<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

Hot avoidance behavior in fruit flies.

Thermosensation is critical for the survival of animals. However, mechanisms through which nutritional status modulates thermosensation remain unclear. Herein, we showed that hungry Drosophila exhibit a strong hot avoidance behavior (HAB) compared to food-sated flies. We identified that hot stimulus increases the activity of α′β′ mushroom body neurons (MBns), with weak activity in the sated state and strong activity in the hungry state. Furthermore, we showed that α′β′ MBn receives the same level of hot input from the mALT projection neurons via cholinergic transmission in sated and hungry states. Differences in α′β′ MBn activity between food-sated and hungry flies following heat stimuli are regulated by distinct Drosophila insulin-like peptides (Dilps). Dilp2 is secreted by insulin-producing cells (IPCs) and regulates HAB during satiety, whereas Dilp6 is secreted by the fat body and regulates HAB during the hungry state. We observed that Dilp2 induces PI3K/AKT signaling, whereas Dilp6 induces Ras/ERK signaling in α′β′ MBn to regulate HAB in different feeding conditions. Finally, we showed that the 2 α′β′-related MB output neurons (MBONs), MBON-α′3 and MBON-β′1, are necessary for the output of integrated hot avoidance information from α′β′ MBn. Our results demonstrate the presence of dual insulin modulation pathways in α′β′ MBn, which are important for suitable behavioral responses in Drosophila during thermoregulation under different feeding states.</div

MBON-α′3 and -β′1 output the converged information from α′β′ MBn.

(A) Optogenetic silencing of MBON-α′3 (labeled by MB027B-GAL4) activity inhibited HAB in both feeding states (Satiety: P-values: P-values: 0.0008, 0.0016, 0.0026, and 0.1663 from left to right). (B) Optogenetic silencing of MBON-β′1 (labeled by MB057B-GAL4) activity inhibited HAB in both feeding states (Satiety: P-values: P-values: 0.0004, 0.0001, C) Hot stimulus induced the calcium response in MBON-α′3 in sated flies (black), whereas an increased hot response was observed in hungry flies (red) (P = 0.0353). The GCaMP intensity changes (ΔF/F0) in MBON-α′3 dendrites were recorded and analyzed. (D) Hot stimulus induced the calcium response in MBON-β′1 in sated flies (black), whereas an increased hot response was observed in hungry flies (red) (P = 0.0134). The GCaMP intensity changes (ΔF/F0) in MBON-β′1 dendrites were recorded and analyzed. The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied. Each N represents either a group of 15 flies analyzed together in behavioral assays (A, B) or the response of a single fly in calcium imaging experiments (C, D). Data are represented as mean ±  SEM with dots representing individual values. The data underlying this figure can be found in S1 Data. Data were analyzed by one-way ANOVA followed by Tukey’s test (A, B) or the unpaired two-tailed t test (C, D). *P < 0.05. HAB, hot avoidance behavior; MBn, mushroom body neuron; MBON, mushroom body output neuron; SEM, standard error of mean.</p

InR manipulations in α′β′ MBn affects HAB.

(A) Genetic expression of InRDN in α′β′ MBn via VT57244-GAL4 > UAS-InRDN increased HAB during both sated and hungry states (Satiety: P-values: 0.0003, P-values: B) Genetic expression of InRDN in γ MBn via VT44966-GAL4 > UAS-InRDN had no effect on HAB during both sated and hungry states (Satiety: P-values: 0.3221, 0.9858, 0.6006, and 0.8822 from left to right; Hunger: P-values: 0.9897, 0.6707, 0.8446, and 0.5868 from left to right). (C) Genetic expression of InRDN in γ MBn via R16A06-GAL4 > UAS-InRDN had no effect on HAB during both sated and hungry states (Satiety: P-values: 0.3765, 0.8375, 0.9045, and 0.9183 from left to right; Hunger: P-values: 0.5816, 0.8305, 0.8359, and 0.7554 from left to right). (D) Genetic expression of InRDN in αβ MBn via VT49246-GAL4 > UAS-InRDN had no effect on HAB during both sated and hungry states (Satiety: P-values: 0.9815, 0.4656, 0.9837, and 0.9286 from left to right; Hunger: P-values: 0.9478, 0.9479, 0.9743, and 0.9928 from left to right). (E) Genetic expression of InRDN in αβ MBn via C739-GAL4 > UAS-InRDN had no effect on HAB during both sated and hungry states (Satiety: P-values: 0.9846, 0.7737, 0.7182, and 0.7518 from left to right; Hunger: P-values: 0.5773, 0.6921, 0.853, and 0.4112 from left to right). (F) Permissive temperature control of Fig 2E. At permissive temperatures, there were no significant differences in HAB between tub-GAL80ts; VT30604-GAL4 > InRDN, tub-GAL80ts; VT30604-GAL4 >+, and UAS-InRDN >+ flies during sated and hungry states (Satiety: P-values: 0.4415, 0.7193, 0.7097, and 0.9129 from left to right; Hunger: P-values: 0.9994, 0.7927, 0.9944, and 0.3847 from left to right). (G) Genetic expression of InRDN in α′β′ MBn via VT30604-GAL4 > UAS-InRDN had no effect on the cold avoidance behavior during sated and hungry states (Satiety: P-values: 0.4082, 0.9822, 0.9626, and 0.7241 from left to right; Hunger: P-values: 0.5038, 0.9253, 0.7114, and 0.6802 from left to right). (H) Genetic expression of InRDN in α′β′ MBn via VT57244-GAL4 > UAS-InRDN had no effect on the cold avoidance behavior during both sated and hungry states (Satiety: P-values: 0.6194, 0.7396, 0.8875, and 0.8173 from left to right; Hunger: P-values: 0.5038, 0.5851, 0.6177, and 0.7691 from left to right). Each N represents a group of 15 flies analyzed together in the behavioral assay. The data underlying this figure can be found in S1 Data. Data are represented as mean ± SEM with dots representing individual values and analyzed by one-way ANOVA followed by Tukey’s test, *P (TIF)</p

Manipulation of <i>dilp2</i> in IPCs affects HAB in sated state.

(A) HAB was not affected in dilp3 mutant flies (dilp3-/-) and dilp5 mutant flies (dilp5-/-) (Satiety: P-values: 0.6672, 0.4866, 0.8345, and 0.4314 from left to right; Hunger: P-values: 0.1133, 0.2915, 0.718, and 0.426 from left to right). (B) dilp2-GAL4 > UAS-dilp2RNAi flies exhibited an increased HAB during the sated state (Satiety: P-values: P-values: 0.6612, 0.7088, 0.2353, and 0.8943 from left to right). (C) Permissive temperature control of Fig 3C. At permissive temperatures, there were no significant differences in HAB between dilp2-GAL4; tub-GAL80ts > UAS-dilp2RNAi, dilp2-GAL4; tub-GAL80ts > +, and UAS-dilp2RNAi > + flies during the sated state (Satiety: P-values: 0.8918, 0.7176, 0.5073, and 0.7762 from left to right). (D) Immunostaining with anti-Dilp2 antibody in dilp2-GAL4 > UAS-mCD8::GFP flies (left panel). Quantification of anti-Dilp2 antibody immunostaining intensity in GFP positive IPCs during sated and hungry states (right panel). The anti-Dilp2 immunostaining signals in IPCs were normalized to the signals in fan-shaped body (P E) Hot stimulus did not induce the calcium response in IPCs during both feeding states. The soma of IPCs were recorded and analyzed. There were no significant differences in GCaMP intensity in IPCs before and after hot stimuli (P-values: 0.8381). The arrow under calcium response curve indicates the time points at which the hot stimuli were applied. (F) Permissive temperature control of Fig 3E. At permissive temperatures, there were no significant differences in HAB between dilp2-GAL4; tub-GAL80ts > UAS-dilp2, dilp2-GAL4; tub-GAL80ts > +, and UAS-dilp2 > + flies during sated and hungry states (Satiety: P-values: 0.5363, 0.3002, 0.8416, and 0.7235 from left to right; Hunger: P-values: 0.3413, 0.8279, 0.9225, and 0.4409 from left to right). Each N represents either a group of 15 flies analyzed together in the behavioral assay (A, B, C, F) or a single fly in Dilp2 immunostaining experiments (D) and calcium imaging experiments (E). The data underlying this figure can be found in S1 Data. Data are represented as mean ± SEM with dots representing individual values and analyzed by one-way ANOVA followed by Tukey’s test (A, B, C, F) or unpaired two-tailed t test (D, E). *P (TIF)</p

Manipulation of <i>dilp6</i> in the fat body affects Dilp2 expression in IPCs.

Immunostaining with anti-Dilp2 antibody in cg-GAL4 > UAS-dilp6RNAi (dilp6RNAi), cg-GAL4 > + (Control), and cg-GAL4 > UAS-dilp6 (dilp6) flies (left panel). Quantification of anti-Dilp2 immunopositive signals in IPC during sated and hungry states (right panel). The anti-Dilp2 immunostaining signals in IPCs were normalized to the signals in the fan-shaped body (Satiety: P-values: P-values: N represents a single fly in Dilp2 immunostaining experiments. Data are represented as mean ± SEM with dots representing individual values. The data underlying this figure can be found in S1 Data. Data were analyzed by unpaired two-tailed t test. *P (TIF)</p

Dilp6 produced by the fat body regulates ERK for HAB during the hungry state.

(A) dilp6 loss-of-function mutant flies (dilp6LOF) showed increased HAB when hungry (Satiety: P-values: 0.6176, 0.4785, 0.8634, and 0.7292 from left to right; Hunger: P-values: 0.0003, 0.0029, 0.0222, and 0.4018 from left to right). (B) α′β′ MBn showed increased hot response in dilp6LOF mutant background specifically when hungry but not when sated (P = 0.2043 for satiety; P = 0.0002 for hunger). (C) Adult-stage-specific knockdown of dilp6 in the fat body increased HAB in hungry but not in sated flies (Satiety: P-values: 0.7112, 0.4332, 0.748, and 0.4753 from left to right; Hunger: P-values: 0.0006, 0.0002, 0.0332, and 0.0732 from left to right). (D) Genetic knockdown of dilp6 in the fat body increased hot response of α′β′ MBn specifically in the hungry state (P = 0.8682 for satiety; P = 0.0328 for hunger). (E) Adult-stage-specific expression of dilp6 in the fat body decreased HAB in the hungry state (Satiety: P-values: 0.2437, 0.2066, 0.3148, and 0.2978 from left to right; Hunger: P-values: F) Genetic expression of dilp6 in the fat body decreased hot response of α′β′ MBn in the hungry state (P = 0.5214 for satiety; P = 0.002 for hunger). (G) Adult-stage-specific knockdown of Erk in α′β′ MBn increased HAB during hunger (Satiety: P-values: 0.9053, 0.6805, 0.4785, and 0.2593 from left to right; Hunger: P-values: H) Genetic knockdown of Erk in α′β′ MBn increased hot responses of α′β′ MBn during hunger but not during satiety (P = 0.2155 for satiety; P = 0.0361 for hunger). (I) Adult-stage-specific expression of Erk in α′β′ MBn decreased HAB during the hungry state (Satiety: P-values: 0.6868, 0.7727, 0.7495, and 0.0552 from left to right; Hunger: P-values: 0.001, 0.0003, 0.0002, and 0.0121 from left to right). (J) Genetic expression of Erk in α′β′ MBn decreased the hot response of α′β′ MBn in the hungry state (P = 0.4394 for satiety; P = 0.0003 for hunger). The arrows under each calcium response curve indicate the time points at which the hot stimulus was applied. Each N represents either a group of 15 flies analyzed together in the behavioral assay (A, C, E, G, I) or a single fly in calcium imaging experiments (B, D, F, H, J). Data are represented as mean ± SEM with dots representing individual values. The data underlying this figure can be found in S1 Data. Data were analyzed by one-way ANOVA followed by Tukey’s test (C, E, G, I) or the unpaired two-tailed t test (A, B, D, F, H, J). *P < 0.05; ns, not significant. HAB, hot avoidance behavior; MBn, mushroom body neuron; SEM, standard error of mean.</p