DOI: 10.5061/dryad.zkh1893h1
title: Sex-specific transgenerational effects of diet on offspring life history and physiology

description: Dietary variation in males and females can shape the expression of
 offspring life histories and physiology. However, the relative
 contributions of maternal and paternal dietary variation to phenotypic
 expression of latter generations are currently unknown. We provided male
 and female Drosophila melanogaster grandparents with diets differing in
 sucrose concentration prior to reproduction, and similarly subjected their
 grandoffspring to the same treatments. We then investigated the phenotypic
 consequences of this dietary variation among the grandsons and
 granddaughters. We observed transgenerational effects of dietary sucrose,
 mediated through the grandmaternal lineage, which mimic the direct effects
 of sucrose on lifespan, with opposing patterns across sexes; low sucrose
 increased female, but decreased male, lifespan. Dietary mismatching of
 grandoffspring-grandparent diets increased lifespan and reproductive
 success, and moderated triglyceride levels of grandoffspring, providing
 insights into the physiological underpinnings of the complex
 transgenerational effects on life histories.
description: <strong>Methods</strong>
 <strong>Study species and generating experimental
 flies</strong> We sourced flies from Dahomey, a
 large laboratory population of <em>D. melanogaster</em>,
 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.
 <strong>Dietary treatments</strong>
 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 <em>et al</em>, 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 <em>D. melanogaster</em>. 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
 <em>et al. </em>(2007). Each vial is 40mL in volume, and
 contained 7mL of food. <strong>Experimental
 design</strong> 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 <em>tester </em>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.
 <strong>Lifespan</strong> 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.
 <strong>Fecundity</strong> 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
 <em>D.melanogaster</em><em>. </em>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). <strong>Lipids
 and protein</strong> 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. <strong>Statistical
 Analyses</strong> 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.
 We used log-likelihood ratio tests that reduce the
 full model, via the sequential removal of highest order interactions that
 did not (significantly) change the deviance of the model, using a
 <em>p </em>value significance level of &lt;0.05. The final
 reduced models (except fecundity measures) were fit by restricted maximum
 likelihood, applying type III ANOVA with Kenwood-Roger’s F test and
 approximation of denominator degrees of freedom. We used sum to zero
 constraints in all models, and we visually inspected diagnostic plots for
 the linear mixed effect models, to ensure that the assumptions of
 normality and equal variances were met.
description: # GENERAL INFORMATION 1. Sex-specific transgenerational effects of diet on
 offspring life history and physiology 2. Author Information A. Principal
 Investigator Contact Information Name: Damian Dowling Institution: Monash
 University Email:
 [damian.dowling@monash.edu](mailto:damian.dowling@monash.edu) B.
 Corresponding Author Name: Tara-Lyn Camilleri Institution: Monash
 University and Oxford University Email:
 [taralyncamilleri@gmail.com](mailto:taralyncamilleri@gmail.com) ## File
 List: Five CSV files as follows: * F0_fecundity_data: Egg laying data over
 a 23 hour period for the female F0 flies * F2_fecundity_data: Egg laying
 data over a 23 hour period for the female F2 flies * Egg_Adult_Viability:
 The F2 female flies laid egg over a 23 hour period and then adult flies
 that emerged (F3 gen) were counted for each treatment combination *
 F2_longevity_data: Data on how long the flies both male and female F2
 lived on the different diets and parental diet combinations ###
 DATA-SPECIFIC INFORMATION FOR: \[F0\_fecundity\_data.csv] 1. Number of
 variables: 10 2. Number of cases/rows: 49 3. Variable List: *
 Gen=generation, * vial no= vial number/replicate, * no_fem= how many
 female flies in the vial, * diet_fem= diet fem was challenged with (HS for
 high sucrose 20%, LS for low sucrose 2.5%), * diet_mal=diet the male was
 challenged with, * diet_combo= diet combination of both males and females;
 female is always first, i.e HSLS means HS females LS males, * photo_label
 = diet_combo + gen + vial (because eggs were counted from photos). *
 egg_count= number of eggs counted in the vial over a 23 hour period,
 egg_per_female= egg_count/no_fem ### DATA-SPECIFIC INFORMATION FOR:
 \[F2\_fecundity\_data.csv] 1. Number of variables: 12 2. Number of
 cases/rows: 161 3. Variable List: * vial no = vial number/replicate *
 grand_diet= diet combination of the grandparents, LS is low sucrose,
 females are always first eg HSLS= high sugar dams and low sugar sires. *
 gdam_diet= diet the gmother of the F2 fly was challenge with prior to
 mating HS or LS (HS for high sucrose 20%, LS for low sucrose 2.5%), *
 gsire_diet= diet the gfather of the F2 fly was challenge with prior to
 mating HS or LS (HS for high sucrose 20%, LS for low sucrose 2.5%), *
 parent= whether diet effects were passed through the dam or sire line, *
 own_diet= diet the F2 ate themselves (HS for high sucrose 20%, LS for low
 sucrose 2.5%) * sex= sex of the F2 fly * photo_label = diet_combo + gen +
 vial (because eggs were counted from photos). * egg_count= amount of eggs
 counted in 23 hour period per vial, * no_female= how many female flies in
 the vial, * no_male= how many male flies in the vial, * eggs_per_female=
 egg_count/no_fem ### DATA-SPECIFIC INFORMATION FOR:
 \[Egg\_Adult\_Viability.csv] 1. Number of variables: 12 2. Number of
 cases/rows: 161 3. Variable List: * tube_no= vial number/replicate, *
 F0_diet= diet combination of the grandparents, LS is low sucrose, females
 are always first eg. HSLS= high sugar dams and low sugar sires *
 gdam_diet= diet the gmother of the F2 fly was challenge with prior to
 mating HS or LS (HS for high sucrose 20%, LS for low sucrose 2.5%) *
 gsire_diet= diet the gfather of the F2 fly was challenge with prior to
 mating HS or LS (HS for high sucrose 20%, LS for low sucrose 2.5%) *
 F1_parent= whether diet effects were passed through the dam or sire line *
 F2_damdiet= what diet the F2's dam ate, HS is high sucrose (20%), LS
 is low sucrose (2.5%) * no_males= how many male flies in the vial *
 no_females= how many female flies in the vial * no_adults_all= how many
 flies overall were in the vial * counter= which person in the lab counted
 the flies * no_femaleF2= number of female flies in the vials that produced
 the F3 * no_maleF2= number of male flies that were placed in the vial for
 the female flies to mate with. ### DATA-SPECIFIC INFORMATION FOR:
 \[F2\_longevity\_data.csv] 1. Number of variables: 8 2. Number of
 cases/rows: 2830 3. Variable List: * vial no= vial number/replicate, *
 grand_diet= diet combination of the grandparents, LS is low sucrose,
 females are always first eg * HSLS= high sugar dams and low sugar sires. *
 gdam_diet= diet the gmother of the F2 fly was challenge with prior to
 mating HS or LS (HS for high sucrose 20%, LS for low sucrose 2.5%), *
 gsire_diet= diet the gfather of the F2 fly was challenge with prior to
 mating HS or LS (HS for high sucrose 20%, parent= whether diet effects
 were passed through the dam or sire line, * own_diet= diet the F2 ate
 themselves (HS for high sucrose 20%, LS for low sucrose 2.5%), * sex= sex
 of the F2 fly, * age= how long the flies lived in days.