Dhr96[1] mutation and maternal tudor[1] mutation increase life span and reduce the beneficial effects of mifepristone in mated female Drosophila

Abstract

Mating and receipt of male Sex Peptide hormone cause increased egg laying, increased midgut size and decreased life span in female Drosophila. Feeding mated females with the synthetic steroid mifepristone decreases egg production, reduces midgut size, and increases life span. Here, several gene mutations were assayed to investigate possible mechanisms for mifepristone action. Drosophila Dhr96 is a hormone receptor, and a key positive regulator of midgut lipid uptake and metabolism. Dhr96[1] null mutation increased female life span, and reduced the effects of mifepristone on life span, suggesting that Dhr96[1] mutation and mifepristone may act in part through the same mechanism. Consistent with this idea, lipidomics analysis revealed that mating increases whole-body levels of triglycerides and fatty-acids in triglycerides, and these changes are reversed by mifepristone. Maternal tudor[1] mutation results in females that lack the germ-line and produce no eggs. Maternal tudor[1] mutation increased mated female life span, and reduced but did not eliminate the effects of mating and mifepristone on life span. This indicates that decreased egg production may be related to the life span benefits of mifepristone, but is not essential. Mifepristone increases life span in w[1118] mutant mated females, but did not increase life span in w[1118] mutant virgin females. Mifepristone decreased egg production in w[1118] mutant virgin females, indicating that decreased egg production is not sufficient for mifepristone to increase life span. Mifepristone increases life span in virgin females of some, but not all, white[+] and mini-white[+] strains. Backcrossing of mini-white[+] transgenes into the w[1118] background was not sufficient to confer a life span response to mifepristone in virgin females. Taken together, the data support the hypothesis that mechanisms for mifepristone life span increase involve reduced lipid uptake and/or metabolism, and suggest that mifepristone may increase life span in mated females and virgin females through partly different mechanisms.

Fig 1. Dhr96[1] mutation effect on life span and response to mifepristone.

A. w[1118] strain, experiment 1. B. Dhr96[1] strain, experiment 1. C. w[1118] strain, experiment 2. D. Dhr96[1] strain, experiment 2. VF, virgin female. MF, mated female. (+), 200μg/ml mifepristone. Statistical summaries presented in Table 1.

Fig 2. Dhr96[1] mutation effect on life span and response to mifepristone, methoprene and HFD.

(A-D) Life span assay and effect of methoprene. VF, virgin female. MF, mated female. (+), 200μg/ml mifepristone. +Met, 100μg/ml methoprene. A. w[1118] strain, experiment 1. B. Dhr96[1] strain, experiment 1. C. w[1118] strain, experiment 2. D. Dhr96[1] strain, experiment 2. Statistical summaries presented in Table 1. (E, F) Life span assay and effect of HFD. Virgin female (VF) life span was assayed in absence and presence of the indicated concentrations of coconut oil (CO). E. w[1118] control strain. F. Dhr96[1] strain. Statistical summaries presented in Table 2.

Fig 3. Dhr96[1] mutation effect on egg laying.

(A, B) Average eggs per fly per day. A. w[1118] strain. B. Dhr96[1] strain. C. Total eggs per fly, estimated by area-under-curve. The statistical test is unpaired, two-sided t test, and the p value for significance with two comparisons is p ≤ 0.025. The p value for pair-wise comparisons is presented above the plots. VF, virgin female. MF, mated female. (+), 200μg/ml mifepristone.

Fig 4. Dhr96[1] mutation effect on maximum midgut diameter.

Maximum midgut diameter was assayed in virgin females, mated females, and mated females treated with mifepristone for both the w[1118] control strain and the Dhr96[1] strain. The statistical test is unpaired, two-sided t test, and the p value for significance with three comparisons is p ≤ 0.0167. The change in mean maximum midgut diameter between samples and the p value for significance is presented above the plots. VF, virgin female. MF, mated female. (+), 200μg/ml mifepristone.

Fig 5. Lipidomics analysis of major lipid classes.

The indicated lipid classes were quantified in flies after 12 days treatment with mifepristone and in no-drug controls. Four replicates of 10 flies each were assayed for each group, and the four replicate values are plotted with the mean. Each control group was compared to the corresponding drug-treated group using unpaired, two-sided t test, and the p value is presented at top of the plot; the p value for significance with one comparison is p < 0.05. A. Free fatty acids (FFA). B. Triglycerides (TAG). C. Fatty acids in triglycerides (FA in TAG). D. Phosphatidylinositol (PI). E. Phosphatidylcholine (PC). F. Lyso phosphatidylcholine (Lyso PC). G. Phosphatidylserine (PS). H. Phosphatidylethanolamine (PE). I. Lyso phosphatidylethanolamine (Lyso PE). VF, virgin females. MF, mated females. VM, virgin males. (-), no drug controls. (+), mifepristone treated (200μg/ml mifepristone).

Fig 6. Lipidomics analysis of phosphatidylglycerol, cardiolipin, cholesterol and additional lipids.

The indicated lipid classes were quantified in flies after 12 days treatment with mifepristone and in no-drug controls. Four replicates of 10 flies each were assayed for each group, and the four replicate values are plotted with the mean. Each control group was compared to the corresponding drug-treated group using unpaired, two-sided t test, and the p value is presented at top of the plot; the p value for significance with one comparison is p < 0.05. A. Phosphatidylglycerol (PG). Cardiolipin. C. Lyso cardiolipin. D. Total cholesterol. E. Free cholesterol. F. Cholesterol ester. G. Ceramide. H. Acylcarnitine. VF, virgin females. MF, mated females. VM, virgin males. (-), no drug controls. (+), mifepristone treated (200μg/ml mifepristone).

Fig 7. Maternal tudor[1] mutation effect on life span, egg laying and response to RH5849 and mifepristone.

(A-D) Life span assays. A. Control mothers, experiment 1. B. tudor[1] mutant mothers, experiment 1. C. Control mothers, experiment 2. D. tudor[1] mutant mothers, experiment 2. VF, virgin female. MF, mated female. (+), 200μg/ml mifepristone. +RH, 1000μg/ml RH5849. Statistical summaries presented in Table 3. E. Egg laying assay. Egg counts were generated at days 8, 12, and 16, and area-under-curve analysis was used to estimate total eggs laid per fly. The statistical test is unpaired, two-sided t test, and the p value for significance with one comparison is p ≤ 0.05. The p value for pair-wise comparisons is presented above the plots. VF, virgin female. MF, mated female. (+), 200μg/ml mifepristone. +RH, 1000μg/ml RH5849.

Fig 8. Maternal tudor[1] mutation effect on maximum midgut diameter.

The statistical test is unpaired, two-sided t test, and the p value for significance with two comparisons is p ≤ 0.025. The change in mean maximum midgut diameter between samples and the p value for significance is presented above the plots. VF, virgin female. MF, mated female. (+), 200μg/ml mifepristone.

Fig 9. White gene mutation effect on life span and egg laying.

A. Virgin females of the indicated genotypes were assayed for life span in absence and presence (+) of 200μg/ml mifepristone. Statistical summary including replicate experiment presented in Table 4. B. Egg laying assay. Virgin females of the indicated genotypes were assayed for egg production every other day from days 2 to 34 (trajectories presented in S4 Fig). Area-under-curve analysis was used to estimate total eggs laid per fly. The statistical test is unpaired, two-sided t test, and the p value for significance with one comparison is p ≤ 0.05. The p value for pair-wise comparisons is presented above the plots. (+), 200μg/ml mifepristone. C. Effect of mifepristone on virgin female life span across strains. The average virgin female (VF) life span for the 5 strains is plotted on the X axis, in relation to the average change in life span caused by mifepristone (Delta) on the Y axis; negative life span effects of mifepristone were treated as zero. Linear regression R[2] = 0.94.