(A-G) Mis-expression of traF feminizes male midguts. (A-F”) PV (A-B”) and R2 (C-F”) morphology in +/traF females (A-F), +/traF males (A’-F’) and NP1>traF males (A”-F”) at 7 and 35 days, reveal female-like pathology in the R2 region of NP1>traF males at 35 days (F”). The NP1 driver is not expressed in the majority of the PV and accordingly, the PV is well-maintained at 35 days (B”). Control females and feminized males increased ISC proliferation over age, but control males did not (n=10–20 guts per condition, student’s t test, p=0.0366 for 3 vs 35 day-old +/traFfemales, p=0.0015 for 3 vs 35 day-old NP1>traF females, p=0.7057 for 3 d vs 35 d +/traFmales, p=0.00022 for 3 vs 35 day-old NP1>traF feminized males). Feminized male guts (NP1>traF) had more mitoses at 35 days than control (+/traF) male guts (p=0.00018) (G). (H-J) barrier dysfunction and systemic AMP expression were increased in feminized males. Barrier dysfunction was significantly higher in feminized males than control (+/traF) males at 42 days (n≥150 per group, Fisher’s exact, p=0.0001) and control (+/traF) females (p=0.0002) (H). Diptericin expression was increased over aging in all genotypes (n≥3 samples per condition, 10 individuals pooled per sample, 2 technical repeats; 2-way ANOVA, age p=0.0487, condition p=0.1031, interaction p=0.3485) and was increased in feminized males relative to control males at 7 days only (t test with Welch’s correction, p=0.0018 for NP1>traFvs +/NP1 at 7 days; p=0.5152 for NP1>traFvs +/NP1 at 42 days; p=0.0011 for NP1>traFvs +/traF at 7 days; p=0.8907 for NP1>traFvs +/traF at 42 days) (I). Doux expression did not increase over aging in any genotype, but was higher in males than females overall (J). (K) Aerobic bacterial load tended to increase between 7 and 21 days for both sexes and genotypes (n≥8 samples per condition, 5 individuals pooled per sample; Monte Carlo Markov Chain Generalised Linear Model with Poisson Error Family, where pMCMC=0.040 for males and pMCMC=0.064 for females). Feminized males had a significantly higher load than control males (pMCMC<0.001). In addition, the direction of bias compared to females was switched in feminized males, such that control males had lower load than females, but feminized males had a higher load. A similar result was obtained for anaerobic load. (L-N) Pathologies in feminized males are responsive to diet and rapamycin treatment. Pathologies were binned into scaled categories and quantified, n≥12 per condition. PV categories as described in Figure 2 legend (see Figure 3—figure supplement 2 for PV scoring). R2 and R4 categories were defined as follows: I = WT, single layer epithelium with low number of basal ISCs. II = sporadic pathology of small nuclei ‘nests’ without significant disruption to the epithelium; III = widespread pathology, majority of epithelium has several layers of nuclei; IV = widespread pathology plus clear tumor formation. Gut feminized males have significantly worse pathology than control males on both diets in R2 (OLR, low-yeast, z=-3.916, p=0.0000899; high-yeast z=-4.339, p=0.0000143) and R4 (low-yeast, z=-4.012, p=0.0000602; high-yeast z=-4.520, p=0.0000617). The incidence of severe pathology and tumors (cat IV) in R2 was greater in feminized males than control females on high yeast diet (p=0.04) but not low yeast diet (p=0.48), suggesting that there was a cost of feminization that was partly alleviated by DR (L-M). Rapamycin treatment decreased mitoses in females and feminized males at 16 days (n≥10 guts per condition, students t test; control (+/traF) females, p=0.0079; control (+/traF) males, p=0.1; control females (NP1/traF), p=0.22; feminized males (NP1/traF), p=0.0001). (N) O-R Feminized males were more sensitive to oral infection, but acquired a lifespan response to dietary restriction. At 42 days males succumbed to Ecc oral infection while females did not. Feminized males died significantly sooner than controls (O). After Ecc oral infection at 7 days, males and females of all genotypes increased gut mitoses compared to sham infected (n≥10 guts per condition, students t test; control (+/NP1) females, p=2.082E-06; control (+/NP1) males, p=0.0011; control females (NP1/traF), p=0.00017; feminized males (NP1/traF), p=0.00045). However, females and feminized males lost the response to infection against a background of high proliferation in unchallenged individuals at 42 days (n≥10 guts per condition, students t test; control (+/NP1) females, p=0.2; control (+/NP1) males, p=0.0088; control females (NP1/traF), p=0.1478; feminized males (NP1/traF), p=0.2344) (P). Systemic dipt expression was increased after 18 hr continuous infection in all genotypes at 42 days (n≥10 guts per condition, t test with Welch’s correction; +/NP1 females, p=0.0571; +/NP1 males, p=0.0132; +/traF females, p=0.0376; +/traF males, p=0.0282; NP1/traF females, p=0.0110; NP1/traF feminized males p=0.0331), but at a higher level in males than females in both sham and infected conditions (sham: +/NP1 females vs males, p=0.0135; +/traF females vs males, p=0.0428; NP1/traF females vs males, p=0.0022. Infected: NP1/+ females vs males, p=0.0012; +/traF females vs males, p=0.0964; NP1/traF females vs males, p=0.0237.) (Q). Lifespan analysis of NP1>traF males and +/NP1 control males and females on two yeast dilutions. NP1>traF males were significantly shorter lived than control males on both standard (low yeast; log rank, p=0.0023) and double (high yeast; log rank, p=2.06E-11) yeast dilutions, whereas +/NP1 control males did not differ between food conditions (log rank, p=0.34). This is a representative lifespan of three with similar outcomes. Cox proportional hazards analysis of the lifespan demonstrated a significantly increased risk of dying on high-yeast vs low-yeast food overall (p=2 x 10–16), and a significant difference in the response to food between control male genotypes and NP1>traF (gut feminized) males (p=0.0298). For full analysis, see Figure 3—source data 1. PV, proventriculus.