bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Sex-specific transgenerational effects of diet on offspring life 2 history and physiology 3 Tara-Lyn Camilleri*1, Matthew D.W. Piper1, Rebecca L. Robker2, 3, Damian K. Dowling1. 4 1 School of Biological Sciences, Monash University, Melbourne, VIC, Australia, 3800 5 2 School of Paediatrics and Reproductive Health, Robinson Research Institute, The 6 University of Adelaide, Adelaide, Australia, 5005 7 3 School of Biomedical Sciences, Monash University, Melbourne, VIC, Australia, 3800 8 9 10 *Corresponding author: Tara-Lyn Camilleri - tara-lyn.carter@monash.edu; Twitter: @TaraLynC 11 12 Abstract 13 Dietary variation in males and females can shape the expression of offspring life histories 14 and physiology. However, the relative contributions of maternal and paternal dietary 15 variation to phenotypic expression of latter generations is currently unknown. We provided 16 male and female Drosophila melanogaster diets differing in sucrose concentration prior to 17 reproduction, and similarly subjected grandoffspring to the same treatments. We then 18 investigated the phenotypic consequences of this dietary variation among grandsons and 19 granddaughters. We demonstrate transgenerational effects of dietary sucrose, mediated 20 through the grandmaternal lineage, which mimic the direct effects of sucrose on lifespan, 21 with opposing patterns across sexes; low sucrose increased female, but decreased male, 22 lifespan. Dietary mismatching of grandoffspring-grandparent diets increased lifespan and 23 reproductive success, and moderated triglyceride levels, of grandoffspring, providing 1 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 24 insights into the physiological underpinnings of the complex transgenerational effects on 25 life histories. 26 27 Keywords 28 Transgenerational effects, sucrose, life history, drosophila 29 30 Main 31 From nematodes to primates, parental environments may shape the phenotypes of their 32 offspring through non-genetic mechanisms that are either condition dependent or 33 epigenetic in origin1–3 4. Consequently, when individuals are subjected to environmental 34 heterogeneity prior to reproduction, their exposure to these environments can shape 35 components of fitness in offspring and subsequent generations (transgenerational effects)5– 36 9 37 predation risk and levels of sexual conflict, among parents may catalyse transgenerational 38 effects that differ in magnitude or direction across sexes, and which may also be lineage 39 (genotype) specific10. Notwithstanding, currently it remains unclear whether such 40 transgenerational effects are consistently instigated across diverse environmental stresses, 41 whether they generally act to enhance or depress offspring performance, and whether they 42 are transferred primarily through maternal or paternal lineages or hinge on interactions 43 between both. 44 Nutrition is a pervasive and critical source of environmental variation that shapes 45 phenotype. Variation in macronutrient balance or caloric content has been shown to confer 46 direct effects on lifespan, fecundity, and underlying physiology11–14. Studies from diverse 47 species have demonstrated that females and males require different diets to maximise their 48 fitness15–19. Female fitness is maximised on a higher relative protein concentration because . Recent experiments have shown that variation in environmental factors, such as 2 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 49 high protein facilitates egg production, while higher relative carbohydrate content for 50 males provides fuel for attracting and locating a mate20–24. Recent studies have also shown 51 dietary-induced intergenerational effects across a variety of species; for example, changes 52 to sugar content of the parental diets in fruit flies25,26 or dietary fat content in mice27 53 induces phenotypic changes in parents that are transmitted to their offspring. Intriguingly, 54 when the sucrose content of both male and female parents are altered, then parental 55 contributions to offspring phenotypes may involve complex dam-by-sire interactions that 56 are non-cumulative and dependent upon the sucrose content of the offspring diet 28. It is 57 less clear, however, whether these dietary-mediated parental effects are epigenetic in 58 origin, and thus inherited across multiple generations 10,25,29–32, and if so, whether 59 phenotypic consequences for males and females are divergent. 60 Here, we experimentally tested the capacity for dietary sucrose variation among male and 61 female fruit flies (Drosophila melanogaster) to precipitate transgenerational effects on 62 components of life-history and physiology in their grandoffspring. Flies were administered 63 one of two diets that varied in the concentration of sucrose (2.5% or 20% sucrose). The diets 64 were administered using a full factorial design: males and females were each assigned to one 65 of the two diets prior to reproduction, and then their grandsons and granddaughters were 66 administered the same dietary treatments. All male-female-grandoffspring dietary 67 combinations were represented, resulting in female-male and grandparent-grandoffspring diet 68 combinations that were either matched or mismatched (Figure 1, panels A and B). This 69 design enabled us to test whether dietary-mediated transgenerational effects exist, to decipher 70 the relative grandmaternal and grandpaternal contributions, and the capacity for interactions 71 between grandparental diets and those of the grandoffspring to shape grandoffspring 72 phenotype, and to determine whether such effects are sex specific. 73 3 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 74 75 Figure 1. A) Diet effects lineage. Diet treatments were administered to both parents in the 76 F0; and they were mated to create the F1 offspring, and received a standard diet (both 77 males and females for each parent), and F1 offspring were mated with flies outside of the 4 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 78 experiment that received a standard diet. This allowed us to track which F1 sex was 79 passing on the diet effects to the F2 generation. B) Experimental design. The F0 80 generation was administered either higher (20% of overall solution) or lower (2.5%) 81 relative sucrose in adulthood, and kept on this diet in sex-specific cohorts for 6 days as 82 virgins before a subsequent three day cohabitation (on common garden media) that 83 allowed mating to occur. Male and female F0 flies were combined in all possible diet 84 combinations. The F1 generation was reared, maintained (6 days again), and cohabited (3 85 days) on common garden media (an intermediate sucrose content of 5%). The F2 86 generation was reared from egg-to-adulthood on common garden media, and then 87 challenged as virgins with either the higher or lower sucrose such that their diet either 88 matched or mismatched one or both of their grandparents (F0). 89 90 Results 91 Direct and indirect effects of dietary sucrose on grandoffspring lifespan are sex- 92 specific 93 The diets of the grandoffspring (F2) flies conferred direct and sex-specific effects on their 94 lifespan (F1,148 = 369.80, p <0.001, Table S2, Figure 2). Female F2 flies assigned to the 95 low sucrose diet lived longer than females or males assigned to any other treatment, and 96 30% longer than females on the high sucrose diet. Females assigned to a high sucrose diet 97 exhibited the shortest lifespan of any group of flies. In contrast to the large negative effect 98 of high sucrose on female lifespan, high dietary sucrose conferred a moderate increase in 99 male lifespan relative to males assigned to a low sucrose diet (Table S2; Figure 2). 100 5 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 101 102 Figure 2. Direct effects of dietary sucrose on the lifespan (± standard error) of F2 103 granddaughters (F) and grandsons (M). HS indicates a high sucrose diet of 20% (P:C ratio 104 1:5.3), LS indicates a low sucrose diet of 2.5% (P:C ratio 1:1.4). 105 106 The lifespan of F2 flies was also in part mediated by the diets of their grandmothers, with 107 the pattern of effects differing across F2 males and females (F1,148 = 9.35, p <0.01, Table 108 S2, Figure 3, panel A). The transgenerational effects of sucrose concentration mimicked 109 the direction of direct effects described above. That is, F2 females descended from 110 grandmaternal lineages assigned to a low sucrose diet lived longer than those descended 111 from high sucrose lineages, while the opposite pattern was observed in F2 males, whereby 112 those descended from high sucrose grandmaternal lineages outlived those from low 113 sucrose lineages (Figure 3, panel A). Additionally, matching combinations of 6 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 114 grandmaternal-grandoffspring dietary sucrose led to shorter F2 lifespan than mismatched 115 combinations (Figure 3, panel B). 116 In our experimental design, grandparental flies were manipulated, and F2 phenotypes 117 measured. This involved transfer of effects across an intermediate generation – the F1 118 parents. Although the diets of F1 parents were never manipulated (they received a standard 119 diet of 5% sucrose, an intermediate sucrose content), our experimental design ensured the 120 grandparental effects were transferred through either male F1 or female F1 flies (but not both, 121 Figure 1). Thus, we could track whether the sex of the transferring F1 parents affected the 122 pattern and direction of the transgenerational effects. Indeed, the interaction between the sex 123 of the F2 flies and the sex of the transferring F1 parents affected F2 lifespan (F1,148 = 4.44, p 124 <0.05, Table S2); female F2 lived longer if the grandparental dietary treatments were 125 transferred through F1 females, while male F2 lived longer when the effects were transferred 126 through F1 males (Figure 3, panel C). The sex of the transferring F1 parent flies also 127 moderated the direct effects of the F2 diet on F2 lifespan, Table S2, Figure 3, panel D, F1,148 128 = 12.42, p <0.001). F2 flies assigned directly to a high sugar diet lived longer if grandparental 129 dietary treatments were transferred through F1males rather than through females, while F2 130 flies assigned to a low sugar diet lived longer if grandparental dietary treatments were 131 transferred through F1 females than males. 7 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 132 133 Figure 3. 134 Effects of high sucrose (20% of overall solution) and low sucrose (2.5% of overall 135 solution) on F2 lifespan. Plots show means, and standard error bars. (A) Lifespan of F2 136 flies (y-axis), their grand dam's diet (colour), their sex (x-axis), (interaction: grand dam diet 137 × F2 sex). (B) Lifespan of F2 flies (y-axis), their grand dam's diet (colour), their diet (x- 138 axis), (interaction: grand dam diet × F2 diet). (C) Lifespan of F2 flies (y-axis), the sex of 139 the parental linage that received a diet treatment, (colour), their sex (x-axis), (interaction: 140 F1 sex × F2 sex). (D) Lifespan of F2 flies (y-axis), the sex of the parental linage that 141 received a diet treatment (colour), their diet (x-axis), (interaction: F1 sex × F2 diet). 142 8 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 143 Grandoffspring fecundity, viability, and triglycerides are mediated by grand 144 maternal and grand paternal diets 145 Fecundity & viability 146 Direct dietary effects were observed in the F2 generation; F2 Females had higher fecundity 147 when ingesting the low sucrose than high sucrose diet. These direct effects of diet were, 148 however, shaped by the grand paternal, but not grand maternal diet (Table S3, F1 = 5.49, p 149 <0.05). Mismatched combinations of grandpaternal-F2 female diet resulted in F2 150 granddaughters producing more eggs than matched combinations (Figure 4, panel A). 151 Female F2 fecundity was also shaped by an interaction between the grand maternal and 152 grand paternal diets (Table S3, Figure 4, panel B, F1= 14.77, p <0.05); F2 females that 153 descended from matched grandmaternal-grandpaternal combinations tended to have lower 154 fecundity than those arising from mismatched combinations, and in particular F2 females 155 descended from grandparents that were each assigned to low sucrose diets exhibited lowest 156 fecundity (Figure 4, panel B). The reproductive success (as gauged by the number of adult 157 offspring produced) of the F2 females was also shaped by a similar interaction between 158 grandmaternal and grandpaternal diet, in which the clutch size was lower for F2 females 159 descended from matched, relative to mismatched, combinations of grandmaternal- 160 grandpaternal diet (Table S4, Figure 4, panel C, F1 = 5.25, p <0.05). 161 9 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 162 163 164 Figure 4 165 Effects of high sucrose (20% of overall solution) and low sucrose (2.5% of overall 166 solution) on female F2 reproductive output. Plots show means, and standard error bars. (A) 167 Number of eggs laid by F2 flies (y-axis), their grand sire's diet (colour), their diet (x-axis), 168 (interaction: grand sire diet × F2 diet). (B) Number of eggs laid by F2 flies (y-axis), their 169 grand sire's diet (colour), their grand dam’s diet (x-axis), (interaction: grand sire diet × 170 grand dam diet). (C) Number of F3 flies eclosed per vial (y-axis), their grand sire's diet 171 (colour), their grand dam’s diet (x-axis), (interaction: grand sire diet × grand dam diet). 172 Triglyceride levels 173 An interaction between the diet of F2 offspring and the grandmaternal diet affected the 174 triglyceride level of the F2 flies (Table S5, Figure 5, panel A, F1,98= 8.56, p <0.01). F2 flies 175 fed high sucrose diets that descended from grandmothers assigned to high sucrose, 176 exhibited much higher triglyceride levels than F2 flies from any other combination of 177 grandmaternal-F2 offspring diet (Figure 5, panel A). Similarly, the interaction between F2 178 diet and grandpaternal diet shaped triglyceride level; however in this case, F2 offspring 179 assigned to a high sucrose diet and descended from grandfathers assigned to low sucrose, 10 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 180 exhibited much higher triglyceride levels than any other combination of grandpaternal-F2 181 diet (Table S5, Figure 5, panel B, F1,98= 12.75, p <0.001). 182 183 Figure 5. 184 Effects of high sucrose (20% of overall solution) and low sucrose (2.5% of overall 185 solution) on F2 whole body triglyceride (TAG) levels divided by their whole body protein 186 levels, per fly. Plots show means, and standard error bars. (A) F2 TAG per fly (y-axis), 187 their grand dam's diet (colour), their diet (x-axis), (interaction: grand dam diet × F2 diet). 188 (B) F2 TAG per fly (y-axis), their grand sire's diet (colour), their diet (x-axis), (interaction: 189 grand sire diet × F2 diet). 190 No direct effect of dietary sucrose on female F0 fecundity 191 Neither the male, nor female diet, affected egg output of the F0 females (Table S1). 192 193 Discussion 194 Here, we show opposing effects of dietary sucrose on the lifespan of each sex, in D. 195 melanogaster—low sucrose enhances female lifespan, but decreases male lifespan relative 11 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 196 to high sucrose. Notably, these effects were observed in both direct and indirect (i.e. 197 transgenerational) contexts. Moreover, the dietary-mediated transgenerational effects on 198 lifespan mimicked the observed direct effects for each sex: a low sucrose grandmaternal 199 diet conferred elevated F2 female lifespan, but decreased male F2 lifespan, relative to a 200 high sucrose grandmaternal diet. We also revealed strong effects of specific combinations 201 of grandparental and grandoffspring diet, and between grandmaternal and grandpaternal 202 diets, in shaping the measured traits; all of which exhibited a similar pattern—a mismatch 203 in diet enhanced trait expression in subsequent generations. We highlight inherent 204 complexity in the nature of the transgenerational effects. The effects are generally sex- 205 specific, and unexpectedly, are affected by the sex of the transferring F1 parent. Finally, 206 we note that interactions between grandparental diet and F2 diet affected triglyceride levels 207 of F2 flies, suggesting that dietary-mediated modifications of triglyceride levels, across 208 generations, may contribute to the observed transgenerational effects on life history 209 phenotypes. 210 Studies investigating sex-specificity of transgenerational effects across a range of taxa 211 have observed instances in which environmental modification such as dietary challenges, 212 presence of predators, or behaviour-modifying drugs of the grandparental environment 213 triggered sex-specific effects on grandoffspring phenotype. Intriguingly, in these cases, 214 transgenerational effects tend to manifest in the opposite sex to that subjected to the 215 grandparental treatment; that is, modification of the grandmaternal environment may 216 enhance or inhibit trait expression among grandsons, or conversely, modification to the 217 grandpaternal environment may enhance or inhibit trait expression among granddaughters 218 10,25,30,32,33 219 directions of sucrose-mediated grandmaternal effects in each of the sexes. Notably, we 220 have uncovered further levels of complexity in the nature of the transgenerational effects. . Our findings are consistent with previous research, revealing opposing 12 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 221 First, we revealed sex differences in the magnitude of transgenerational effect (the effect 222 transmitted from dietary-treated F0 flies to F2 flies) are dependent on the sex of the 223 transferring F1 parent. Second, we observed that the outcomes of transgenerational effects 224 depend on interactions between the diets of the grandparents and those of the 225 grandoffspring; dietary mismatching across generations tends to enhance lifespan 226 (mediated by a grandmaternal-by-grandoffspring interaction) and fecundity (mediated by a 227 grandpaternal by granddaughter diet interaction). Whether or not these effects are mediated 228 by underlying triglyceride levels of the experimental flies remains unclear; yet one pattern 229 was notable, suggestive of a possible transgenerational link between physiology and 230 lifespan. F2 offspring assigned to a high sucrose treatment, and descended from high 231 sucrose grandmaternal lineages exhibited the highest triglyceride levels and the shortest 232 lifespans. 233 Investigations into life history traits are imperative in assessing the adaptive significance of 234 transgenerational effects on offspring, given the close link between these traits and lifetime 235 fitness5. Our experiments, across three generations, with the diet challenge also given to 236 the F2 generation had the requisite power to address these previous knowledge gaps. Our 237 finding that dietary mismatching (between both grandparents and between grandparents 238 and grandoffspring) tends to enhance trait expression adds new insight to studies 239 investigating transgenerational effects of diet, and of transgenerational effects of 240 environmental change more generally. Previous studies of dietary-mediated 241 transgenerational effects have tended to focus on changes in metabolite profiles and 242 physiology across generations, rather than on changes to expression of life history 243 traits25,26. Moreover, these designs typically do not have the requisite power to partition 244 relative influences of (grand)maternal and (grand)paternal effects on transgenerational 13 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 245 phenotypes, nor the factorial design required to determine whether transgenerational 246 mismatches enhance or depress performance 9. 247 The prevailing prediction is that a matching of environment between grandparents and 248 grandoffspring may augment offspring fitness-related traits, because the matching 249 environments may allow parents to prime offspring to cope with environments that their 250 parents faced (anticipatory effects). The evidence for anticipatory effects across contexts 251 and taxa is, however, mixed and weak9,29, and many studies that have leveraged 252 experimental designs with the power to test for these effects have primarily focused on 253 intergenerational effects (from F0-F129), with very few studies classified as 254 transgenerational where grandoffspring should have no direct experience of the 255 grandparental environment34. Our study generally revealed patterns that were contrary to 256 the predicted pattern – dietary mismatching, rather than matching, between grandparents 257 and F2 offspring tended to augment offspring performance. This begs the question of 258 whether cross-generational dietary mismatching may be a general phenomenon that 259 extends across the diets used in our study. 260 Two recent studies shed some light on this question. Deas et al. (2019) manipulated dietary 261 quality across three generations (F0 to F2) in D. melanogaster, providing flies of each 262 generation with a ‘rich’ diet (rich in calories and supplemented with yeast) or a poor diet 263 (calorie diluted, with no yeast supplementation), in all combinations, and then measuring 264 phenotypic expression in the grandoffspring (F2). They reported that a mismatch between 265 the diet quality (“poor vs “good” diet) of granddams and granddaughters led to a faster 266 development time in the pupal stage of the granddaughters, but this effect did not hold for 267 the entire development time35. This study also focused on females, and therefore was not 268 able to capture sex specificity in any generation. On the other hand, Camilleri et al. (2022) 269 tested effects of dietary mismatching of F0 flies and their F1 offspring, manipulating the 14 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 270 diets of parents of each sex and their offspring, and utilising the same sucrose diets used in 271 the current study. We found that dietary mismatching between parents and F1 offspring led 272 to an increase in lifespan, and fecundity of the offspring28. Here, we advance these 273 findings by demonstrating that these effects of dietary mismatch are carried over for 274 multiple generations, are also dependant on the sex of F1 lineage. Because the effects are 275 unambiguously transgenerational (extending from F0 to F2), they are less likely to result 276 from differences in condition of the grandparents, suggesting instead possible epigenetic 277 mechanisms regulating the effects. 278 In sum, our work uncovers dietary-mediated transgenerational effects that are on the one 279 hand remarkably consistent across generations – transgenerational effects of sucrose tend 280 to mimic the direct effects. We have also extended previous work to demonstrate that 281 dietary mismatching across generations tends to augment phenotype in a manner that is 282 unlikely to be directly linked to condition-dependence. Future work should focus on 283 uncovering the ecological and evolutionary significance of these results, and the 284 underpinning mechanistic drivers. We suggest that a process in which transgenerational 285 dietary mismatching promotes fitness of future generations could buffer populations from 286 future changes in environment, and be particularly adaptive for species that live and 287 depend on ephemeral resources for their source of nutrients. If this is the case, then 288 populations evolving in fluctuating environments may be more likely to evolve 289 mechanisms that promote the fitness of offspring encountering novel environments. 290 291 292 15 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 293 Methods 294 Study species and generating experimental flies 295 We sourced flies from Dahomey, a large laboratory population of D. melanogaster, 296 originally sourced from Benin West Africa36. The flies have been maintained in large 297 population cages, with overlapping generations in the Piper laboratory, Monash 298 University, Australia, since 2017, and prior to that in the Partridge laboratory, University 299 College London 37. Prior to the beginning of the experiment, we collected ~3000 eggs from 300 the cages, and distributed them into 250mL bottles containing 70mL of food. Food 301 comprised 5% sucrose (50 grams sucrose, 100 grams yeast, 10 grams agar per 1 litre 302 solution with an estimated protein to carbohydrate [P:C] ratio of 1:1.9, and 480.9 kcal per 303 litre (see Supplementary Material Figure S4 for further diet details). Every generation (for 304 7 generations), adult flies eclosing from multiple bottles were admixed prior to 305 redistributing the flies across new bottles. To control for potential sources of variation in 306 their environment, during these 7 generations we strictly controlled both the age of flies at 307 the time of ovipositioning—all flies were within 24 h of eclosion into adulthood when 308 producing the eggs that propagated the subsequent generation, and their population density 309 was 300-320 adult flies within each bottle in each generation. 310 311 Dietary treatments 312 313 The diet media we used consists of sucrose, autolysed brewer’s yeast powder (sourced 314 from MP Biomedicals SKU 02903312-CF), and agar (grade J3 from Gelita Australia), as 315 well as preservatives—propionic acid, and nipagin. We prepared two dietary treatments, 316 differing in relative sucrose concentration; 2.5% sucrose (that we refer to as a lower 16 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 317 sucrose treatment relative to the 5% concentration usually provided to the population of 318 flies used in this experiment), and 20% sucrose (that we refer to as a higher sucrose 319 treatment) of overall food solution. The 2.5% sucrose diet contains 25 grams of sucrose, 320 100 grams of yeast and 10 grams of agar per litre of food prepared, with an estimated P:C 321 ratio of 1:1.4 and 380.9kcal per litre of food. The 20% sucrose treatment contains 200 322 grams of sucrose, 100 grams of yeast, and 10 grams of agar per litre of food prepared, with 323 an estimated P:C ratio of 1:5.3 and 1080.9kcal per litre of food. The diets thus differed not 324 only in sucrose concentration, but overall macronutrient balance and their total caloric 325 content, resembling differences typically observed between obesogenic and healthy diets in 326 humans. The higher sucrose concentration was selected based on preliminary experiments 327 that we conducted, and which elicited an obese-like phenotype in the flies, consistent with 328 results from previous work in D. melanogaster 11,25,38. All diets contained 3ml/l of 329 propionic acid and 30ml/l of a Nipagin solution (100g/l methyl 4-hydroxybenzoate in 95% 330 ethanol) and were cooked according to the protocol described in Bass et al. (2007) 39. 331 Each vial is 40mL in volume, and contained 7mL of food. 332 Experimental design 333 Male and female virgin flies were assigned to one of two of the dietary treatments prior to 334 mating (we refer to this generation of flies as F0), and then the grandoffspring produced (F2 335 generation) were also assigned to one of the two treatments. All possible combinations of 336 grand dam × grand sire × grandoffspring diet treatment were represented (= 2 × 2 × 2 = 8 337 combinations). Specifically, we collected 1280 flies of the F0 generation as virgins and 338 placed them onto either the high sucrose (20%) or the low sucrose (2.5%) diets for the first 6 339 days of their adult life. They were in vials of 10 flies across 64 vial replicates per treatment, 340 and per sex (High sucrose: 32 vials of males and 32 vials of females; low sucrose, 32 vials of 341 males and 32 vials of females, 128 vials in total;1280 flies, 640 of each sex). They were kept 17 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 342 in their respective sexes. We transferred flies to vials containing fresh food of the designated 343 diet every 48 hours during this 6 day period. 344 345 At day 6, we randomly sampled six vials from each treatment, and snap froze (using liquid 346 nitrogen) the flies of these vials, storing them at -80°C for subsequent measures of 347 triglyceride levels. Cohorts of flies in the remaining vials then entered a cohabitation phase to 348 enable female and male F0 flies to mate. Cohorts of males and female flies were combined, in 349 vials of 10 pairs, in each of all four possible diet combinations: lower sucrose females × 350 lower sucrose males; higher sucrose females × higher sucrose males; lower sucrose females × 351 higher sucrose males; higher sucrose females × lower sucrose males. During this phase, flies 352 cohabited for 96 hours. They were transferred to a new vial with fresh food of standard 5% 353 sucrose diet every 24 hours during this time. 354 355 The vials from the 6 day old F0 flies (i.e., the vials from Day 1 of the 96 h cohabitation 356 phase) were retained, and the eggs that had been laid by females of the respective vials were 357 trimmed to 80 per vial by removing excess eggs with a spatula. The remaining eggs were left 358 to develop into adult offspring over 10 days at 25°C (on a 12:12 light/dark cycle in a 359 temperature-controlled cabinet; Panasonic MLR-352H-PE incubator). These adult flies 360 constituted the F1 offspring in the experiment, and F1 flies developed on standard 5% 361 sucrose media. We collected 2080 virgin F1 flies from each of the four combinations of 362 parental diet treatments, and placed them in sex-specific cohorts of 10 individuals per vial, on 363 standard 5% sucrose media for 6 days. We then allowed these F1 males and F1 females to 364 cohabit and mate with male or female tester flies (creating 10 pairs per vial) that had been 365 collected from the same Dahomey stock population (but not subjected to a dietary sucrose 366 treatment) to create the F2 generation. The diet treatments applied to the F0 flies were thus 18 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 367 transferred to the F2 generation via either F1 males or F2 females, but never through both 368 sexes. The F1 flies were 6 days of adult age when laying the eggs that produced the F2 369 generation. 370 371 We then collected virgin F2 flies – the grandoffspring of the F0 flies – from each of the four 372 combinations of F0 diet treatments (per sex), and placed them in their respective sexes in 373 vials of 10 flies, across vial replicates per treatment per sex (4080 flies, 2040 male, 2040 374 female). We then assigned these F2 flies, produced by each dietary treatment combination of 375 F0 flies, to either the lower sucrose or higher sucrose diet. At day 6 of adulthood, we snap 376 froze F2 flies of six randomly chosen vials per grand dam × grand sire × grandoffspring 377 combination. On the same day, 10 virgin focal F2 flies of each grand dam × grand sire × 378 grandoffspring combination and each sex were placed together with 10 age-matched tester 379 flies of the opposite sex from the Dahomey population, entering into a cohabitation phase of 380 96 h (during which time the number of eggs laid by females of each vial was assessed). After 381 96 hours flies were separated again into their respective sexes (in vials of 20 flies), and 382 assigned back onto either the lower sucrose or higher sucrose diets that they had been on 383 prior to cohabitation, and a lifespan assay carried out. 384 385 Lifespan 386 We scored the lifespan of experimental flies of the F2 generation. Each vial in the assay 387 commenced with 20 flies of single sex in each, and we included 10 vial replicates per 388 treatment (grand dam × grand sire × grandoffspring) (3400 flies total, the original amount 389 collected, minus the snap frozen samples). The number of dead flies per vial was scored three 390 times per week (Monday, Wednesday, Friday), and surviving flies at each check transferred 391 to vials with fresh food of the assigned diet treatment—until all flies were deceased. During 19 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 392 the lifespan assay, vials were stored in boxes (of 85 vials per box) that were moved to 393 randomised locations in a (25°C) control temperature cabinet every few days to decrease the 394 potential for confounding effects of extraneous sources of environmental variation within the 395 cabinet from affecting the results. 396 397 Fecundity 398 We measured the egg output of female flies from generations F0 and F2 at eight days 399 following eclosion, as a proxy of female fecundity. On day eight, female flies oviposited for a 400 23 hour period, and were then transferred to fresh vials. Day eight was selected because 401 fecundity over 24 hours at this age has been shown to correlate with total lifetime fecundity 402 of females in this Dahomey population39 and early, short term measures of reproduction of 403 between one and seven days can be used to accurately predict total lifelong fecundity in D. 404 melanogaster 40. Moreover, data shows that varying the range of sucrose concentrations did 405 not alter the timing reproductive peaks between treatments 39. 406 407 For the F0 generation, we counted eggs from vials, each containing 10 female flies, that had 408 been mated with 10 male flies, across 2 different sucrose levels (2.5% and 20% sucrose), and 409 different mate combinations, as above. For the F2 generation, we counted eggs from each 410 grand dam × grand sire × grandoffspring dietary treatment combination; each combination 411 was represented by 10 vial replicates, each containing 10 focal females (females from the 412 experiment) combined with 10 tester male flies. Additionally, we counted the number of 413 adult flies that eclosed within 10.5 days from the eggs laid by F2 females (a composite of 414 clutch viability and juvenile developmental speed). F2 females cohabited and mated with 415 age-matched tester males of the Dahomey population (in the experimental process described 416 above, rather the standard medium of 5% sucrose), for 24 hours at 6 days of life, and the vials 20 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 417 containing these eggs were left to develop into adult offspring, for 10 days at 25°C; 12:12 418 light/dark cycle in a temperature-controlled cabinet (Panasonic MLR-352H-PE incubator). 419 420 Lipids and protein 421 Whole-body triglyceride levels were measured in adult flies from the F2 generation (six days 422 of adult age, corresponding with six days of exposure to the relevant F2 dietary treatment, 423 prior to mating) and normalized to protein content (full protocols reported in the 424 Supplementary Material). Three biological replicates per treatment level, with three technical 425 replicates per biological replicate were used. Five female flies and eight male flies 426 respectively, were used for each biological replicate in the assay. 427 428 429 Statistical Analyses 430 431 We used R (Version 3.6.1) and RStudio (Version 1.2.1335) (R Core Team, 2019) for 432 statistical analyses. To test the effects of F0 female diet, F0 male diet, F2 diet, and sex on 433 lifespan, TAG, and F2 offspring production, we fitted linear mixed effects models, using the 434 R package lme441, to the lifespan data for the F2 generation. We use the term lifespan to 435 denote the age of recorded death for each individual fly within a margin of 72 hours (for 436 example, a lifespan of 30 days indicates that a fly died between 27-30 days post eclosion). To 437 test the effects of grand maternal diet, grand paternal diet, grand-offspring diet, and sex on 438 female fecundity, we fit a linear model to the egg output data for both generations. 439 We included F0 male, F0 female, F2 diets, and F2 sex as fixed effects in each model, 440 exploring interactions between these factors. We included the vial identification number as a 21 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 441 random effect in the lifespan models. The fecundity models only included one observation 442 per vial because we counted eggs per vial, and divided by the number of females in the vial 443 (approx. 10 females); therefore no random effects were included in this model. We used log- 444 likelihood ratio tests that reduce the full model, via the sequential removal of highest order 445 terms that did not (significantly) change the deviance of the model, using a p value 446 significance level of <0.05. The final reduced models (except fecundity measures) were fit by 447 restricted maximum likelihood, applying type III ANOVA with Kenwood-Roger’s F test and 448 approximation of denominator degrees of freedom. We used sum to zero constraints in all 449 models, and we visually inspected diagnostic plots for the linear mixed effect models, to 450 ensure that the assumptions of normality and equal variances were met. 451 452 Funding 453 The School of Biological Sciences at Monash University supported this work. 454 455 Conflicts of Interest 456 The authors have no conflicts of interest to declare. 457 458 Author contributions 459 TLC, DKD, MDWP & RLR designed the experiment, TLC planned and carried out the 460 experiment, and wrote the initial draft of the manuscript, and TLC, DKD, MDWP & RLR 22 bioRxiv preprint doi: https://doi.org/10.1101/2022.05.23.492998; this version posted May 26, 2022. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 461 all contributed to the writing and editing of the manuscript. TLC performed statistical 462 analysis under the guidance of DKD. 463 Acknowledgements 464 The authors are grateful for the help they received in the laboratory from: Pavani 465 Manchanayake, James Wang, Skye Bulka, Natalie Wagan, Rebecca Koch, and Winston 466 Yee. Indispensable guidance with molecular work was provided from Amy Dedman. 467 Additional invaluable assistance was provided by Caleb Carter. 468 469 References 470 1. inheritance for evolution in changing environments. Evol. Appl. 5, 192–201 (2012). 471 472 Bonduriansky, R., Crean, A. J. & Day, T. The implications of nongenetic 2. Gluckman, P. D., Hanson, M. A. & Low, F. M. Evolutionary and developmental 473 mismatches are consequences of adaptive developmental plasticity in humans and 474 have implications for later disease risk. Philos. Trans. R. Soc. B Biol. Sci. 374, 475 (2019). 476 3. 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