Feeding a rich diet supplemented with the translation inhibitor cycloheximide decreases lifespan and ovary size in Drosophila

Drosophila has been an excellent model system to dissect the genetic pathways and molecular mechanisms controlling processes as diverse as behavior, development and cell differentiation. Females contain a pair of ovaries made up of ∼20 ovarioles (Fig. 1A,B). Each ovariole is a string of developing follicles, with a specialized structure at the anterior called the germarium, which houses the germline stem cells (Fig. 1C). Each follicle is composed of 16 interconnected cells: one cell becomes the oocyte, whereas the other fifteen develop as nurse cells (Fig. 1C) (Spradling, 1993). Oogenesis is energy dependent and highly sensitive to nutrition (Armstrong, 2020). Females fed a diet supplemented with wet yeast paste have maximum germline stem cell division and egg production, with all stages of follicle development present (Fig. 1C). However, when females are fed a poor diet, oogenesis is quickly affected to conserve energy. Clear indications of nutritional stress are apoptosis that occurs at stage 8 and missing follicle stages, although there are other problems including mitochondrial mislocalization, changes to metabolism and apoptosis in the germarium (Drummond-Barbosa and Spradling, 2001). This is a conundrum for researchers wishing to disrupt various pathways and study the effect on oogenesis by feeding flies various compounds. The most common method of drug feeding is using a sucrose solution or spiking the standard fly medium, neither of which constitutes a rich diet and adversely affects oogenesis (Belozerov et al., 2014; Jeong et al., 2022; Kim et al., 2023; Lagasse et al., 2009; Tully et al., 1994).

To circumvent these problems, we developed a method to feed Drosophila a rich diet of yeast paste supplemented with cycloheximide (CHX), a translation inhibitor. CHX is a popular compound widely used to study intracellular events and physiological effects by reducing protein synthesis (Darvishi and Woldemichael, 2016; Jevtov et al., 2015; Kim et al., 2023; Lin et al., 2008; Moutaoufik et al., 2014; Sheth and Parker, 2003). This method would allow examining how decreased translation and ribosome stalling affects lifespan and oogenesis without the confounding effects of decreased nutrition. Published articles examining the effects of CHX have used food with limited sources of nutrients when flies were fed with drugs (Kim et al., 2023; Lagasse et al., 2009; Lee et al., 2018; Tully et al., 1994). To support that this feeding method is effective, females fed yeast paste with CHX must readily and reliably consume the drug and exhibit deleterious effects on the fly. Here, we show that feeding CHX in a rich diet reduces lifespan in males and females and females have reduced protein synthesis in the ovary. We also show that feeding CHX using this method does not affect the number of eggs laid but does affect their quality, resulting in reduced number of progeny. In addition, ovary size is reduced and there is an increased amount of apoptotic and missing follicle stages. Overall, our data support that CHX treatment directly reduces lifespan and disrupts oogenesis in flies supported with a rich diet. In addition, this method could also be used to feed other water-soluble drugs to Drosophila, provided that the drug is not repellent to the fly, in order to test the effects on development and lifespan without the added stress of a poor diet.

To ensure that any CHX effects were due to decreased protein synthesis rather than directly due to poor nutrition that could result from food avoidance by the flies due to drug taste, we fed flies varying concentrations of CHX mixed in their preferred food source: wet yeast paste. To test whether flies would accept and consume this source of CHX, they were fed freshly made CHX-supplemented yeast paste containing increasing concentrations of CHX for 24 or 48 h. We supplemented the food with the dye FD&C Blue No. 1 to track food ingestion (Fig. 1D). Females readily ate the yeast paste as indicated by the blue guts visible in their abdomens (Fig. 1E). To ensure that CHX was having the desired effect and suppressing protein synthesis, we coupled CHX feeding with puromycin (Fig. 2A). Puromycin is a naturally occurring aminonucleoside that mimics tyrosyl-tRNA and incorporates into nascent polypeptides. As it is not amino acid specific, incorporation terminates all translation. Well-fed flies were fed CHX-containing fresh yeast paste for 24 h, then transferred to fresh yeast paste containing both CHX and puromycin and allowed to feed for an additional 24 h (Fig. 2A). Fly extracts were then probed with anti-puromycin antibodies that recognize the terminated nascent peptides (Fig. 2B, upper panels). If CHX decreased translation, we would expect a decrease in the amount of puromycin incorporation. Flies fed 3.5, 7.0 and 10 mM CHX had decreased puromycin incorporation, supporting that this method of feeding CHX is effective (Fig. 2B).

We next tested the effect of dietary CHX on lifespan (Fig. 3A). It is possible that CHX treatment would be quickly lethal to flies if our feeding regimen immediately blocked global translation. However, we found that the flies tolerated daily dietary CHX when fed freshly made yeast paste supplemented with CHX (Fig. 3B,C). We found that continually feeding yeast paste supplemented with 3.5, 7.0 or 10 mM CHX was not immediately lethal but did significantly decrease lifespan in males and females (Fig. 3B,C). Males exhibited a dose-dependent decrease in lifespan, whereas females did not (Fig. 3B versus Fig. 3C). We noticed that the larvae, which hatched from the eggs laid on CHX-supplemented yeast paste, appeared small and delayed. To test whether larvae that grew on CHX-supplemented yeast had delayed development, we counted how many pupae developed (Fig. 3D). CHX strongly suppressed pupation levels compared to those for larvae not reared on CHX (Fig. 3E). These data show that constant exposure to CHX in a nutrient-rich diet decreases lifespan and that larvae exposed to CHX struggle to pupate.

Once we established that feeding the flies a rich diet with CHX was having an effect, we wanted to determine how decreased protein synthesis affects oogenesis with optimal nutrition. We found that egg laying was not affected, even at the highest dose of CHX (10 mM) (Fig. 4A,B). To test the quality of the eggs laid, we again performed a pupation assay but transferred the CHX-fed females to a standard food vial, letting these females lay eggs in the vial without CHX (Fig. 4C). With this assay, fewer larvae pupated from females fed CHX in yeast paste compared to controls, with a dose-dependent effect (Fig. 4D). These data suggest that, although the number of eggs being laid was unaffected with dietary CHX, egg quality was reduced.

Although egg-laying levels remained the same as the progeny number was reduced, it was possible that there were defects during oogenesis. To identify ovary defects, well-fed females were fed CHX-supplemented yeast paste for 24 h before ovary dissection (Fig. 5A). Whole ovaries dissected from females fed 3.5, 7.0 and 10 mM CHX did not look grossly abnormal but did look slightly smaller compared to controls (Fig. 5B-E). Females fed CHX for 48 h had noticeably smaller ovaries (Fig. S1). To determine any decrease in ovary size after 24 h of feeding, we measured the area and perimeter of each ovary (Fig. 5F,G). Females fed all three doses of CHX had decreased ovary size, with 10 mM CHX causing the smallest ovaries (Fig. 5F,G). Oogenesis is metabolically and energy-intensive and the smaller ovaries might have been caused by less material being made due to reduced protein synthesis. Thus, we extracted and measured total protein from ovaries exposed to CHX. Normalizing for size differences using ovary area, we did not observe a decrease in total protein after 24 h of dietary CHX (Fig. S1G). To determine whether a rich CHX diet altered follicle stage development, we used indirect immunofluorescence. In well-fed females, all stages (2-14) of follicle development in the ovariole are present (Fig. 1C). One of the two developmental checkpoints during oogenesis when follicle death occurs is at stage 8 (Fig. 1C, asterisk). This occurs in response to several stressors, of which the best characterized is nutritional stress (Drummond-Barbosa and Spradling, 2001). To identify any missing follicle stages, we labeled ovaries with the antibody against the germplasm protein Vasa and 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei. Well-fed females had ovarioles that contained the expected follicle stages, with no stage 8 follicles containing condensed nuclei (Fig. 6A-A″). In contrast, females fed a rich diet supplemented with CHX were frequently missing stages and had increased numbers of condensed nuclei, signifying apoptotic follicles (Fig. 6B-D″). Increasing the dose of CHX increased the number of defective ovarioles (Fig. 6E). In the follicles present, we did not detect changes to the number of germ cells per cyst, nor an increase in the loss of the oocyte. These observations support that, with a rich diet, CHX decreases ovary size by increasing apoptotic follicles.

Dietary CHX has been administered to flies in several contexts, but not for its effects on the Drosophila ovary and not by feeding a rich diet (Cervantes-Sandoval et al., 2016; Jelen et al., 2023; Jeong et al., 2022; Lagasse et al., 2009; Lee et al., 2018; Takakura et al., 2021; Tully et al., 1994). A previous study examined the effects of CHX feeding on various developmental stages and found a dose-dependent effect on lifespan (Marcos et al., 1982). Behavioral studies have found that CHX feeding affects certain types of memory in flies (Jelen et al., 2023; Lagasse et al., 2009; Lee et al., 2018; Takakura et al., 2021; Tully et al., 1994). These feeding studies used a standard method that relies on a mixture of sucrose and the desired drug and did not use a rich diet, which is critical for supporting optimal ovary development (Drummond-Barbosa and Spradling, 2001; Wei and Lilly, 2014). A sugar-only diet is considered a poor food source for females and does not support robust ovary development and egg laying. As we wanted to observe the effects of CHX on oogenesis without the confounding effects of a poor diet, we fed flies CHX mixed in live yeast paste, a rich diet that Drosophila prefer. As the yeast in the paste is active and likely metabolizing the CHX, the paste was made fresh daily. This ensured that CHX levels could be maintained at as steady a level as possible, although there was likely a drop in concentration over 24 h as wet yeast paste contains active yeast cultures that will presumably also metabolize any compound, thus reducing the concentration of drug. Using this method, we saw reduced survival, with females more affected than males. Males, however, exhibited a dose-dependent decrease in lifespan, whereas females did not. Somewhat counterintuitively, female egg laying was not affected. Given that there was increased loss of follicle stages, it is possible that the rich diet combined with stalled translation increased germline stem cell division, making up for any deficit in ovariole development, but we did not test germline stem cell division rates directly. Although egg laying was normal, progeny number was reduced from females fed CHX, indicating that the eggs laid were less competent to develop. The most parsimonious explanation for this observation is that dietary CHX reduces the levels of specific maternal transcripts critical for larval development. Another possibility is that global maternally loaded protein reduction, although not severe, falls below a threshold that cannot support larval development. However, further experiments must be done to determine the specific mechanisms underlying decreased egg competency. Dietary CHX reduced ovary size, likely due to increased apoptosis at a well-known checkpoint, but normalized total protein levels were unchanged. Thus, any action of CHX on disrupting follicle development appears to be independent of decreased global protein levels. Rather, these defects could be due to reduced translation of specific transcripts or signaling pathways while the robust signaling pathways responding to a rich food source remain intact. Overall, these data demonstrate that CHX treatment and stalled ribosomes specifically trigger follicle loss and increase apoptosis. In addition, this feeding method could be used for other compounds to distinguish specific effects on ovary development, as opposed to decreased nutrition.

The fly stock w¹¹¹⁸ was maintained on standard cornmeal fly medium. Animals were grown at room temperature. A newly eclosed fly was considered day 0.

CHX (Sigma-Aldrich, Burlington, MA, USA; C7698) was dissolved in water to prepare a 10 mM stock solution. Briefly, 140 mg of CHX powder was dissolved in 50 ml of water at room temperature in a dark box by rotating for 30 min, which was aliquoted and stored at −20°C until use. The desired concentration of CHX was prepared by serial dilution of the stock solution. To make fresh yeast paste containing CHX, 0.3 g of active dry yeast (Red Star Yeast) was mixed with 450 µl of CHX solution to create a yeast paste, which was provided daily as needed.

Blue dye feeding was performed as described in Wong et al. (2009). Newly eclosed flies were fattened with yeast paste for 3-7 days. Thirty female flies were then transferred to a vial with fresh fly food containing wet yeast paste (control) or freshly made yeast paste with varying concentrations of CHX prepared with 2.5% (w/v) of FD&C Blue No. 1 (Sigma-Aldrich, Burlington, MA, USA; 80717).

Twenty female or male day 0 adult flies were collected in standard fly food vials containing fresh wet yeast paste. After 24 h, the flies were transferred to freshly made wet yeast paste containing CHX. Fresh yeast paste with CHX was provided daily until no flies survived. Flies were maintained at room temperature, and the dead and missing animals were counted every day. All individual experiments had three replicates. Survival percentage was plotted using GraphPad Prism (GraphPad Software, Boston, MA, USA; https://www.graphpad.com/), and data were analyzed with the Kaplan–Meier test and for significant differences using the log-rank test using the OASIS2 online application with combined triplicates (Han et al., 2016).

Five newly eclosed female flies and three newly eclosed male flies were collected and fed with wet yeast paste for 24 h in an egg-laying cup containing an agar-corn syrup plate. The flies were transferred to a plate supplemented with wet yeast paste supplemented with varying concentrations of CHX. Every 24 h, the plate was replaced with fresh CHX-supplemented wet yeast paste. The eggs laid on the plate were counted every other day. Each experimental condition had three replicates. The bar graph was generated using GraphPad Prism.

Ten female day 0 adult flies and five male day 0 adult flies were collected in a standard food vial with wet yeast paste. Flies were fed yeast paste for 4 days. On day 4, flies were transferred to a standard food vial with wet yeast paste containing CHX. In 24 or 48 h after CHX feeding, flies were transferred to a new standard food vial that was kept at room temperature. After 48 h, flies were removed and the vials were kept at room temperature. The resulting pupae was counted daily and normalized with the number of parent females. All experimental conditions had three biological replicates. The line graph was generated using GraphPad Prism, and the data were analyzed statistically with two-way ANOVA with multiple comparisons followed by Tukey's post hoc test.

Ten female day 0 adult flies and ten male day 0 adult flies were collected in a standard food vial with wet yeast paste. The food vial with fresh wet yeast paste was switched every day for 4 days. On day 4, all female flies in each vial were transferred to a standard food vial containing freshly made yeast paste with varying concentrations of CHX. Fly ovaries were dissected in Grace's Insect Medium (Invitrogen, Waltham, MA, USA; 11595030) 24 h after CHX feeding. To measure whole ovary size, the dissected ovaries were immediately imaged under a stereomicroscope using an Accu-Scope 3076 digital microscope 0.67×-4.5× (Accu-Scope, Commack, NY, USA). After imaging, ovaries were washed with phosphate-buffered saline twice, frozen, and kept at −80°C for protein analysis. Ovary area and perimeter were measured using FIJI/ImageJ (Schindelin et al., 2012). The violin plot was generated using GraphPad Prism, and the data were analyzed statistically using one-way ANOVA with multiple comparisons followed by Tukey's post hoc test. To evaluate CHX effect on oogenesis, the ovaries were immunostained for Vasa and DNA and imaged as described below. Ovarioles were considered having developmental defects if they contained an apoptotic follicle and/or were missing stage 8-11 egg chambers. The following numbers of ovarioles were counted per individual experimental condition: experiment 1–78 (0 mM CHX), 92 (3.5 mM CHX), 97 (7 mM CHX), 72 (10 mM); experiment 2–44 (0 mM CHX), 72 (3.5 mM CHX), 89 (7 mM CHX), 84 (10 mM CHX); experiment 3–68 (0 mM CHX), 81 (3.5 mM CHX), 59 (7 mM CHX), 84 (10 mM CHX). The relative percentage of defective ovarioles compared to normal ovarioles was calculated within each experimental condition. The bar graph was generated by combining the results from three independent experiments using GraphPad Prism, and the data were analyzed statistically using one-way ANOVA with multiple comparisons followed by Tukey's post hoc test. All individual experiments had three biological replicates.

Fifteen day 0 adult flies and fifteen male day 0 adult flies were collected in a standard food vial with wet yeast paste. The food vial with fresh wet yeast paste was switched every day for 4 days. On day 4, all adult flies in each vial were transferred to a standard food vial containing a wet yeast paste with varying CHX concentration. After 24 h, flies were transferred to a standard vial containing a wet yeast paste with both 600 µM puromycin (Gibco, Waltham, MA, USA; A1113803) and varying CHX concentrations. After feeding puromycin and CHX, the female flies were collected and kept at −80°C until preparing and quantifying protein amounts as described below. 40 µg of each sample was separated by SurePAGE Bis-Tris gel (GenScript, Piscataway, NJ, USA; M00653) and transferred to a nitrocellulose membrane. The nitrocellulose membrane was probed with mouse anti-puromycin (3RH11) antibody (Kerafest, Shirley, MA, USA; EQ0001; 1:2000) following a staining with Ponceau S solution (Sigma-Aldrich; P7170) for imaging of total protein loading.

Dissected and frozen ovaries or frozen whole animals were homogenized with a whole-cell lysis buffer composed of 50 mM Tris (pH 8.0), 150 mM sodium chloride, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 0.1% sodium dodecyl sulfate and Complete-mini EDTA-free protease inhibitor (Roche Applied Science, Indianapolis, IN, USA; 11836170001) by repeated freeze-thaw cycles and 30 times of strokes with a pestle. Insoluble material was removed by centrifugation (12,000 g for 20 min at 4°C) and the subsequent supernatant was collected as a fraction of whole cellular proteins. Protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA; 23227).

Ovaries were dissected with Grace's Insect Medium and fixed for 20 min (4% paraformaldehyde and 20 mM formic acid in Grace's Insect Medium). After washing with antibody wash buffer (0.1% Triton X-100 and 1% BSA in phosphate-buffered saline) three times for 20 min, the tissue was stained with the primary antibody rat anti-Vasa (1:14, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), overnight at 4°C. After washing with antibody wash buffer three times for 20 min, the tissues were stained with the secondary antibody goat anti-rat IgM Cross-Adsorbed Secondary Antibody, DyLight 488 (1:500, Invitrogen, SA510010) overnight at 4°C, then incubated with antibody wash buffer twice for 20 min and stained with 5 ng/ml DAPI solution for 10 min. After removing the DAPI solution, the tissues were mounted in VectaShield Antifade Mounting Medium (Vector Laboratories, Newark, CA, USA; H-1000). Images were obtained using a Zeiss LSM 980 confocal laser scanning microscope at Plan Apochromat 10× objective (Carl Zeiss Microscopy, White Plains, NY, USA) or a Nikon Eclipse Ti2 spinning disk microscope at Plan Apo λ 10× objective (Nikon Corporation, Tokyo, Japan) with z-stacks for a depth of 20 µm.

We would like to thank the Uniformed Services University of the Health Sciences (USUHS) Biomedical Instrumentation Core and Dr Dennis McDaniel for imaging support. Antibodies obtained from the Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development of the National Institutes of Health (NIH) and maintained at the University of Iowa, Department of Biology, were used in this study. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Cartoons in Figs 1-5 and Fig. S1 are courtesy of BioRender [(Cox, R. (2023) https://BioRender.com/o94j079]. The contents of this publication are the sole responsibility of the authors and do not necessarily reflect the views, assertions, opinions or policies of USUHS, the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF), the Department of Defense (DoD), or the Departments of the Army, Navy or Air Force. Mention of trade names, commercial products or organizations does not imply endorsement by the US Government.