Fig 1.
Sulfakinin (NlSK) and its receptor (SKR) signal satiety and inhibit feeding in the rice planthopper.
(A) Injection of N. lugens sulfated sulfakinins inhibits food intake in the brown planthopper. Fifty nanoliter of PBS (as control) and four sulfakinins (20 pmol/insect) were injected into 4rd instar nymph of brown planthopper. Non-sulfated Sulfakinins (nsNlSK1 and 2) have no effect. All data are presented as means ± s.e.m in this manuscript. ns: not significant, ***p < 0.001, ****p < 0.0001; One-way ANOVA followed by Tukey’s multiple comparisons test. (B) Refeeding for 5 hr after 24 hr starvation increases Nlsk mRNA transcript. *p < 0.05; Mann–Whitney test. (C) NlSK expression in the brain is revealed by anti-NlSK1/2 (green) and anti-nc82 (red). Two pairs of MP1 (medial protocerebrum), two pairs of MP3 neurons, and median neurosecretory cells (MNCs) are indicated. Bar: 50 μm. (D and E) Refeeding after 24h starvation increases NlSK peptide expression as revealed by anti-NlSK1/2 immunolabeling. Scale bars, 50 μm. **p < 0.01; Student’s t test. Note that we measure SK levels in neuronal MP1 cell bodies where production occurs. (F) Downregulation of Nlsk gene using Nlsk-RNAi leads to a reduction in mRNA expression level. ****p < 0.0001; Student’s t test. (G and H) Downregulation of Nlsk gene using Nlsk-RNAi (dsNlsk) leads to a reduction in NlSK1/2 immunoreactivity. In (G) we show NlSK1/2 immunoreactivity in brains of dsgfp- or dsNlsk-injected planthoppers. Scale bar: 50 μm. (H) Intensity of anti-NlSK1/2 immunoreactivity in brain. All data are presented as means ± s.e.m. ****p < 0.0001; Student’s t test. (I) Downregulation of Nlsk gene using Nlsk-RNAi (dsNlsk) increases the food intake. **p < 0.01; Mann–Whitney test. (J) Simplified model showing that NlSK inhibits feeding. Note that this model and those in subsequent figures of planthopper data are highly simplified and serve to summarize our data, rather that presenting accurate signaling pathways. The question mark indicates one or more possibly additional signals.
Fig 2.
Global gene expression profile in the rice planthopper after Nlsk gene knockdown.
(A) Heatmap showing the square of correlation value from four biological replicates of dsNlsk and dsgfp groups analyzed by RNA-seq. The square of correlation value was assessed by using the Pearson correlation. (B) Volcano plot of differentially expressed genes comparing the dsgfp and dsNlsk treated brown planthoppers. The red dots indicate significantly (p < 0.05 and > 2-fold) upregulated genes. The black dots indicate significantly (p < 0.05 and > 2-fold) downregulated genes. (C) WEGO (Web Gene Ontology Annotation Plotting) output for Nlsk regulated genes. The histogram shows the percent of genes with GO terms enriched in each category. Arrows point to interesting GO categories affected by Nlsk knockdown. Hypergeometric test (FDR-adjusted): black arrow indicated p < 0.05. (D) The pie chart shows the percentage of regulated transcripts distribution in different pathways/processes identified by KEGG pathway analysis.
Fig 3.
Takeout (to) is a downstream signal of SK that inhibits food intake in the rice planthopper.
(A) Knockdown of Nlsk results in downregulation of Nlto gene in the brown planthopper. All data are presented as means ± s.e.m. **p < 0.01; Mann–Whitney test. (B) Injection of sNlSK2 increases gene expression level of Nlto. *p < 0.05; Mann–Whitney test. (C) Model of the NlSK promotes Nlto expression. (D) Downregulation of Nlto gene using Nlto-RNAi leads to a reduction in mRNA expression level. ****p < 0.0001; Student’s t test. (E) Knockdown of Nlto have no impacts on expression of Nlsk gene in brown planthopper. All data are presented as means ± s.e.m. ns, no significant difference; Mann–Whitney test. (F) Model showing that Nlto has no effects on Nlsk expression. (G) Food intake after silencing Nlto gene. The dsNlto-injected nymphs eat three times more food than dsgfp-injected nymphs in the normal conditions. All data are presented as means ± s.e.m. ***p < 0.001; Mann–Whitney test. (H) Food intake after silencing the Nlto gene with injection of pbs or sNlSK2. No difference was observed between these two conditions. All data are presented as means ± s.e.m. ns, no significant difference; Student’s t test. (I) Model of the Takeout in the food inhibition. (J) Simplified model of the Takeout as a downstream signal of NlSK involved in the feeding inhibition. The question mark indicates one or more possibly additional signals.
Fig 4.
Sulfakinin inhibits expression of the sweet gustatory receptor Gr64f, which promotes food ingestion in the rice planthopper.
(A) Downregulation of Nlsk gene using Nlsk-RNAi (dsNlsk) leads to up-regulation of transcript of sweet sensing NlGr64f. *p < 0.05; Mann–Whitney test. (B) Injection of sNlSK2 leads to down-regulation of NlGr64f gene. *p < 0.05; Mann–Whitney test. (C) Model showing that NlSK inhibits NlGr64f expression. (D) Downregulation of Nlto gene using Nlto-RNAi (dsNlto) leads to up-regulation of transcript of sweet sensing NlGr64f. *p < 0.05; Mann–Whitney test. (E) Model showing that Nlto inhibits NlGr64f expression. (F) Refeeding for 5 hr after 24 hr starvation decreases NlGr64f transcript. *p < 0.05; Mann–Whitney test. (G) Downregulation of NlGr64f gene using NlGr64f-RNAi (dsNlGr64f) leads to a reduction in mRNA expression level. ****p < 0.0001; Student’s t test. (H) Downregulation of NlGr64f gene decreases the food intake of brown planthopper. ****p < 0.0001; Mann–Whitney test. (I) Double knockdown of Nlto and NlGr64f does not rescue the increased food consumption seen with Nlto knockdown alone. All data are presented as means ± s.e.m. ns, no significant difference; Student’s t test. (J) Simplified model showing that the feeding state regulates SK signaling which in turn modulates Nlto and NlGr64f signaling (sweet sensing) and thereby feeding. The question mark indicates one or more possibly additional signals.
Fig 5.
Drosulfakinin (DSK) signals satiety and inhibits feeding in Drosophila.
(A) Refeeding after 24h starvation increases Dsk transcript in the head of Drosophila. ****p < 0.0001; Student’s t test. (B and C) Refeeding after 24h starvation increases DSK peptide expression as revealed by anti-DSK1/2 immunolabeling. Yellow arrows indicated the MP1 and MP3 neurons. Scale bars, 50 μm. **p < 0.01; Student’s t test. (D) Representative optical recordings of spontaneous membrane activity in median protocerebrum (MP1) neurons (Somata) in the starved (0.5% agar for 24 hours) or in the refed (fed 1.5 h after 24 h starvation in 0.5% agar) of flies that Dsk-GAL4 drive the voltage indicator ArcLight maintained in 12 hr:12 hr light:dark conditions. (E) Standard deviations (SDs) over the recording trial were computed for each field. Refed SD is significantly greater than starved. *p < 0.05; Student’s t test. (F) Power spectrum was computed for each terminal field using fast Fourier transform with 0.05 Hz bin width. Re-fed power is significantly greater than starved power between 1 Hz to 2.5 Hz. n = 14 for starved and n = 30 for refed. *p < 0.05, **p < 0.01; Student’s t test. (G) Representative images showing CaLexA signals in DSK expressing MP1 neurons of flies exposed to starvation and refeeding. Starved: flies were raised in 0.5% agar for 24 hours; Refed: flies were raised in fly food 1.5 h after 24 h starvation in 0.5% agar. Red: maximal intensity of CaLexA signals. Scale bar, 50 μm. (H) Quantification of the signal intensity of CaLexA signals in DSK expressing MP neurons from flies treated with conditions shown in (G). **p < 0.01; Student’s t test. (I) Optogenetic activation (19 μm/mm2) of Dsk-GAL4 neurons expressing Chrimson is sufficient to inhibit proboscis responses. A fly immobilized in a pipet tip was stimulated on the labellum with different concentrations of sucrose in the presence of light stimulus. We used two values to score the PER assay. A score of 1.0 indicates a fly that extended its proboscis and ingested after being presented with the probe. The score was 0 if the fly failed to extend its proboscis. n = 10 trials. **p < 0.01, ****p < 0.0001; Mann–Whitney test. (J) Three Dsk mutants show increased food consumption compared to wildtype in the CAFE essay. **p<0.01, ***p<0.001, ****p<0.0001, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (K) Simplified model showing that DSK inhibits feeding. Note that this model and those in subsequent figures with Drosophila data are highly simplified and serve to summarize our data, rather that presenting complete and accurate signaling pathways. The question mark indicates one or more possibly additional signals.
Fig 6.
In Drosophila, Gr64f promotes feeding.
(A) Refeeding after 24h starvation decreases Gr64f transcript. ****p < 0.0001; Student’s t test. (B) The sugar blind mutant (all eight known sugar receptors mutated) show almost no motivation to feed in proboscis extension reflex (PER). n = 10 trials. ***p < 0.001; Mann–Whitney test. (C) Gr64f mutant (Gr64fLexA) flies show less motivation to feed in PER. n = 10 trials. ***p < 0.001, **p < 0.01, ns, no significant; Mann–Whitney test. (D) Silencing Gr64f-GAL4 neurons by expressing the hyperpolarizing channel Kir2.1 also caused loss of motivation to feed in PER. n = 10 trials. ***p < 0.001, **p < 0.01, *p < 0.05; Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (E) Silencing the Gr64f gene using the Gr5a-GAL4 driver leads to decreased food consumption compared to wildtype flies in the CAFE essay. *p < 0.05; Student’s t test. Food intake is the estimated food consumption (μl) of one female per day. (F) Gr64f mutant (Gr64fLexA) flies show decreased food consumption compared to wildtypes in the CAFE essay. **p < 0.01; Student’s t test. (G) Silencing Gr64f-GAL4 neurons by expressing the hyperpolarizing channel Kir2.1 also leads to decreased food consumption in the CAFE assay. ****p < 0.0001; Mann–Whitney test. (H) Dynamics of light-induced proboscis extension after photoactivation in a fly expressing Gr64f-Gal4, UAS-CsChrimson. (I) Relationship between the intensity of the stimulating light and PER rate using the indicated flies. A fly immobilized in a pipet tip was stimulated with red light at the indicated intensity and the light-induced proboscis extension of the fly was recorded. We used two values to score the PER assay. A score of 1.0 indicates a fly that extended its proboscis after being presented with the light. The score was 0 if the fly failed to extend its proboscis. n = 8 trials. **p < 0.01, *p < 0.05; Mann–Whitney test. (J) Increasing the baseline activity of sweet GRNs (UAS-Chrimson (X)/+;; +/Gr5a-GAL4) by photo-activation with 0.78 μW/mm2 of red light increases the probability that the fly shows PER upon sugar stimulation (1 mM sucrose) compared with either red light stimulation (0.78 μW/mm2) or 1 mM sucrose alone. *p < 0.05; Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (K) Simplified model showing that Gr64f promotes feeding.
Fig 7.
In Drosophila DSK and TO inhibits Gr64f gene transcription and optogenetic activation of Dsk-GAL4 neurons decreases the sensitivity of gustatory neurons in starved flies.
(A and B) Relative expression levels of Gr64f transcripts in DSK deficient files. Depletion of Dsk, either globally in all cells (Actin5C-GAL4) or in the Dsk-GAL4-expressing cells increased Gr64f transcripts in starved flies. ****p < 0.0001; Student’s t test. (C) Silencing only Dsk in the IPCs with RNAi (Dilp2-GAL4/Dsk-RNAi; red bars) did not affect Gr64f transcript levels. ns: not significant; Mann–Whitney test. (D) Relative expression levels of to transcripts in Dsk deficient files. Depletion of Dsk in all cells (Actin5C-GAL4) decreased to transcripts in flies. *p < 0.05; Mann–Whitney test. (E) Relative expression levels of Gr64f transcripts in TO deficient files. Depletion of to in all cells (Actin5C-GAL4) increased Gr64f transcripts in flies. **p < 0.01; Mann–Whitney test. (F) Expression pattern of Dsk-GAL4 in the brain revealed by anti-GFP (F1) and anti-DSK (F2). Scale bars, 50 μm. (G) Electrophysiological recordings reveal that starved flies (24 h) show increased sensitivity of GRNs compared with refed flies (fed 1.5 h after starvation) and optogenetic activation of Dsk-GAL4 neurons (30 min) decreases the sensitivity of gustatory neurons in starved flies (24 h). **p < 0.001; *p < 0.05; Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (H) Simplified model of the feeding state, DSK, TO and Gr64f interaction.
Fig 8.
In Drosophila the DSK receptor, CCKLR-17D3, is expressed in Gr64fLexA expressing neurons.
(A) 17D3GAL4, UAS-Stinger-GFP (green) superimposes with Gr64fLexA, LexAop > tdTomato (magenta) expressing cells in the labellum (A1-A4) and proleg tarsi (A5-A8) of flies. Overlap is in white. The white arrowheads (A1 and A5) indicate the positive neural cells co-labeled by 17D3GAL4 and Gr64LexA. Scale bar: 50 μm. (B) Double labeling of 17D3GAL4-expressing neurons and Gr64fLexA-expressing sweet neurons in the brain (B1), SEZ (B2), and VNC (B3-B5). SEZ: subesophageal zone; VNC: ventral nerve cord; T1-T3: thoracic ganglion 1–3. Scale bar: 50 μm.
Fig 9.
In Drosophila activity of the DSK receptor (CCKLR-17D3) is necessary for PER.
(A and B) Refeeding after 24h starvation decreases 17d3 mRNA transcript in the labellum and proleg tarsi. All data are presented as means ± s.e.m. **p < 0.01; Mann–Whitney test. (C and D) Silencing of the 17d3 gene in sweet GRNs leads to downregulation of Gr64f and upregulation of the to gene. All data are presented as means ± s.e.m. **p < 0.01, ***p < 0.001; Student’s t test. (E) 17D3 mutants show decreased motivation to feed in PER. **p < 0.01; Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (F) Silencing of 17D3 gene in Gr64f-GAL4-expressing neurons showed decreased motivation to feed in PER. *p < 0.05; Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (G) Simplified model of SK as satiety signal that reflects internal states and inhibits sweet sensation (an external stimulus). Note that this model is simplified and does not exclude additional parallel signaling pathways that modulate gustation and feeding. The question mark indicates one or more possibly additional signals.