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) mRNA levels of AstA (p=0.5039; Student’s t-test) in guts were not significantly different between 5-day-old w1118 females and males. n=7 biological replicates. (L-N) mRNA levels of AstC (p=0.0002; Student’s t-test) (L), Tk (p<0.0001; Student’s t-test) (M), NPF (p=0.0006; Mann-Whitney test) (N) in guts were significantly higher in 5-day-old w1118 females compared to males. n=7 biological replicates. (O) mRNA levels of Dh31 (p=0.7517; Student’s t-test) 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.

For all data, cell type-specific RNAi was considered to have a significant effect only if the experimental genotype (e.g., tissue-GAL4>UAS-RNAi) significantly differed from both parental strains (e.g., tissue-GAL4>+ and +>UAS-RNAi) with the same direction of effect. (A-E) mRNA levels of AstA (A), AstC (B), Tk (C), NPF (D), and Dh31 (E) in the gut were measured in voila-GAL4>UAS-traF flies and respective genetic controls (voila-GAL4>+ and +>UAS-traF) in females and males. Tra expression did not alter AstA (female: pGAL4>0.9999 and pUAS>0.9999; male: pGAL4>0.9999 and pUAS=0.0084), AstC (female: pGAL4=0.6814 and pUAS=1.0; male: pGAL4=0.9965 and pUAS=0.0463), Tk (female: pGAL4=0.2258 and pUAS>0.9999; male: pGAL4=0.0006 and pUAS=0.0004), NPF (female: pGAL4=0.1579 and pUAS=0.3389; male: pGAL4=0.6639 and pUAS=0.9043) or Dh31 (female: pGAL4=0.0439 and pUAS=0.9745; male: pGAL4=0.9953 and pUAS=0.1370) levels in either sex. Sex:genotype interaction: AstA (p<0.0001), AstC (p=0.1078), Tk (p=0.0004), NPF (p=0.1655), Dh31 (p=0.1945). Data were analyzed by two-way ANOVA with Bonferroni or Tukey’s HSD post-hoc tests as appropriate (aligned rank transform applied for non-parametric data in B and E); n=5 biological replicates. (F–J) mRNA levels of AstA (F), AstC (G), Tk (H), NPF (I), and Dh31 (J) in the head were measured in voila-GAL4>UAS-traF flies and respective genetic controls (voila-GAL4>+ and +>UAS-traF) in females and males. Tra expression did not alter AstA (female: pGAL4>0.9999 and pUAS=0.3344; male: pGAL4>0.9999 and pUAS>0.9999), AstC (female: pGAL4>0.9999 and pUAS<0.0001; male: pGAL4=0.6687 and pUAS=0.0236), NPF (female: pGAL4>0.9999 and pUAS=0.0006; male: pGAL4=0.5030 and pUAS=0.6158) or Dh31 (female: pGAL4>0.9999 and pUAS=0.0003; male: pGAL4>0.9999 and pUAS>0.9999) levels in either sex. In females, Tk (pGAL4>0.9999 and pUAS=0.0044) levels did not alter, but in males, Tk was increased compared with both controls (pGAL4=0.0120 and pUAS=0.0156). Sex:genotype interaction: AstA (p=0.2471), AstC (p=0.0189), Tk (p=0.0003), NPF (p=0.1408). Dh31 (p=0.0403). Data were analyzed by two-way ANOVA with Bonferroni post-hoc tests; n=8 biological replicates. (K–O) mRNA levels of AstA (K), AstC (L), Tk (M), NPF (N), and Dh31(O) in the gut were measured in elav-GAL4>UAS-traF flies and respective genetic controls (elav-GAL4>+ and +>UAS-traF) in females and males. Neuronal Tra expression did not alter AstA (female: pGAL4>0.9999 and pUAS=0.0534; male: pGAL4=0.5496 and pUAS=0.1858), AstC (female: pGAL4=0.5948 and pUAS=0.0878; male: pGAL4=0.1745 and pUAS=0.1745), Tk (female: pGAL4>0.9999 and pUAS=0.0269; male: pGAL4>0.9999 and pUAS=0.2110), NPF (female: pGAL4>0.9999 and pUAS=0.1158; male: pGAL4>0.9999 and pUAS=0.6652), or Dh31 (female: pGAL4=0.5442 and pUAS=0.8086; male: pGAL4=0.1650 and pUAS=0.5258) levels in either sex. Sex:genotype interaction: AstA (p=0.6125), AstC (p=0.4992), Tk (p=0.5212), NPF (p=0.6546), Dh31 (p=0.9566). Data were analyzed by two-way ANOVA with Bonferroni or Tukey’s HSD post-hoc tests as appropriate (aligned rank transform applied for non-parametric data in L and O); n=6 biological replicates. (P–T) mRNA levels of AstA (P), AstC (Q), Tk (R), NPF (S), and Dh31 (T) in the head were measured in elav-GAL4>UAS-traF flies and respective genetic controls (elav-GAL4>+ and +>UAS-traF) in females and males. Neuronal Tra expression did not alter AstA (female: pGAL4=0.9843 and pUAS=0.7086; male: pGAL4=0.0628 and pUAS=0.9936), AstC (female: pGAL4=0.1253 and pUAS=0.8540; male: pGAL4=0.9086 and pUAS=0.0188), Tk (female: pGAL4=0.6051 and pUAS=0.9999; male: pGAL4=0.3600 and pUAS=0.2760), or Dh31 (female: pGAL4>0.9999 and pUAS>0.9999; male: pGAL4=0.2918 and pUAS=0.5990) levels in either sex. NPF was reduced in females compared with both controls (pGAL4=0.0347 and pUAS=0.0273) but was unchanged in males (pGAL4=0.0656 and pUAS=0.6253). Sex:genotype interaction: AstA (p=0.0171), AstC (p=0.0198), Tk (p=0.2324), NPF (p=0.6872), Dh31 (p=0.4360). Data were analyzed by two-way ANOVA with Tukey’s HSD or Bonferroni post-hoc tests as appropriate (aligned rank transform applied for non-parametric data in P, Q, R, S); 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.

For all data, cell type-specific RNAi was considered to have a significant effect only if the experimental genotype (e.g., tissue-GAL4>UAS-RNAi) significantly differed from both parental strains (e.g., tissue-GAL4>+ and +>UAS-RNAi) with the same direction of effect. (A) Whole-body triglyceride levels were significantly lower in AstC-GAL4>UAS-AstC-RNAi,R57C10-GAL80 females compared with AstC-GAL4>+,R57C10-GAL80 and +>UAS-AstC-RNAi control females (pGAL4<0.0001 and pUAS<0.0001), an effect that was not observed in males (pGAL4=0.4527 and pUAS=0.1056) (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 not significantly different in Tk-GAL4>UAS-Tk-RNAi,R57C10-GAL80 females and males compared with controls (female: pGAL4=0.1109 and pUAS=0.0118; male: pGAL4<0.0001 and pUAS=0.5704) (sex:genotype interaction p<0.0001) though we note a trend toward lower body fat in females. Two-way ANOVA followed by Bonferroni post-hoc test; n=8 biological replicates. (C) Whole-body triglyceride levels were not significantly different in NPF-GAL4>UAS-NPF-RNAi,R57C10-GAL80 females and males compared with controls (female: pGAL4>0.9999 and pUAS>0.9999; male: pGAL4>0.9999 and pUAS=0.4134) (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.

For all data, cell type-specific RNAi was considered to have a significant effect only if the experimental genotype (e.g., tissue-GAL4>UAS-RNAi) significantly differed from both parental strains (e.g., tissue-GAL4>+ and +>UAS-RNAi) with the same direction of effect. (A–C) Whole-body triglyceride levels were measured in elav-GAL4>UAS-RNAi flies targeting AstC-R2 (A), TkR99D (B), and NPFR (C), along with respective genetic controls (elav-GAL4>+ and +>UAS-RNAi) in females and males. Knockdown of AstC-R2 (female: pGAL4=0.0001 and pUAS=0.0013; male: pGAL4=0.0564 and pUAS>0.9999; sex:genotype interaction p=0.0631) and TkR99D (female: pGAL4<0.0001 and pUAS<0.0001; male: pGAL4>0.9999 and pUAS>0.9999; sex:genotype interaction p<0.0001) significantly reduced triglyceride levels in females compared with no significant differences in males. Knockdown of NPFR did not change triglyceride levels in either females or males compared with controls (female: pGAL4<0.0001 and pUAS>0.9999; male: pGAL4<0.0001 and pUAS>0.9999; sex:genotype interaction p=0.2470). Data were analyzed by two-way ANOVA followed by Bonferroni post-hoc tests; n=8 biological replicates. (D) Whole-body triglyceride levels were not significantly different in dilp2-GAL4>UAS-AstC-R2-RNAi females and males compared with controls (female: pGAL4<0.0001 and pUAS>0.9999; male: pGAL4=0.0156 and pUAS=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 not significantly different in dilp2-GAL4>UAS-TkR99D-RNAi females compared with controls (pGAL4=0.0321 and pUAS=0.0724). Whole-body triglyceride levels were significantly higher in dilp2-GAL4>UAS-TkR99D-RNAi males compared with dilp2-GAL4>+ and +>UAS-TkR99D-RNAi control males (pGAL4<0.0001 and pUAS=0.0003; 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 and males compared with controls (female: pGAL4=0.3817 and pUAS=0.0181; male: pGAL4=0.1229 and pUAS>0.9999; 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 and males compared with controls (female: pGAL4=0.4601 and pUAS>0.9999; male: pGAL4>0.9999 and pUAS=0.8744; 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.

Model of sex differences in fat storage.

Schematic representation of complex mechanisms that promote higher fat storage in Drosophila females. Profound female-biased EE cell-derived Tk and AstC promote female fat storage, an effect mediated by neurons but independent of the IPC or APC. EE cell-derived factors therefore act alongside a greater insulin/insulin-like growth factor signaling pathway activity, lower Adipokinetic hormone (Akh) signaling, and lower expression of triglyceride lipase brummer (bmm) to specify higher adiposity in female flies. In males, we detected no contribution of gut-derived factors to fat storage, which we previously showed is kept at a lower level than in females by higher bmm expression and Akh signaling. Thus, multiple complex mechanism specify higher fat storage in unmated female flies compared with males. IPCs: insulin-producing cells; APCs: adipokinetic hormone-producing cells; CNS: central nervous system; bmm: brummer; EC: enterocyte; EB: enteroblast; EE: enteroendocrine; ISC: intestinal stem cells; AstC: Allatostatin C; Tk: Tachykinin; Dilps: Drosophila insulin-like peptides; Akh: Adipokinetic hormones. Created in BioRender. Rideout, E. (Created with https://BioRender.com/9z1gko5).