Fig 1.
Natural variation of starvation-induced hyperactivity in the DGRP.
(A) Representative plots of locomotor activity responses to starvation. Beam crosses in 30 min were plotted as a function of time. Black lines represent activities under the food condition (F) and red lines represent activities under the starvation condition (S). Black and white bars at the bottom of panel A represent night and day cycles, respectively. Each plot was from one DGRP strain (n = 8 per condition). (B) Natural variation occurred in SIH across DGRP. Error bars represent SEM.
Fig 2.
Manhattan plot for GWA, genomic location of significant SNPs, and GO analysis.
(A) Manhattan plot of SIH. The gray dotted line is the significant cutoff line used for gene nomination (P < 1 × 10-5). (B) Genomic location of significant SNPs/Indels. (C) GO terms associated with more than four genes. BP: Biological Process; CC: Cellular Component; MF: Molecular Function.
Table 1.
SNPs/Indels significantly associated with SIH variation at P < 1 × 10-5.
Table 2.
Known or predicted molecular functions of nominated genes.
Fig 3.
Knocking down of syp in neurons reduces SIH.
(A) Locomotor activity responses to starvation from neuron-specific syp knockdown flies (genotype: UAS-syp RNAi/nSyb-Gal4) and two parental control lines (genotypes: UAS-syp RNAi/+ and nSyb-Gal4/+, respectively). Gray lines represent activities under the food condition (F) and purple lines represent activities under the starvation condition (S). Black and white bars at the bottom of the panel represent night and day cycles, respectively. n = 16-20 per genotype per condition. (B) Summary plot of SIH in panel A. (C) Locomotor activity responses to starvation from neuron-specific syp knockdown flies generated from a second RNAi line (genotype: UAS-syp RNAi-2/+; nSyb-Gal4/+) and two parental control lines (genotypes: UAS-syp RNAi-2/+; +/+ and +/+; nSyb-Gal4/+, respectively). n = 20-24 per genotype per condition. (D) Summary plot of SIH in panel C. ** P < 0.01, *** P < 0.001, unpaired t-test with Bonferroni Correction. Error bars represent SEM.
Fig 4.
Syp in adult neurons regulates SIH.
(A) Breeding and testing scheme. S: starvation condition; F: food condition. (B) Locomotor activity responses to starvation from each genotype under the condition shown in panel A. Gray lines represent activities under the food condition (F) and green lines represent activities under the starvation condition (S). n = 24-30 per genotype per condition. (C) Summary plot of SIH from each genotype in panel B. (D) Breeding and testing scheme. S: starvation condition; F: food condition. (E) Locomotor activity responses to starvation from each genotype under the condition shown in panel D. n = 8-28 per genotype per condition. (F) Summary plot of SIH from each genotype in panel E. (G) Western blot of adult fly head homogenate from Tub-Gal80ts/+;nSyb-Gal4/UAS-syp RNAi (ts-syp-KD) and +/+;nSyb-Gal4/UAS-syp RNAi (syp-KD) flies. Four biological replicates per genotype per condition. Tubulin is the loading control. (H) Quantification of Syp from four biological replicates. * P < 0.05, ** P < 0.01, *** P < 0.001, n.s.: P > 0.05, unpaired t-test with Bonferroni Correction. Error bars represent SEM.
Fig 5.
syp is alternatively spliced under starvation.
(A) Differential usage of exons in syp under food and starvation conditions. Four exons involved in the alternative splicing were shown and referred to as exon a, exon b, exon c, and exon d. PSI: Percentage Sliced In. (B) Various splice variants of syp (www.flybase.org). Arrows indicate splice variants that were either downregulated (red color) or upregulated (purple) under starvation. (C) Relative expression of targeted exons measured by qPCR. The targeted locations of each pair of primers were indicated in panel A. * P < 0.05, n.s.: P > 0.05, unpaired t-test, n = 4 per condition. Error bars represent SEM.
Fig 6.
Deletion of exons b and c in syp leads to reduced SIH.
(A) Generation of syp exons b and c deletion lines using CRISPR/Cas9. Two sgRNAs that target the intronic regions adjacent to exon b and exon c (symbol X in red) cut each side of the targeted region, respectively. The sequences of sgRNAs were show in the rectangles. A pair of primers (genoF and genoR) were used to amplify the targeted region for sequencing. Exons b and c were deleted in the syp1 allele based on sequencing results. (B) Locomotor activity responses to starvation from each genotype under either food (F) or starvation (S) conditions. n = 16—26 per genotype per condition. Df-1: Df(3R)BSC124; Df-2: Df(3R)BSC141. (C) Summary plot of SIH from each genotype in panel B. ** P < 0.01, unpaired t-test with Bonferroni Correction. Error bars represent SEM.