Quantification of diapause in Drosophila Genetic Reference Panel (DGRP) lines measured as the ratio of post-diapause to non-diapause fecundity.

(A) Schematic of experimental workflow. (B) Average number of progenies produced (fecundity) in a 4-day individual female fly mating experiment of DGRP lines either as non-diapausing (yellow) or after a 35-day post-diapause (blue) virgin flies. Each dot represents the average fecundity and the line above represents the standard error. DGRP line numbers are indicated wherever the post-diapause fecundity exceeds the non-diapause fecundity. (C) Normalized post-diapause fecundity [average of (individual post-diapause fecundity/mean non-diapause fecundity)] of each DGRP line. (D) Correlation of post-diapause to non-diapause fecundity. Pearson’s r=0.5682.r2=0.3228. (E-F) Frequency distribution of DGRP lines fecundity under non-diapause (E) and of the normalized post-diapause fecundity (F). (G) Average 4-day fecundity of single female flies, each crossed with 2 young Canton S male flies, aged for 1, 35, or 42 days in non-diapause conditions or kept in diapause conditions for 35 or 42 days followed by recovery. 1-way ANOVA and Tukey’s multiple comparison test, compact letter display shows comparisons. n is the number of individual female fly fecundity measured.

Genome-wide association of Drosophila diapause.

(A) Manhattan plot for genome-wide association distribution. The position of each point along the y-axis indicates -log10(P-value) of association of a SNP, insertion, or deletion. Points above the blue line have a P value < 1e-5. The red line represents Bonferroni corrected P value = 4.8e-8. (B) Q-Q plot of P values from the DGRP single variant GWAS with the red line representing expected P-value and observed P values deviating (black dots) from expected. (C) Numbers of genetic variants and candidate genes associated with diapause according to the GWAS. (D) Subnetworks from the Cytoscape analysis showing q-value (using the Benjamini-Hochberg procedure) for each subnetwork identified.

Common genes to diapause-GWAS hits and other behavior-associated genes.

(A-T) Venn diagrams illustrate the intersection of genes associated with diapause identified through Genome-Wide Association Studies (diapause-GWAS) with genes from other behavior-related gene lists obtained from various studies. The diapause-GWAS gene set is represented as the first set throughout the figure, while subsequent sets represent different behavior-related gene lists identified in separate studies. The percentage of common genes compared to the total genes from different respective behavior-associated gene lists are provided for each Venn diagram. p-values of overlap to the diapause gene list determined by Fisher’s exact tests are also provided. Venn diagrams are arranged in the order of p-values.

RNAi-mediated loss of function study to identify genes involved in diapause.

(A-C) Mat-ɑ-tub-Gal4 driving expression of UASp-F-tractin.tdTomato (red) at the indicated temperatures. Scale bars are 100µm. (D) Quantification of zpg RNAi knockdown in Stage-3 egg chambers normalized to the level of Zpg in the germarium. (1-way ANOVA and Tukey’s multiple comparison test, compact letter display shows comparisons). The numbers (n) of Stage 3 egg chambers quantified are shown. (E-H) Representative images of egg chambers stained with anti-Zpg antibody (green) from either control (no-knockdown), (E, E’) or knockdown of zpg (F-H’) driven by Mat-ɑ-tub-Gal4 at different temperatures. (E’-H’) are higher magnification, single channel views of the ovarioles shown in (E-H). Scale bars are 100 µm for (E-H) and 20 µm in (E’-H’). All flies in (A-H’) were kept at respective temperatures for 3 weeks. (I) Experimental design for RNAi knockdown specifically during recovery for the experiment shown in (J). The temperature-sensitive Gal80 repressor of Gal4 prevented RNAi expression during development and diapause. Incubation at 30°C during recovery inactivates Gal80, allowing Gal4-mediated RNAi knockdown. (J) Ubiquitous knockdown of Dip-γ or sbb with tub Gal4 specifically during recovery as shown in (I) significantly reduces post-diapause/non-diapause fecundity compared to the control (tubGal80TS;tubGal4 > Ctrl RNAi #9331). (K) Pan-neuronal RNAi knockdown of Dip-γ and sbb with nSybGal4 significantly reduces post-diapause/non-diapause fecundity compared to the control (nSyb Gal4> Ctrl RNAi #54037). (L) Glia-specific knockdown of Dip-γ or sbb with Repo Gal4 causes little or no reduction in post-diapause/non-diapause fecundity (Control-Repo Gal4> Ctrl RNAi #54037). In (J-L), 1-way ANOVA and Tukey’s multiple comparison test, compact letter display shows comparisons. n is the number of individual female flies tested.

Neural control of diapause.

(A) Effect of antenna removal on recovery of fecundity post-diapause. Arista removal was used as a control for the surgery. n is the number of individual female flies tested. 1-way ANOVA and Tukey’s multiple comparison test, compact letter display shows comparisons. (B) Effect of antenna removal on GSC recovery after 5 weeks of diapause. n is the number of germaria counted (there are typically 2-3 GSCs/germarium). 1-way ANOVA and Tukey’s multiple comparison test with compact letter display to show comparisons. (C) Role of antenna in lifespan extension in diapause. Control flies were maintained at 25°C and diapause flies were moved to 10°C. Median survival for flies with intact antenna in diapause (+A_Diap) - 142.5 days; antennaless flies in diapause (-A_Diap) - 7 days; Control with intact antenna in optimal conditions (+A_Ctrl) - 75 days; and antennless flies in optimal conditions (-A_Ctrl) - 72 days. Survival curves are compared pairwise using the Log-rank (Mantel-Cox) test and ꭓ2 values are: Diap +/- antenna = 98.89, Ctrl +/- antenna = 11.69, Diap +antenna vs Ctrl +antenna = 163.5, and Diap-antenna vs Ctrl-antenna = 0.5. (D) Control (+A_Diap) and antennaless (-A_Diap) flies were maintained at 25°C for 2 weeks post-surgery to allow for wound healing and shifted to the diapausing conditions. Median survival for +A_Diap - 142 days; -A_Diap - 95 days. Survival curves are compared pairwise using the Log-rank (Mantel-Cox) test and ꭓ2 values =86. (E-F) Effects on diapause of inactivating neuronal transmission (by driving tetanus toxin using UAS-TeTxLC.tdt, (E) in odor responsive neurons using the indicated Gal4 lines. Orco is the co-receptor for all of the odorant receptors. (F) Ir8a is a co-receptor involved in organic acid detection. Ir25a is a co-receptor involved in chemo- and thermo-sensation. Ir76b is involved in detection of various amines and salt. Ir84a is involved in detection of phenylacetic acid and male courtship behavior. Hot Cells are heat-sensitive cells in the arista.