Sex differences in expression of gut-derived peptide hormones.

(A-E) mRNA levels of AstA (p<0.0001; Student’s t-test) (A), AstC (p=0.0002; Mann-Whitney test) (B), Tk (p<0.0001; Student’s t-test) (C), NPF (p=0.0001; Student’s t-test) (D), Dh31 (p=0.0002; Mann-Whitney test) (E) in whole-body were significantly higher in 5-day-old w1118 males compared to females. n=7-8 biological replicates. (F-J) mRNA levels of AstA (p<0.0001; Student’s t-test) (F), AstC (p=0.001; Student’s t-test) (G), Tk (p<0.0001; Student’s t-test) (H), NPF (p=0.0015; Student’s t-test) (I), Dh31 (p<0.0001; Student’s t-test) (J) in heads were significantly higher in 5-day-old w1118 males compared to females. n=8-10 biological replicates. (K-O) mRNA levels of AstC (p=0.0002; Student’s t-test) (K), Tk (p<0.0001; Student’s t-test) (L), NPF (p=0.0006; Mann-Whitney test) (M) in guts were significantly higher in 5-day-old w1118 females compared to males. n=7 biological replicates. (N-O) mRNA levels of AstA (p=0.5039; Student’s t-test) (N), Dh31 (p=0.7517; Student’s t-test) (O) in guts were not significantly different between 5-day-old w1118 females and males. n=7 biological replicates. All data plotted as mean ± SEM. ns indicates not significant with p>0.05; ** p<0.01, *** p<0.001, **** p<0.0001. See also Figure S1.

Sex determination gene transformer does not regulate sex differences in EE cell-derived peptide mRNA levels.

(A) mRNA levels of AstA in gut were not significantly different between voila-GAL4>UAS-traF females and voila-GAL4>+ and +>UAS-traF control females (p>0.9999 and p>0.9999, respectively). mRNA levels of AstA in gut were significantly higher in voila-GAL4>UAS-traF males compared to +>UAS-traF control males, but were not significantly different from voila-GAL4>+ control males (p=0.0084 and p>0.9999, respectively) (sex:genotype interaction p<0.0001). Two-way ANOVA followed by Bonferroni post-hoc test; n=5 biological replicates. mRNA levels of AstC in gut were not significantly different between voila-GAL4>UAS-traF females and voila-GAL4>+ and +>UAS-traF control females (p=0.6814 and p=1.0, respectively). mRNA levels of AstC in gut were significantly higher in voila-GAL4>UAS-traF males compared to +>UAS-traF control males, but were not significantly different from voila-GAL4>+ control males (p=0.0463 and p=0.9965, respectively) (sex:genotype interaction p=0.1078). Two-way ANOVA followed by Tukey HSD on data processed using the aligned rank transform for non-parametric data; n=5 biological replicates. (B) mRNA levels of Tk in gut were not significantly different between voila-GAL4>UAS-traF females and voila-GAL4>+ and +>UAS-traF control females (p=0.2258 and p>0.9999, respectively). mRNA levels of Tk in gut were significantly higher in voila-GAL4>UAS-traF males compared to +>UAS-traF control males, but were significantly lower compared to voila-GAL4>+ control males (p=0.0004 and p=0.0006, respectively) (sex:genotype interaction p=0.0004). Two-way ANOVA followed by Bonferroni post-hoc test; n=5 biological replicates. (C) mRNA levels of NPF in gut were not significantly different between voila-GAL4>UAS-traF females and voila-GAL4>+ and +>UAS-traF control females (p=0.1579 and p=0.3389, respectively). mRNA levels of NPF in gut were not significantly different between voila-GAL4>UAS-traF males and voila-GAL4>+ and +>UAS-traF control males (p=0.6639 and p=0.9043, respectively) (sex:genotype interaction p=0.1655). Two-way ANOVA followed by Bonferroni post-hoc test; n=5 biological replicates. (D) mRNA levels of Dh31 in gut were significantly lower in voila-GAL4>UAS-traF females compared to voila-GAL4>+ control females, but were not significantly different from +>UAS-traF control females (p=0.0439 and p=0.9745, respectively). mRNA levels of Dh31 in gut were not significantly different between voila-GAL4>UAS-traF males and voila-GAL4>+ and +>UAS-traF control males (p=0.9953 and p=0.1370, respectively) (sex:genotype interaction p=0.1945). Two-way ANOVA followed by Tukey HSD on data processed using the aligned rank transform for non-parametric data; n=5 biological replicates. (E) mRNA levels of AstA in head were not significantly different between voila-GAL4>UAS-traF females and voila-GAL4>+ and +>UAS-traF control females (p>0.9999 and p=0.3344, respectively). mRNA levels of AstA in head were not significantly different between voila-GAL4>UAS-traF males and voila-GAL4>+ and +>UAS-traF control males (p>0.9999 and p>0.9999, respectively) (sex:genotype interaction p=0.2471). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (F) mRNA levels of AstC in head were significantly lower in voila-GAL4>UAS-traF females compared to +>UAS-traF control females, but were not significantly different from voila-GAL4>+ control females (p<0.0001 and p>0.9999, respectively). mRNA levels of AstC in head were significantly lower in voila-GAL4>UAS-traF males compared to +>UAS-traF control males, but were not significantly different from voila-GAL4>+ control males (p=0.0236 and p=0.6687, respectively) (sex:genotype interaction p=0.0189). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (G) mRNA levels of Tk in head were significantly lower in voila-GAL4>UAS-traF females compared to +>UAS-traF control females, but were not significantly different from voila-GAL4>+ control females (p=0.0044 and p>0.9999, respectively). mRNA levels of Tk in head were significantly higher in voila-GAL4>UAS-traF males compared to both voila-GAL4>+ and +>UAS-traF control males (p=0.0120 and p=0.0156, respectively) (sex:genotype interaction p=0.0003). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (H) mRNA levels of NPF in head were significantly lower in voila-GAL4>UAS-traF females compared to +>UAS-traF control females, but were not significantly different from voila-GAL4>+ control females (p=0.0006 and p>0.9999, respectively). mRNA levels of NPF in head were not significantly different between voila-GAL4>UAS-traF males and voila-GAL4>+ and +>UAS-traF control males (p=0.5030 and p=0.6158, respectively) (sex:genotype interaction p=0.1408). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (I) mRNA levels of Dh31 in head were significantly lower in voila-GAL4>UAS-traF females compared to +>UAS-traF control females, but were not significantly different from voila-GAL4>+ control females (p=0.0003 and p>0.9999, respectively). mRNA levels of Dh31 in head were not significantly different between voila-GAL4>UAS-traF males and voila-GAL4>+ and +>UAS-traF control males (p>0.9999 and p>0.9999, respectively) (sex:genotype interaction p=0.0403). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (J) mRNA levels of AstA in gut were not significantly different between elav-GAL4>UAS-traF females and elav-GAL4>+ and +>UAS-traF control females (p>0.9999 and p=0.0534, respectively). mRNA levels of AstA in gut were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p=0.5496 and p=0.1858, respectively) (sex:genotype interaction p=0.6125). Two-way ANOVA followed by Bonferroni post-hoc test; n=6 biological replicates. (K) mRNA levels of AstC in gut were not significantly different between elav-GAL4>UAS-traF females and elav-GAL4>+ and +>UAS-traF control females (p=0.5948 and p=0.0878, respectively). mRNA levels of AstC in gut were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p=0.1745 and p=0.1745, respectively) (sex:genotype interaction p=0.4992). Two-way ANOVA followed by Tukey HSD on data processed using the aligned rank transform for non-parametric data; n=6 biological replicates. (L) mRNA levels of Tk in gut were significantly lower in elav-GAL4>UAS-traF females compared to +>UAS-traF control females, but were not significantly different from elav-GAL4>+ control females (p=0.0269 and p>0.9999, respectively). mRNA levels of Tk in gut were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p>0.9999 and p=0.2110, respectively) (sex:genotype interaction p=0.5212). Two-way ANOVA followed by Bonferroni post-hoc test; n=6 biological replicates. (M) mRNA levels of NPF in gut were not significantly different between elav-GAL4>UAS-traF females and elav-GAL4>+ and +>UAS-traF control females (p>0.9999 and p=0.1158, respectively). mRNA levels of NPF in gut were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p>0.9999 and p=0.6652, respectively) (sex:genotype interaction p=0.6546). Two-way ANOVA followed by Bonferroni post-hoc test; n=6 biological replicates. (N) mRNA levels of Dh31 in gut were not significantly different between elav-GAL4>UAS-traF females and elav-GAL4>+ and +>UAS-traF control females (p=0.5442 and p=0.8086, respectively). mRNA levels of Dh31 in gut were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p=0.1650 and p=0.5258, respectively) (sex:genotype interaction p=0.9566). Two-way ANOVA followed by Tukey HSD on data processed using the aligned rank transform for non-parametric data; n=6 biological replicates. (O) mRNA levels of AstA in head were not significantly different between elav-GAL4>UAS-traF females and elav-GAL4>+ and +>UAS-traF control females (p=0.9843 and p=0.7086, respectively). mRNA levels of AstA in head were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p=0.0628 and p=0.9936, respectively) (sex:genotype interaction p=0.0171). Two-way ANOVA followed by Tukey HSD on data processed using the aligned rank transform for non-parametric data; n=5-6 biological replicates. (P) mRNA levels of AstC in head were not significantly different between elav-GAL4>UAS-traF females and elav-GAL4>+ and +>UAS-traF control females (p=0.1253 and p=0.8540, respectively). mRNA levels of AstC in head were significantly lower in elav-GAL4>UAS-traF males compared to +>UAS-traF control males, but were not significantly different from elav-GAL4>+ control males (p=0.0188 and p=0.9086, respectively) (sex:genotype interaction p=0.0198). Two-way ANOVA followed by Tukey HSD on data processed using the aligned rank transform for non-parametric data; n=5-6 biological replicates. (Q) mRNA levels of Tk in head were not significantly different between elav-GAL4>UAS-traF females and elav-GAL4>+ and +>UAS-traF control females (p=0.6051 and p=0.9999, respectively). mRNA levels of Tk in head were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p=0.3600 and p=0.2760, respectively) (sex:genotype interaction p=0.2324). Two-way ANOVA followed by Tukey HSD on data processed using the aligned rank transform for non-parametric data; n=5-6 biological replicates. (R) mRNA levels of NPF in head were significantly lower in elav-GAL4>UAS-traF females compared to both elav-GAL4>+ and +>UAS-traF control females (p=0.0347 and p=0.0273, respectively). mRNA levels of NPF in head were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p=0.0656 and p=0.6253, respectively) (sex:genotype interaction p=0.6872). Two-way ANOVA followed by Tukey HSD on data processed using the aligned rank transform for non-parametric data; n=5-6 biological replicates. (S) mRNA levels of Dh31 in head were not significantly different between elav-GAL4>UAS-traF females and elav-GAL4>+ and +>UAS-traF control females (p>0.9999 and p>0.9999, respectively). mRNA levels of Dh31 in head were not significantly different between elav-GAL4>UAS-traF males and elav-GAL4>+ and +>UAS-traF control males (p=0.2918 and p=0.5990, respectively) (sex:genotype interaction p=0.4360). Two-way ANOVA followed by Bonferroni post-hoc test; n=5-6 biological replicates. All data plotted as mean ± SEM. ns indicates not significant with p>0.05; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. See also Figure S2.

Gut-derived Tachykinin and Allatostatin C promote female fat storage.

(A) Whole-body triglyceride levels were significantly lower in AstC-GAL4>UAS-AstC-RNAi,R57C10-GAL80 females compared to AstC-GAL4>+,R57C10-GAL80 and +>UAS-AstC-RNAi control females (p<0.0001 and p<0.0001, respectively). Whole-body triglyceride levels were not significantly different between AstC-GAL4>UAS-AstC-RNAi,R57C10-GAL80 males and AstC-GAL4>+,R57C10-GAL80 and +>UAS-AstC-RNAi control males (p=0.4527 and p=0.1056, respectively) (sex:genotype interaction p<0.0001). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (B) Whole-body triglyceride levels were significantly lower in Tk-GAL4>UAS-Tk-RNAi,R57C10-GAL80 females compared to +>UAS-Tk-RNAi control females (p=0.0118) but were not significantly different from Tk-GAL4>+,R57C10-GAL80 control females (p=0.1109). Whole-body triglyceride levels were significantly higher in Tk-GAL4>UAS-Tk-RNAi,R57C10-GAL80 males compared to Tk-GAL4>+,R57C10-GAL80 control males (p<0.0001) but were not significantly different from +>UAS-Tk-RNAi control males (p=0.5704) (sex:genotype interaction p<0.0001). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (C) Whole-body triglyceride levels were not significantly different between NPF-GAL4>UAS-NPF-RNAi,R57C10-GAL80 females and NPF-GAL4>+,R57C10-GAL80 and +>UAS-NPF-RNAi control females (p>0.9999 and p>0.9999, respectively). Whole-body triglyceride levels were not significantly different between NPF-GAL4>UAS-NPF-RNAi,R57C10-GAL80 males and NPF-GAL4>+,R57C10-GAL80 and +>UAS-NPF-RNAi control males (p>0.9999 and p=0.4134, respectively) (sex:genotype interaction p=0.2890). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. All data plotted as mean ± SEM. ns indicates not significant with p>0.05; ** p<0.01, **** p<0.0001.

Allatostatin C receptor and Tachykinin receptor in neurons promote fat storage in females but not males.

(A) Whole-body triglyceride levels were significantly lower in elav-GAL4>UAS-AstC-R2-RNAi females compared to elav-GAL4>+ and +>UAS-AstC-R2-RNAi control females (p=0.0001 and p=0.0013, respectively). Whole-body triglyceride levels were not significantly different between elav-GAL4>UAS-AstC-R2-RNAi males and elav-GAL4>+ and +>UAS-AstC-R2-RNAi control males (p=0.0564 and p>0.9999, respectively) (sex:genotype interaction p=0.0631). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (B) Whole-body triglyceride levels were significantly lower in elav-GAL4>UAS-TkR99D-RNAi females compared to elav-GAL4>+ and +>UAS-TkR99D-RNAi control females (p<0.0001 and p<0.0001, respectively). Whole-body triglyceride levels were not significantly different between elav-GAL4>UAS-TkR99D-RNAi males and elav-GAL4>+ and +>UAS-TkR99D-RNAi control males (p>0.9999 and p>0.9999, respectively) (sex:genotype interaction p<0.0001). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (C) Whole-body triglyceride levels were significantly lower in elav-GAL4>UAS-NPFR-RNAi females compared to elav-GAL4>+ control females (p<0.0001) but were not significantly different from +>UAS-NPFR-RNAi control females (p>0.9999). Whole-body triglyceride levels were significantly lower in elav-GAL4>UAS-NPFR-RNAi males compared to elav-GAL4>+ control males (p<0.0001) but were not significantly different from +>UAS-NPFR-RNAi control males (p>0.9999) (sex:genotype interaction p=0.2470). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (D) Whole-body triglyceride levels were significantly lower in dilp2-GAL4>UAS-AstC-R2-RNAi females compared to dilp2-GAL4>+ control females (p<0.0001) but were not significantly different from +>UAS-AstC-R2-RNAi control females (p>0.9999). Whole-body triglyceride levels were significantly higher in dilp2-GAL4>UAS-AstC-R2-RNAi males compared to dilp2-GAL4>+ control males (p=0.0156) but were not significantly different from +>UAS-AstC-R2-RNAi control males (p=0.3419) (sex:genotype interaction p<0.0001). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (E) Whole-body triglyceride levels were significantly lower in dilp2-GAL4>UAS-TkR99D-RNAi females compared to dilp2-GAL4>+ control females (p=0.0321) but were not significantly different from +>UAS-TkR99D-RNAi control females (p=0.0724). Whole-body triglyceride levels were significantly higher in dilp2-GAL4>UAS-TkR99D-RNAi males compared to dilp2-GAL4>+ and +>UAS-TkR99D-RNAi control males (p<0.0001 and p=0.0003, respectively) (sex:genotype interaction p<0.0001). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (F) Whole-body triglyceride levels were not significantly different in Akh-GAL4>UAS-AstC-R2-RNAi females compared to Akh-GAL4>+ control females (p=0.3817) but were significantly lower than +>UAS-AstC-R2-RNAi control females (p=0.0181). Whole-body triglyceride levels were not significantly different in Akh-GAL4>UAS-AstC-R2-RNAi males compared to Akh-GAL4>+ and +>UAS-AstC-R2-RNAi control males (p=0.1229 and p>0.9999, respectively) (sex:genotype interaction p=0.0241). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (G) Whole-body triglyceride levels were not significantly different in Akh-GAL4>UAS-TkR99D-RNAi females compared to Akh-GAL4>+ and +>UAS-TkR99D-RNAi control females (p=0.4601 and p>0.9999, respectively). Whole-body triglyceride levels were not significantly different in Akh-GAL4>UAS-TkR99D-RNAi males compared to Akh-GAL4>+ and +>UAS-TkR99D-RNAi control males (p>0.9999 and p=0.8744, respectively) (sex:genotype interaction p=0.0595). Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. All data plotted as mean ± SEM. ns indicates not significant with p>0.05; * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.