Methods

Study species and generating experimental flies

We sourced flies from Dahomey, a large laboratory population of D. melanogaster, originally sourced in 1970 from Benin West Africa. The flies have been maintained in large population cages, with overlapping generations in the Piper laboratory, Monash University, Australia, since 2017, and prior to that in the Partridge laboratory, University College London(33). Prior to the beginning of the experiment, we collected ~3000 eggs from the cages, and distributed them into 250mL bottles containing 70mL of food. Food comprised 5% sucrose (50 grams sucrose, 100 grams yeast, 10 grams agar per litre) solution with an estimated protein to carbohydrate [P:C] ratio of 1:1.9, and 480.9 kcal per litre (see Supplementary Material Figure S4 for further diet details). Every generation (for seven generations), adult flies eclosing from multiple bottles were admixed prior to redistributing the flies across new bottles. To control for potential sources of variation in their environment, during these seven generations we strictly controlled both the age of flies at the time of ovipositioning—all flies were within 24 hours of eclosion into adulthood when producing the eggs that propagated the subsequent generation, and their population density was 300-320 adult flies within each bottle in each generation.

Dietary treatments

The diet media we used consists of sucrose, autolysed brewer’s yeast powder (sourced from MP Biomedicals SKU 02903312-CF), and agar (grade J3 from Gelita Australia), as well as preservatives—propionic acid, and nipagin. We prepared two dietary treatments, differing in relative sucrose concentration; 2.5% sucrose (that we refer to as a lower sucrose treatment relative to the 5% concentration usually provided to the population of flies used in this experiment), and 20% sucrose (that we refer to as a higher sucrose treatment) of overall food solution. The 2.5% sucrose diet contains 25 grams of sucrose, 100 grams of yeast and 10 grams of agar per litre of food prepared, with an estimated P:C ratio of 1:1.4 and 380.9kcal per litre of food. The 20% sucrose treatment contains 200 grams of sucrose, 100 grams of yeast, and 10 grams of agar per litre of food prepared, with an estimated P:C ratio of 1:5.3 and 1080.9kcal per litre of food. The diets thus differed not only in sucrose concentration, but overall macronutrient balance and their total caloric content.

We used varying levels of sucrose in our experiments because it is ecologically relevant to do so, as fruit flies in a natural environment can experience both spatial and temporal heterogeneity in their diet, i.e., depending on what food is available to them at that time in that place. Fruit flies usually feed on rotting fruits, which depending upon the type of fruit and varying levels of decomposition, will produce differing levels of sucrose (but very low levels of fats). Additionally, the higher sucrose concentration was selected based on preliminary experiments that we conducted (see supplementary material in Camilleri et al, 2022, which caused flies to accumulate more body fat (measured by measuring whole-body triglyceride levels), than diets of regular sugar, consistent with results from previous work in D. melanogaster. A total sugar concentration of over 20% tends to result in flies that are too unhealthy to conduct a transgenerational experiment with, i.e. flies either are low in fertility, die before they have a chance to mate, or their offspring are not viable enough to experiment on cross-generationally. It is also estimated that the percentage of overall daily caloric needs from added sugars in the average American diet could be around 13-20% in children, and as high as 57% in adults, and those added sugars are often consumed in the form of ultra processed foods, which are also usually made up of around 20% added sugar, therefore our choice of 20% sucrose for fly food is a fair (and conservative) analog sugar content in a western diet. All diets contained 3ml/l of propionic acid and 30ml/l of a Nipagin solution (100g/l methyl 4-hydroxybenzoate in 95% ethanol) and were cooked according to the protocol described in Bass et al. (2007). Each vial is 40mL in volume, and contained 7mL of food.

Experimental design

Male and female virgin flies were assigned to one of two of the dietary treatments prior to mating (we refer to this generation of flies as F0), and then the grandoffspring produced (F2 generation) were also assigned to one of the two treatments. All possible combinations of grand dam × grand sire × grandoffspring diet treatment were represented (= 2 × 2 × 2 = 8 combinations). Specifically, we collected 1280 flies of the F0 generation as virgins and placed them onto either the high sucrose (20%) or the low sucrose (2.5%) diets for the first 6 days of their adult life. They were kept in vials of 10 flies across 64 vial replicates per treatment, and per sex (high sucrose: 32 vials of males and 32 vials of females; low sucrose, 32 vials of males and 32 vials of females, 128 vials in total;1280 flies, 640 of each sex). They were kept in their respective sexes. We transferred flies to vials containing fresh food of the designated diet every 48 hours during this six-day period. On day six, we randomly sampled six vials from each treatment, and snap froze (using liquid nitrogen) the flies of these vials, storing them at -80°C for subsequent measures of triglyceride levels. Cohorts of flies in the remaining vials then entered a cohabitation phase to enable female and male F0 flies to mate. Cohorts of males and female flies were combined, in vials of ten pairs, in each of all four possible diet combinations: lower sucrose females × lower sucrose males; higher sucrose females × higher sucrose males; lower sucrose females × higher sucrose males; higher sucrose females × lower sucrose males. During this phase, flies cohabited for 96 hours. They were transferred to a new vial with fresh food of standard 5% sucrose diet every 24 hours during this time.

The vials from the six-day-old F0 flies (i.e., the vials from Day one of the 96-hour cohabitation phase) were retained, and the eggs that had been laid by females of the respective vials were trimmed to 80 per vial by removing excess eggs with a spatula. The remaining eggs were left to develop into adult offspring over 10 days at 25°C (on a 12:12 light/dark cycle in a temperature-controlled cabinet; Panasonic MLR-352H-PE incubator). These adult flies constituted the F1 offspring in the experiment, and F1 flies developed on standard 5% sucrose media. We collected 2080 virgin F1 flies from each of the four combinations of parental diet treatments, and placed them in sex-specific cohorts of ten individuals per vial, on standard 5% sucrose media for six days. We then allowed these F1 males and F1 females to cohabit and mate with male or female tester flies (creating ten pairs per vial) that had been collected from the same Dahomey stock population (but not subjected to a dietary sucrose treatment) to create the F2 generation. The diet treatments applied to the F0 flies were thus transferred to the F2 generation via either F1 males or F2 females, but never through both sexes. The F1 flies were six days of adult age when laying the eggs that produced the F2 generation. We then collected virgin F2 flies (the grandoffspring of the F0 flies) from each of the four combinations of F0 diet treatments (per sex), and placed them in their respective sexes in vials of ten flies, across 102 vial replicates per diet treatment per sex (4080 flies, 2040 male, 2040  female). We assigned these F2 flies, produced by each dietary treatment combination of F0 flies, to either the lower sucrose or higher sucrose diet. At day six of adulthood, we snap froze F2 flies of six randomly chosen vials per grand dam × grand sire × grandoffspring combination. On the same day, ten virgin focal F2 flies of each grand dam × grand sire × grandoffspring combination and each sex were placed together with ten age-matched tester flies of the opposite sex from the Dahomey population, entering into a cohabitation phase of 96 hours (during which time the number of eggs laid by females of each vial was assessed). After 96 hours flies were separated again into their respective sexes (in vials of 20 experimental flies of the same sex), and assigned back onto either the lower sucrose or higher sucrose diets that they had been on prior to cohabitation, and a lifespan assay carried out.

Lifespan

We scored the lifespan of experimental flies of the F2 generation. Each vial in the assay commenced with 20 same-sex flies in each, and we included ten vial replicates per treatment (grand dam × grand sire × grandoffspring) (3400 flies total, the original amount collected, minus the snap frozen samples). The number of dead flies per vial was scored three times per week (Monday, Wednesday, Friday), and surviving flies at each check transferred to vials with fresh food of the assigned diet treatment—until all flies were deceased. During the lifespan assay, vials were stored in boxes (of 85 vials per box) that were moved to randomised locations in a (25°C) control temperature cabinet every few days to decrease the potential for confounding effects of extraneous sources of environmental variation within the cabinet from affecting the results.

Fecundity

We measured the egg output of female flies from generations F0 and F2 at eight days following eclosion, as a proxy of female fecundity. On day eight, female flies oviposited for a 22 hour period, and were then transferred to fresh vials. Day eight was selected because fecundity over 24 hours at this age has been shown to correlate with total lifetime fecundity of females in this Dahomey population and early, short term measures of reproduction of between one and seven days can be used to accurately predict total lifelong fecundity in D.melanogaster. Moreover, previous data shows that varied the range of sucrose concentrations did not alter the timing of reproductive peaks between treatments(38). For the F0 generation, we counted eggs from vials, each containing 10 female flies that had been mated with ten male flies, across 2 different sucrose levels (2.5% and 20% sucrose), and four different mate diet combinations (dam diet x sire diet combinations). For the F2 generation, we counted eggs from each grand dam × grand sire × grandoffspring dietary treatment combination; each combination was represented by ten vial replicates, each containing 10 focal females (females from the experiment) combined with ten tester male flies. Additionally, we counted the number of adult flies that eclosed within ten days from the eggs laid by F2 females (a composite measure of clutch viability and juvenile developmental speed). F2 females cohabited and mated with age-matched tester males of the Dahomey population, for 24 hours at six days of life, and the vials containing these eggs were left to develop into adult offspring, for ten days at 25°C; 12:12 light/dark cycle in a temperature-controlled cabinet (Panasonic MLR-352H-PE incubator).

Lipids and protein

Whole-body triglyceride levels were measured in adult flies from the F2 generation (six days of adult age, corresponding with six days of exposure to the relevant F2 dietary treatment, prior to mating) and normalized to protein content (full protocols reported in the Supplementary Material). Three biological replicates per treatment level, with three technical replicates per biological replicate were used. Five female flies and eight male flies respectively, were used for each biological replicate in the assay, to standardize weight for each sample. We chose to measure whole-body triglycerides in flies as a proxy measure of overall accumulation of body fat under the differing diet treatments, as previous studies have shown increasing sucrose levels in fruit fly diets tend to also increase triglycerides.

Statistical Analyses

We used R (Version 3.6.1) and RStudio (Version 1.2.1335) (R Core Team, 2019) for statistical analyses. To test the effects of F0 female diet, F0 male diet, F2 diet, and sex on F2 lifespan, TAG, and offspring production, we fitted linear mixed effects models (separate models per trait), using the R package lme4 (V 1.1-27.1). We use the term lifespan to denote the age of recorded death for each individual fly within a margin of 72 hours (for example, a lifespan of 30 days indicates that a fly died between 27-30 days post eclosion). To test the effects of grand maternal diet, grand paternal diet, grand offspring diet, and sex on female fecundity, we fit a general linear model to the egg output data for both generations.

We included F0 male, F0 female, F2 diets, and F2 sex as fixed effects in each model respectively, exploring interactions between these factors. We included the vial identification number as a random effect in the lifespan models. The fecundity models only included one observation per vial because we counted total eggs per vial, and divided by the number of females in the vial (approx. 10 females) therefore, no random effects were included in this model. The viability of the F2 grand offspring (how many F3 eclosed) included ‘Counter’ (the person who counted the flies) as a random effect. The model that investigated the F2 whole-body TAG included, plate reading replicate, technical replicate and vial ID as random effects.