Altered Sxl function in either sex causes significant lethality due to effects on the dosage compensation machinery (Cline, 1978; Cline and Meyer, 1996). We therefore asked whether the presence of Tra in females, which promotes female sexual development, contributes to the elevated whole-body triglyceride levels observed in females (Sieber and Spradling, 2015; Wat et al., 2020). In 5-day-old virgin females lacking tra function (tra¹/Df(3L)st-j7), we found that whole-body triglyceride levels were significantly lower than in age-matched w¹¹¹⁸ control females (Figure 1A). Because we observed no significant difference in fat storage between tra¹/Df(3L)st-j7 mutant males and w¹¹¹⁸ control males (Figure 1—figure supplement 1A), the sex difference in whole-body triglyceride storage was reduced. While previous studies show the ovaries store a small amount of triglyceride (Sieber and Spradling, 2015; Wat et al., 2020), Tra’s effect on whole-body triglyceride storage was not explained by the absence of ovaries in females lacking Tra function: whole-body fat storage was still significantly lower in tra¹/Df(3L)st-j7 mutant females with excised gonads compared with w¹¹¹⁸ control females with excised ovaries (Figure 1B). Given that we reproduced this finding in females carrying a distinct combination of tra mutant alleles (Figure 1C; Hudry et al., 2016), our findings suggest Tra regulates the sex difference in whole-body triglyceride levels by promoting fat storage in females.

We next asked whether Tra function also contributes to the reduced fat breakdown phenotype post-starvation in females (Wat et al., 2020). To quantify fat breakdown, we measured whole-body triglyceride levels at 0 hr, and 24 hr after food withdrawal, and calculated the percent change in whole-body triglyceride levels between time points. While female flies normally have reduced fat breakdown post-starvation compared with males (Wat et al., 2020), the magnitude of fat breakdown post-starvation was not significantly different between tra¹/Df(3L)st-j7 mutants and sex-matched w¹¹¹⁸ controls (genotype:time interactions p=0.6298 [females], p=0.3853 [males]; Supplementary file 1; Figure 1—figure supplement 1B). Tra function is therefore required to promote elevated fat storage in females, but does not regulate fat breakdown post-starvation.

Given that males normally lack a functional Tra protein (Belote et al., 1989; Boggs et al., 1987; Inoue et al., 1990; Sosnowski et al., 1989), we next asked whether the absence of Tra in males explains their reduced whole-body triglyceride levels and rapid triglyceride breakdown post-starvation (Wat et al., 2020). To test this, we ubiquitously overexpressed Tra using daughterless (da)-GAL4, an established way to feminize male flies (Ferveur et al., 1995; Rideout et al., 2015), and examined whole-body fat metabolism. In 5-day-old da-GAL4>UAS-tra^F males, whole-body triglyceride levels were significantly higher than in age-matched da-GAL4>+ or +>UAS-tra^F control males (Figure 1D). No increase in whole-body fat storage was observed in age-matched da-GAL4>UAS-tra^F females compared with da-GAL4>+ or +>UAS-tra^F control females (Figure 1—figure supplement 1C); therefore, the sex difference in fat storage was reduced. Because high levels of Tra overexpression affected viability in one study (Siera and Cline, 2008), we also measured fat storage in males carrying an allele of tra that directs the production of physiological Tra levels (tra^{F K-IN} allele) (Hudry et al., 2019). As in da-GAL4>UAS-tra^F males, whole-body triglyceride levels were significantly higher in tra^{F K-IN} males compared with w¹¹¹⁸ control males (Figure 1E). While these data indicate that gain of a functional Tra protein in males promotes whole-body fat storage, we note that the magnitude of the increase in fat storage was higher in tra^{F K-IN} males. The reason for this discrepancy between tra-expressing males is not clear, therefore, future studies will need to compare tra expression levels and tissue distribution between da-GAL4>UAS-tra^F males and tra^{F K-IN} males.

Importantly, the presence of rudimentary ovaries in tra^{F K-IN} males did not explain their increased fat storage, as whole-body fat storage was still higher in tra^{F K-IN} males lacking gonads compared with gonadless control males (Figure 1F). The elevated fat storage in tra^{F K-IN} males also cannot be attributed to ecdysone production by the rudimentary ovaries, as no ecdysone target genes were upregulated (Figure 1—figure supplement 1D; Sieber and Spradling, 2015); however, future studies will need to address why these tra^{F K-IN} males showed significant ecdysone target gene downregulation. Taken together, these data indicate that lack of Tra function contributes to the reduced whole-body triglyceride levels normally observed in males. In males, this role for Tra may also extend to the regulation of fat breakdown, as triglyceride mobilization post-starvation was significantly reduced in da-GAL4>UAS-tra^F males compared with da-GAL4>+ or +>UAS-tra^F controls during a 24-hr starvation period (genotype:time p<0.0001 [males]; Supplementary file 1; Figure 1—figure supplement 1E), a finding we reproduced in tra^{F K-IN} males (Figure 1—figure supplement 1F). While this effect of Tra on fat breakdown in males does not perfectly align with our data from tra mutant females, we note a trend toward increased fat breakdown in tra mutant females that was not statistically significant (Figure 1—figure supplement 1B). Taken together, these data support a clear role for Tra in regulating the sex difference in fat storage, and suggest that a role for Tra in regulating fat breakdown cannot be ruled out.

Tra function is required in many cell types, tissues, and organs to promote female sexual development (Anand et al., 2001; Baker et al., 2001; Billeter et al., 2006; Brown and King, 1961; Burtis and Baker, 1989; Camara et al., 2008; Christiansen et al., 2002; Clough et al., 2014; Dauwalder, 2011; Demir and Dickson, 2005; Goodwin et al., 2000; Hall, 1994; Heinrichs et al., 1998; Hoshijima et al., 1991; Inoue et al., 1992; Ito et al., 1996; Nagoshi et al., 1988; Neville et al., 2014; Nojima et al., 2014; Pavlou et al., 2016; von Philipsborn et al., 2014; Pomatto et al., 2017; Rezával et al., 2014; Rezával et al., 2016; Rideout et al., 2007; Rideout et al., 2010; Ryner et al., 1996; Sturtevant, 1945). To determine the cell types and tissues in which Tra function is required to influence fat metabolism, we overexpressed Tra using a panel of GAL4 lines that drive expression in subsets of cells and/or tissues. To rapidly assess potential effects on fat metabolism, we measured starvation resistance, an established readout for changes to fat storage and breakdown (Beller et al., 2010; Bi et al., 2012; Choi et al., 2015; Grönke et al., 2003; Grönke et al., 2005; Grönke et al., 2007; Gutierrez et al., 2007).

Normally, adult females have elevated starvation resistance compared with age-matched males due to higher fat storage and reduced fat breakdown (Wat et al., 2020). Indeed, loss of tra reduced starvation resistance in females (Figure 2A) whereas gain of Tra function enhanced starvation resistance in males (Figure 2B), in line with their effects on fat metabolism (Figure 1A and D). From our survey of different GAL4 lines (Figure 2—figure supplement 1A-F; Figure 2—figure supplement 2A-D), we found that neurons were the cell type in which gain of Tra most strongly extended male starvation resistance (Figure 2C). Specifically, starvation resistance in males with Tra overexpression in neurons (elav-GAL4>UAS-tra^F) was significantly extended compared with elav-GAL4>+ and +>UAS-tra^F controls (Figure 2C), with no effect in females (Figure 2—figure supplement 3A). Because the increase in starvation resistance upon neuron-specific Tra expression was similar in magnitude to the increase in survival observed upon global Tra expression (Figure 2B and C), this finding suggests a key role for neuronal Tra in regulating starvation resistance.

To determine whether increased starvation resistance in elav-GAL4>UAS-tra^F males was due to altered fat metabolism, we measured whole-body triglyceride levels in males and females with neuronal Tra overexpression. We found that elav-GAL4>UAS-tra^F males (Figure 2D), but not females (Figure 2—figure supplement 3B), showed a significant increase in whole-body fat storage compared with sex-matched elav-GAL4>+ and +>UAS-tra^F controls. This suggests that the male-specific increase in starvation resistance (Figure 2C) was due to increased fat storage in elav-GAL4>UAS-tra^F males, which we confirm by showing that the rate of fat breakdown in elav-GAL4>UAS-tra^F males and females was not significantly different from sex-matched elav-GAL4>+ and +>UAS-tra^F controls (Figure 2—figure supplement 3C) (genotype:time interaction p=0.2789 [males], p=0.7058 [females]; Supplementary file 1). Neurons are therefore one cell type in which Tra function influences the sex difference in whole-body triglyceride storage.

To identify specific neurons that mediate Tra’s effects on starvation resistance and whole-body fat storage, we overexpressed Tra in neurons known to affect fat metabolism and measured starvation resistance (Figure 2—figure supplement 4A-E; Al-Anzi and Zinn, 2018; Al-Anzi et al., 2009; Chung et al., 2017; Li et al., 2016; May et al., 2020; Min et al., 2016; Mosher et al., 2015; Zhan et al., 2016). One group of neurons that significantly augmented starvation resistance upon Tra expression was the APCs (Figure 2E), a group of neuroendocrine cells in the corpora cardiaca that produce Akh and other peptide hormones such as Limostatin (Lst; FBgn0034140) (Alfa et al., 2015; Lee and Park, 2004). Although we note that Tra expression in additional neurons and in glia affected starvation resistance (Figure 2—figure supplement 2D; Figure 2—figure supplement 4D), suggesting the regulation of fat metabolism by Tra function in neurons is complex, the central role of the APCs in regulating fat metabolism prompted a more detailed investigation into Tra’s function in these neurons. Flies with APC-specific Tra expression (Akh-GAL4>UAS-tra^F) had significantly increased starvation resistance compared with sex-matched Akh-GAL4>+ and +>UAS-tra^F controls (Figure 2E; Figure 2—figure supplement 5A). To determine whether the starvation resistance phenotype indicated altered fat storage, we compared whole-body triglyceride levels in Akh-GAL4>UAS-tra^F males and females with sex-matched Akh-GAL4>+ and +>UAS-tra^F controls. There was a significant increase in whole-body fat storage in males (Figure 2F) but not females (Figure 2—figure supplement 5B) with APC-specific Tra expression. This indicates Tra function in the APCs promotes fat storage, revealing a previously unrecognized role for the APCs in regulating the sex difference in fat storage. Indeed, fat breakdown was unaffected in Akh-GAL4>UAS-tra^F males and females compared with sex-matched Akh-GAL4>+ and +>UAS-tra^F controls (Figure 2—figure supplement 5C) (genotype:time interaction p=0.1201 [males] and p=0.0596 [females]; Supplementary file 1).

Given that the sexual identity of the APCs impacts whole-body fat storage, we compared the regulation of Akh, APC activity, and Akh signaling between adult males and females. We first examined Akh and AkhR mRNA levels in both sexes using quantitative real-time polymerase chain reaction (qPCR). We found that mRNA levels of both Akh and AkhR were significantly higher in 5-day-old w¹¹¹⁸ males than in females (Figure 3A and B). This male bias in Akh mRNA levels did not reflect an increased APC number in males, as we found no sex difference in the number of APCs (Figure 3C). Because Akh release from the APCs is regulated by APC activity (Kubrak et al., 2020; Oh et al., 2019), we next measured APC activity in males and females by driving APC-specific expression of calcium-responsive chimeric transcription factor LexA-VP16-NFAT (Akh-GAL4>UAS-LexA-VP16-NFAT [called UAS-CaLexA]) (Masuyama et al., 2012). Sustained APC activity triggers nuclear import of LexA-VP16-NFAT, where it drives expression of a GFP reporter downstream of a LexA-responsive element (Masuyama et al., 2012). Monitoring GFP levels in the APCs therefore provides a straightforward way to monitor APC activity.

In 5-day-old Akh-GAL4>UAS-CaLexA males, GFP levels were significantly higher than in age- and genotype-matched females (Figure 3D–H). Because GAL4 mRNA levels were not significantly different between males and females carrying the Akh-GAL4 transgene (Figure 3—figure supplement 1A), and the number of APCs did not differ between the sexes (Figure 3C), these findings indicate that the APCs are more active in males than in females. To determine whether the male bias in Akh/AkhR mRNA levels and APC activity affected Akh pathway activity, we next compared levels of phosphorylated Inositol-requiring enzyme-1 (Ire1; FBgn0261984) between males and females. Because levels of phosphorylated Ire1 (p-Ire1) are higher in Drosophila cells stimulated with Akh peptide, regulation that was dependent on the presence of AkhR, high p-Ire1 levels indicate increased Akh pathway activity (Song et al., 2017). We found that the ratio of p-Ire1 to loading control actin was higher in 5-day-old w¹¹¹⁸ males compared with age- and genotype-matched females in three out of four biological replicates (Figure 3I–K; Figure 3—figure supplement 1B), a finding that aligns with the sex difference in Akh/AkhR mRNA levels and APC activity. Taken together, our data suggest a previously unrecognized male bias in the Akh pathway.

Given that high Akh pathway activity limits fat storage via an established intracellular signaling cascade that culminates in lipase recruitment and fat mobilization (Baumbach et al., 2014; Grönke et al., 2007; Lee and Park, 2004; Mochanová et al., 2018), we wanted to determine whether the male bias in Akh pathway activity influences the sex difference in fat metabolism by restricting fat storage in males. We therefore used a published approach to ablate the APCs (Akh-GAL4>UAS-reaper (rpr)) (Lee and Park, 2004; White et al., 1996), and measured whole-body triglyceride levels in each sex. Because the sexual identity of the APCs affects fat storage and not fat breakdown (Figure 2F; Figure 2—figure supplement 5C), we focused our analysis on triglyceride storage rather than mobilization. Triglyceride levels were significantly higher in 5-day-old Akh-GAL4>UAS-rpr males than in Akh-GAL4>+ and +>UAS-rpr control males (Figure 4A). In contrast, triglyceride levels in 5-day-old Akh-GAL4>UAS-rpr females were not significantly different from Akh-GAL4>+ and +>UAS-rpr control females (Figure 4—figure supplement 1A). This suggests that the male bias in Akh pathway activity normally contributes to the sex difference in fat storage by limiting triglyceride accumulation in males via the established intracellular signaling cascade known to regulate lipid droplet breakdown (Arrese et al., 2008; Heier and Kühnlein, 2018; Heier et al., 2021; Kühnlein, 2012; Patel et al., 2005). Importantly, we reproduced the male-biased effects on fat storage in flies carrying loss-of-function Akh and AkhR alleles (Akh^A and AkhR¹, respectively) (Figure 4B and C; Figure 4—figure supplement 1B, C), and show that APC-specific knockdown of Lst had no effect on fat storage in either sex (Figure 4—figure supplement 1D, E). These findings support a model in which it is Akh production by the APCs that plays a role in regulating the male-female difference in fat storage. While the mechanisms underlying the regulation of intracellular fat breakdown by Akh in the fat body have been well-documented (Bharucha et al., 2008; Gáliková et al., 2015; Grönke et al., 2007; Heier and Kühnlein, 2018; Heier et al., 2021; Kubrak et al., 2020; Lee and Park, 2004; Lehmann, 2018; Scopelliti et al., 2019; Zhao and Karpac, 2017), our findings reveal a new role for Akh in regulating the sex difference in fat storage. Notably, this Akh-mediated regulation of the male-female difference in fat storage operates in a parallel pathway to the previously described sex-specific role of triglyceride lipase bmm (Figure 4—figure supplement 2A, B; Wat et al., 2020).

Beyond the APC ablation or complete loss of Akh, we next wanted to test whether the sex-specific Akh regulation we uncovered contributes to the male-female difference in fat storage. To this end, we used a genetic approach to manipulate Akh mRNA levels or APC activity, and measured whole-body fat storage in both sexes. To determine whether the male bias in Akh mRNA levels contributes to the sex difference in fat storage, we measured whole-body triglyceride levels in flies with APC-specific expression of Akh-RNAi (Akh-GAL4>UAS-Akh-RNAi). Importantly, this manipulation effectively reduced Akh mRNA levels in both sexes (Figure 4—figure supplement 3A,B). In males, whole-body triglyceride levels were significantly higher in Akh-GAL4>UAS-Akh-RNAi flies compared with Akh-GAL4>+ and +>UAS-Akh-RNAi control flies (Figure 4D). Akh-GAL4>UAS-Akh-RNAi female flies, in contrast, showed no significant change in whole-body fat storage compared with Akh-GAL4>+ and +>UAS-Akh-RNAi control females (Figure 4—figure supplement 3C). This indicates a strongly male-biased effect on fat storage due to reduced Akh mRNA levels, suggesting that the sex difference in Akh mRNA levels contributes to the male-female difference in whole-body fat storage.

To determine whether the male bias in APC activity also influences the sex difference in fat storage, we silenced the APCs by APC-specific overexpression of an inwardly rectifying potassium channel Kir2.1 (Baines et al., 2001) and measured whole-body triglyceride levels. Whole-body fat storage in Akh-GAL4>UAS-Kir2.1 adult males was significantly higher compared with Akh-GAL4>+ and +>UAS-Kir2.1 control males (Figure 4E). In females, while we observed significantly elevated whole-body fat storage in Akh-GAL4>UAS-Kir2.1 adults compared with Akh-GAL4>+ and +>UAS-Kir2.1 controls (Figure 4—figure supplement 3D), the magnitude of this increase was larger in males (sex:genotype interaction p=0.0455; Supplementary file 1). Taken together, these data suggest that the male bias in APC activity contributes to the sex difference in fat storage by limiting triglyceride accumulation in males. Indeed, augmenting APC activity in females using a bacterial voltage-gated sodium channel (UAS-NaChBac) significantly reduced fat storage in females (Figure 4F; Figure 4—figure supplement 3E). While Akh affects food-related behaviors in some contexts (Choi et al., 2015; Hentze et al., 2015; Huang et al., 2020), we observed no significant effects of altered APC activity on feeding behavior in either sex (Figure 4—figure supplement 4A-D). This suggests that the male-biased effect of APC manipulation on fat storage cannot be fully explained by effects on food intake. Thus, in addition to the contribution of elevated Akh mRNA levels in males to the sex difference in fat storage, we also identify a role for the male bias in APC activity in the sex-specific regulation of whole-body triglyceride levels.

Given that Tra function and the Akh pathway both contribute to the male-female difference in fat storage, we asked whether the presence of Tra affects the sex bias in Akh pathway activity. In 5-day-old tra¹/Df(3L)st-j7 females, levels of p-Ire1 were higher than in w¹¹¹⁸ control females in three out of four biological replicates (Figure 5A–C; Figure 5—figure supplement 1A). This suggests the presence of Tra in females normally represses Akh pathway activity. Indeed, loss of Tra significantly increased Akh mRNA levels in females (Figure 5D). Given Tra’s effects on Akh pathway activity, we next tested whether the change in Akh pathway function was significant for Tra’s effects on whole-body triglyceride levels. We predicted that if increased Akh pathway activity caused the lower fat storage in tra mutant females, genetic manipulations that reduce Akh pathway activity should block this reduction in whole-body triglyceride levels. While all female genotypes lacking tra function had reduced fat storage compared with control females (Figure 5E), APC ablation in tra mutant females rescued this decrease in whole-body triglyceride levels (Figure 5E). Indeed, fat storage in tra mutant females lacking APCs was not significantly different from w¹¹¹⁸ control females (p=0.9384; Supplementary file 1; Figure 5E), indicating that the increased Akh pathway activity we observed in tra mutant females was one reason for their reduced fat storage. Given that APC activation in males expressing physiological levels of Tra similarly rescued the Tra-induced increase in whole-body triglyceride levels (Figure 5F), these findings suggest that the sex-specific regulation of Akh pathway activity represents one way tra influences the male-female difference in fat storage.

Our results suggest that adult females show lower Akh pathway activity and higher fat storage, whereas males maintain a higher level of Akh activity and lower fat storage. Because the correct regulation of fat storage in each sex influences reproduction (Buszczak et al., 2002; Grönke et al., 2005; Sieber and Spradling, 2015; Wat et al., 2020), we tested how complete loss of this critical regulator of the sex difference in fat storage impacted offspring production in each sex. In Akh^A mutant males, we found that the proportion of males copulating with a Canton-S (CS) virgin female was lower than in control w¹¹¹⁸ males at each 10 min interval during a 60-min observation period (Figure 6A). When we counted viable offspring from these copulation events, we found that Akh^A mutant males had significantly fewer overall progeny than w¹¹¹⁸ control males (Figure 6B). These results suggest that Akh function normally promotes reproductive success in males; however, it is important to note that Akh function is not absolutely required for male fertility, as a prolonged 24 hr period of contact between Akh^A mutant males and CS females allowed the production of normal progeny numbers (Figure 6C).

In contrast to males, Akh loss-of-function mutations in females increased fecundity (Figure 6D). Specifically, Akh^A mutant females produced a significantly higher number of offspring compared with w¹¹¹⁸ controls (Figure 6D). Thus, in females, a low level of Akh pathway activity promotes fecundity. Given that a change in one life-history trait such as reproduction often affects traits such as longevity (Chapman et al., 1995; Flatt, 2011; Fowler and Partridge, 1989; Hansen et al., 2013), we also measured lifespan in females with reduced Akh pathway function. We found that lifespan was significantly shorter in Akh-GAL4>UAS-Kir2.1 females compared with Akh-GAL4>+ and +>UAS-Kir2.1 control females (Figure 6E). In contrast, male lifespan was not significantly different between Akh-GAL4>UAS-Kir2.1 flies and Akh-GAL4>+ and +>UAS-Kir2.1 controls (Figure 6F). Our findings are in agreement with a previous study that demonstrated a female-specific lifespan reduction in response to whole-body loss of Akh (Bednářová et al., 2018). This suggests that while low Akh activity in females promotes fertility, this benefit comes at the cost of a shorter lifespan, a possibility that will be explored in future studies using additional strains to genetically augment, or inhibit, Akh pathway activity (e.g., APC activation, Akh mutants).

Fly stocks were maintained at 25°C in a 12:12 light:dark cycle. All larvae were reared at a density of 50 larvae per 10 ml of fly media (recipe in Supplementary file 4). Males and females were separated either as early pupae by gonad size, or late pupae by the presence of sex combs. Sex-transformed males and females were distinguished by the presence (males) or absence (females) of B^SY. Single-sex groups of 20 pupae were transferred to damp filter paper within a food vial until eclosion. Unless otherwise stated, all experiments used 5- to 7-day-old flies.

We obtained the following strains from the Bloomington Drosophila Stock Center: Canton-S (#64349), w¹¹¹⁸ (#3605), UAS-nGFP (#4775), UAS-Akh-RNAi (#27031), UAS-tra^F (#4590), tra¹ (#675), Df(3L)st-j7 (#5416), UAS-NaChBac (#9468), UAS-Kir2.1 (#6595), UAS-reaper (#5823), and UAS-CaLexA (#66542). We obtained Akh^A, AkhR^rev, AkhR¹, bmm¹, and AkhR¹;bmm¹ as kind gifts from Dr. Ronald Kühnlein (Gáliková et al., 2015; Grönke et al., 2005; Grönke et al., 2007), tra^KO and tra^{F K-IN} as kind gifts from Dr. Irene Miguel-Aliaga (Hudry et al., 2016; Hudry et al., 2019), and Mex-GAL4 as a kind gift from Dr. Claire Thomas (Phillips and Thomas, 2006). The authors acknowledge critical resources and information provided by Flybase (Thurmond et al., 2019). The following GAL4 lines were used for tissue-specific expression: da-GAL4 (ubiquitous), cg-GAL4 (fat body), r4-GAL4 (fat body), Lsp2-GAL4 (fat body), Myo1A-GAL4 (enterocytes), Mex-GAL4 (enterocytes), dMef2-GAL4 (skeletal muscle), repo-GAL4 (glia), elav-GAL4 (neurons), c587-GAL4 (somatic cells of the gonad), tj-GAL4 (somatic cells of the gonad), nos-GAL4 (germ cells of the gonad), dimmed-GAL4 (peptidergic neurons), TH-GAL4 (dopaminergic neurons), Tdc2-GAL4 (octopaminergic neurons), VT030559-GAL4 (mushroom body neurons), dilp2-GAL4 (insulin-producing cells), and Akh-GAL4 (APCs). All transgenic stocks were backcrossed into a w¹¹¹⁸ background for a minimum of five generations.

To measure adult weight, groups of 10 flies were weighed in 1.5 ml microcentrifuge tubes on an analytical balance (Mettler-Toledo, ME104).

One biological replicate consisted of five flies homogenized in 200 μl of TRIzol. RNA was extracted following the manufacturer’s instructions, as previously described (Wat et al., 2020). cDNA was synthesized from RNA using the Quantitect Reverse Transcription Kit (Qiagen, 205311). qPCR was used to quantify relative mRNA transcript levels as previously described (Wat et al., 2020). See Supplementary file 3 for a full list of primers.

One biological replicate consisted of five flies homogenized in 200 μl of 0.1% Tween (AMresco, 0777-1L) in 1× phosphate-buffered saline (PBS) using 50 μl of glass beads (Sigma-Aldrich, 11079110) agitated at 8 m/s for 5 s (OMNI International Bead Ruptor 24). Assay was performed according to established protocols (Tennessen et al., 2014) as previously described (Wat et al., 2020).

Five-day-old adult flies were individually anesthetized with CO₂. The gonads or ovaries were removed from the distal end of the abdomen in cold 1× PBS and the carcass was snap-frozen in a 1.5 ml microcentrifuge tube on dry ice.

One biological replicate consisted of 10 flies homogenized in extraction buffer (females=200 μl, males=125 μl) containing 20 mM Hepes (pH 7.8), 450 mM NaCl, 25% glycerol, 50 mM NaF, 0.2 mM EDTA, 0.5% Triton X-100, 1 mM PMSF, 1 mM DTT, 1× cOmplete Protease Inhibitor Cocktail (Roche), and 1× PhosSTOP (Roche) using 50 μl of glass beads (Sigma-Aldrich, 11079110) agitated at 8 m/s for 5 s (OMNI International Bead Ruptor 24). Samples were incubated on ice for 5 min before cellular debris was pelleted by centrifugation at 10,000 rpm for 5 min at 4 °C and supernatant was removed (Thermo Fisher Scientific, Heraeus Pico 21 centrifuge). Centrifugation was repeated two times more to remove fat from the samples. Protein concentration of each sample was determined by a Bradford Assay (Bio-Rad, 550-0205); 20 μg of protein per sample was loaded onto a 12% SDS-PAGE gel. Immunoblotting was performed as previously described (Millington et al., 2021). Primary antibodies used were rabbit anti-p-Ire1 (1:1000; Abcam #48187) and mouse anti-actin (1:200; Santa Cruz #sc-8432). Secondary antibodies used were goat anti-rabbit (1:5000; Invitrogen #65-6120) and horse anti-mouse (1:2000; Cell Signaling Technology #7076).

To isolate the APCs, individual flies were anesthetized on ice, and the brain and foregut were removed in cold 1× PBS. Samples were fixed in 4% paraformaldehyde for 30 min, followed by two 30 min washes in cold 1× PBS. Samples were incubated with Hoechst (Sigma-Aldrich, 33342) at a concentration of 1:500 for 5 min and mounted in SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific, S36967). Images were captured using a Leica TCS SP5 Confocal Microscope and processed using Fiji (ImageJ; Schindelin et al., 2012). To visualize APC neuronal activity (Akh-GAL4>UAS-CaLexA), the mean GFP intensity of one APC cluster was quantified by measuring average pixel intensity within the region of interest using Fiji (ImageJ; Schindelin et al., 2012). To determine APC cell number (Akh-GAL4>UAS-nGFP), GFP punctae were counted manually using Fiji (ImageJ; Schindelin et al., 2012). One biological replicate consists of one cluster of APCs, where only one APC cluster was measured per individual.

One biological replicate consisted of 10 flies placed into a specialized 15 ml conical vial with access to two capillary tubes. Capillary tubes were filled with fly food media containing 5% sucrose, 5% yeast extract, 0.3% propionic acid, and 0.15% nipagin. Approximately 0.5 μl of mineral oil was added to the top of each capillary tube to prevent evaporation. All vials were placed into fitted holes in the lid of a large plastic container. A shallow layer of water was poured into the base of the container to maintain high humidity throughout the experiment. The meniscus of the fly food media was marked before the start of the experiment and again after 24 hr. The distance between the marks is used to quantify the volume of fly food media that was consumed (1 mm=0.15 μl). The volume of fly food consumed was normalized to the weight of individual flies (protocol adapted from Stafford et al., 2012).

One singly housed male was placed with a group of three virgin Canton-S (CS) females and allowed to interact for 60 min. At 10 min intervals during the 60-min observation period, we recorded whether a copulating male-female pair was present in the vial. After the 60-min observation period, the male was removed from the vial and the females were allowed to lay eggs for 72 hr (flies were transferred to new food every 24 hr). After 72 hr, the females were removed and progeny were allowed to develop. After 10 days, we counted the number of pupae in each vial. For the 24 hr mating assay, one singly housed male was allowed to interact with three virgin CS females for 24 hr before the male was removed and females were allowed to lay eggs for 72 hr as described above.

One virgin female was placed with a group of three virgin CS males for 24 hr. The females were then transferred onto fresh food every 24 hr for 3 days and the number of pupae was counted as described above.

Five-day-old flies were transferred to vials containing 2 ml of starvation media (0.75 agar in 1× PBS). To measure fat breakdown post-starvation, biological replicates consisting of five flies each were collected in 1.5 ml microcentrifuge tubes and snap-frozen on dry ice at 0 hr and 24 hr post-starvation. The percent change in fat storage between time points was calculated to determine fat breakdown over time. For starvation resistance, the number of deaths was recorded every 12 hr until no living flies remained in the vial.

Flies were transferred to new vials with 2 ml of fresh food every 2–3 days until no living flies remained in the vial. Deaths were recorded when the flies were transferred.

All figures and data were generated and analyzed using GraphPad Prism (v9.1.2). For experiments with two groups, a Student’s t-test was performed. For experiments with three or more groups, a one-way ANOVA with Tukey HSD post hoc test was performed. For fat breakdown experiments, a two-way ANOVA was used to determine the interaction between genotype and time. Starvation resistance and lifespan statistics were performed using RStudio and a script for a log-rank test with Bonferroni’s correction for multiple comparisons. Note, the lowest p-value given by RStudio is 2.0×10^–16. The below packages and script were used: library ("survminer") library ("survival") data <- read.csv(“xxx.csv”) survfit(Surv(time, event)~ genotype, data) pairwise_survdiff(Surv(time, event)~ genotype, data, p.adjust.method = “bonferroni”) summary (data).