowheads indicate the somata of DPM neurons. The scale bar represents 50 μm. Genotype was as follow: <i>UAS-mCD8</i>::<i>GFP/+; VT64246-GAL4/UAS-mCD8</i>::<i>GFP</i>. (D) Overexpression of the <i>amn</i> transgene (<i>amn</i>^<i>+</i>) in DPM neurons reversed the deficiency of egg-laying decisions in <i>amn</i>^<i>1</i> background. Each value represents mean ± SEM (n = 22–24, ***P < 0.001, n.s., not statistically significant). Genotypes were as follows: (1) +/+, (2) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i>, (3) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; +/UAS-amn</i>^<i>+</i>, (4) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; C316-GAL4/+</i>, (5) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; VT6412-GAL4/+</i>, (6) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; VT64246-GAL4/+</i>, (7) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; C316-GAL4/UAS-amn</i>^<i>+</i>, (8) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; VT6412-GAL4/UAS-amn</i>^<i>+</i>, and (9) <i>amn</i>^<i>1</i><i>/amn</i>^<i>1</i><i>; +/+; VT64246-GAL4/UAS-amn</i>^<i>+</i>. (E) Effects of acute silencing of neuronal activity in DPM neurons on egg-laying decisions. The schematics of the temperature shift protocols are shown above each graph. Each value represents mean ± SEM (n = 17–26, ***P < 0.001. n.s., not statistically significant). Genotypes were as follows: (1) <i>+/+; VT6412-GAL4/+</i>, (2) <i>tubP-GAL80</i>^<i>ts</i><i>/+; UAS-Kir2</i>.<i>1/+</i>, and (3) <i>tubP-GAL80</i>^<i>ts</i><i>/+; VT6412-GAL4/UAS-Kir2</i>.<i>1</i>.</p

Midgut acid suppression induces an obese-like phenotype in flies.

<p>(a and b) <i>m2</i> homozygous mutant flies (red) and lansoprazole-treated flies (orange) show increased triglyceride accumulation (a) and body weight (b) compared to control flies (<i>w</i>^<i>1118</i>, blue). (c and d) Food intake and female fecundity are not altered in <i>m2</i> homozygous mutant flies (red) compared to control flies (<i>w</i>^<i>1118</i>, blue). Data were collected from 8–10 replicates for each group. Each replicate contained 10 flies for triglyceride (a), body weight (b) and feeding assays (c), and 3 mated pairs for female fecundity measurements (d). <b>*</b>, <i>P</i> < 0.05, compared to the control group.</p

The effects of acid suppression on starvation resistance and life span (LS) in <i>Drosophila melanogaster</i>.

<p>* <i>P</i> value<0.01 compared to genetic matched control or vehicle control by log-rank test.</p><p>The effects of acid suppression on starvation resistance and life span (LS) in <i>Drosophila melanogaster</i>.</p

Midgut acid suppression shortens life span in flies.

<p>(a) Shortened life spans in <i>m2</i> homozygous mutant flies (red) and <i>EP2372</i> heterozygous mutant flies (green) compared to control flies (<i>w</i>^<i>1118</i>, blue). (b) Shortened life spans in lansoprazole-treated flies (orange) compared to control flies (<i>w</i>^<i>1118</i>, blue). Statistical analysis is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139722#pone.0139722.t001" target="_blank">Table 1</a>.</p

Gastric acid suppression induces an obese-like phenotype in mice.

<p>(a) Increased stomach pH in mice receiving lansoprazole treatment. (b and c) Chronic lansoprazole treatments do not affect food intake (b) or water consumption (c) of mice. (d-f) Chronic lansoprazole treatments gradually induce increased body weight, and elevated levels of serum triglycerides and cholesterol. Data were collected from 7 mice for each group. <b>*</b>, <i>P</i> < 0.05, compared to the saline group.</p

<i>vha16-1</i> mutation induces starvation resistance in flies.

<p>(a) Enhanced survival of <i>m2</i> homozygous (red) and <i>EP2372</i> heterozygous (green) mutant flies on starvation challenge compared to control flies (<i>w</i>^<i>1118</i>, blue). Statistical analysis is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139722#pone.0139722.t001" target="_blank">Table 1</a>. (b) Schematic diagram of <i>vha16-1</i> gene illustrating the insertion site of P-elements. (c) The <i>P[GawB]</i> insertion in <i>m2</i> mutant flies was further verified by specific primer sets. The GT primer set detected a PCR product spanning the <i>vha16-1</i> gene and <i>P[GawB]</i> fragment, and P generated a PCR product within the <i>P[GawB]</i>. (d) <i>vha16-1</i> mRNA is significantly down regulated in <i>m2</i> homozygous mutant flies (red) and <i>EP2372</i> heterozygous (green) mutant flies compared to control flies (<i>w</i>^<i>1118</i>, blue). Experiments were done in triplicate and each replicate contained more than 30 flies for each group. <b>*</b>, <i>P</i> < 0.05, compared to the control group.</p

<i>vha16-1</i> mutation reduces gut acidity in flies.

<p>(a and b) <i>m2>UAS-GFP</i> reporter flies showing that <i>vha16-1</i> was highly expressed in the <i>Drosophila</i> midgut region. Scale bars = 100 μm. (c-f) Representative images showing that <i>m2</i> homozygous mutant flies (e) displayed reduced midgut acidity compared to control flies (c, <i>w</i>^<i>1118</i>). The midgut acidity can also be diminished by feeding both control flies (d) and <i>m2</i> homozygous mutant flies (f) with carbonic anhydrase inhibitor (acetazolamide). Scale bars = 0.5 mm. The anterior and posterior regions of <i>Drosophila</i> midgut are marked as “A” and “P”, respectively. (g) Quantitative results showing that majority of <i>m2</i> homozygous, <i>EP2372</i> heterozygous mutant flies, and acetazolamide- or lansoprazole-treated flies had diminished midgut acidity compared to control flies. (h) <i>vha16-1</i> overexpression specifically in copper cells (<i>Cop-Gal4/;UAS-vha16-1</i>) did not affect the midgut acidity of flies, but did recover the midgut acidity of <i>EP2372</i> heterozygous mutant flies to normal level. (i) Effectiveness of the <i>UAS-vha16-1</i> constructs used for <i>vha16-1</i> overexpression was verified by crossing <i>daughterless (Da)-Gal4</i> with <i>UAS-vha16-1</i> flies. <i>vha16-1</i> mRNA significantly increased in <i>Da-Gal4;UAS-vha16-1</i> flies compared to control (<i>UAS-vha16-1</i>) flies. Experiments were done in triplicate and each replicate contained more than 30 flies for each group. <b>*</b>, <i>P</i> < 0.05, compared to the control group.</p