The authors have declared that no competing interests exist.
An organism's phenotype is the product of its environment and genotype, but an ancestor’s environment can also be a contributing factor. The recent increase in caloric intake and decrease in physical activity of developed nations' populations is contributing to deteriorating health and making the study of the longer term impacts of a changing lifestyle a priority. The dietary habits of ancestors have been shown to affect phenotype in several organisms, including humans, mice, and the fruit fly. Whether the ancestral dietary effect is purely environmental or if there is a genetic interaction with the environment passed down for multiple generations, has not been determined previously. Here we used the fruit fly,
The effect of environment on an organism’s phenotype has been well documented, however, the effect on descedants has only recently been examined and shows that an ancestor's behavior and environment can affect the health of future generations. Research on human subjects has revealed sex-specific and non-sex specific effects of food resources on multiple generations’ mortality, BMI, and risk of death from diabetes[
In addition to traditional overall direct transgenerational effects of the environment, we should also consider the role of genotype-by-environment interactions. An extensive genotype-by-diet interaction has been revealed in fruit flies [
The effect of diet on subsequent generations can be quickly and easily modeled in Drosophila, which share basic metabolic functions with mammals, including the insulin/TOR signaling pathway and lipid storage, making them a useful model organism for transgenerational effects of diet [
Some of the transgenerational dietary studies on Drosophila have revealed the effects of protein deficient [
With these considerations in mind, we sought to determine the genetic variation for sex-specific transgenerational effects of a larval high fat diet. We studied the genotype-by-diet transgenerational effects for male and female pupal weight in ten wild-derived inbred lines [
We also analyzed three homozygous genetic lines derived from an outcrossed population [
In this study, we asked three primary questions. What is the overall effect of genotype and sex of descendants on transgenerational effects (
To survey transgenerational weight phenotypes, ten genetic lines, here after referred to as the "
Flies were maintained in a controlled environment at 25°C on a 12:12 hr light:dark cycle at 50% humidity for the duration of the experiment. Dietary treatments consisted of a standard cornmeal-molasses diet (normal) and an experimental diet (high fat) used in previous dietary studies [
No specific effort was made to control for gut microbiota. Flies acquire their gut microbes from their diet, which is seeded by other flies (
For each generation, following two days of adult mating, eggs were collected from laying chambers, allowed to hatch, and fifty first-instar larvae were used to seed each food vial. The parental generation (P) was placed on normal food and high fat food. A crossing scheme (
In the first generation (P), larvae were fed a normal diet (Control) and a high fat diet (High Fat). P adults within each genetic line were crossed to create three treatment groups for the second generation (F1). The Control treatment consisted of both males and females from P:Control. F1:MA were offspring from P:Control males and P:High Fat females. F1:PA contained offspring from P:Control females and P:High Fat males. F1 adults were mated to individuals from the same treatment group to create the third generation (F2). All F1 and F2 larvae were reared on a normal diet.
The F1 and F2 generations were raised on a normal diet. To prevent the confounding effects of the adult diet in the P generation P pupae were removed from the vials, sexed, and placed in empty bottles until eclosion. Within 12 hours of eclosion, P adults were crossed according to the scheme in
Experimental procedures were synchronized within the DSPR and the DGRP genetic lines such that for all genotypes the treated and control groups for a given generation were run simultaneously. This allowed for a robust estimate of genotype-by-diet interaction effect within a given generation, but limited the power of statistical comparisons between generations.
All pupae in a vial for the weight phenotypes, known to be quite stable throughout the pupal stage [
Fifteen vials from each treatment for each generation were used for quantifying trehalose, triglyceride, and protein levels. Third instar larvae were collected just prior to wandering, pooled across vials, and fasted for 3–4 hours on plain agar plates then flash frozen in liquid nitrogen in groups of ten, with three or more biological replicates per treatment for each phenotype. The larvae were not sexed thus the samples could have exhibited additional variance due to differences in sex ratio. Samples were stored at -20°C until metabolic testing could be completed.
Larval trehalose levels were measured using homogenized larvae and the Sigma Glucose Determination Kit after overnight treatment with trehalase [
Egg size was measured using 25 randomly selected eggs laid on the first plate retrieved from the laying chambers for a given genetic line and generation. Eggs were measured under a light microscope using a Moticam® 2000. The Motic® Images Plus 2.0 Multilanguage Software package was used to measure the length and width of each egg. Egg volume was then calculated using the formula for an oblate ellipsoid,
Egg size and pupal weight data were normally distributed under a Shapiro-Wilk test. The trehalose and triglyceride data were log transformed, and then normalized for technical effects by calculating residuals from a linear model with a batch effect. For each generation, all normalized phenotypes were analyzed by analysis of variance (ANOVA) according to the model:
To test the robustness of the p-values determined in the ANOVA, data was permutated 1024 times in Excel. Data was permuted within genotype and sex groups to maintain the genetic and sex based data structure but randomized relative to the F1 and F2 generations, treatments, and, in the case of the
Post-hoc tests were conducted using Student’s t-test. The correlation between ancestral and descendant pupal weight was determined by calculating the phenotypic difference from Control for the treated ancestor (P or F1) of the appropriate sex and the difference between the Control and treated weights in the descendent (F1 or F2). All statistical analyses were completed using JMP® (version 10.0.0; SAS Institute, Cary, NC). The threshold for statistical significance was p<0.05 unless otherwise noted. Significance values are converted to negative log p-values (NLP) for ease of interpretation in some of the analyses.
We tested the effect of a high fat diet (3% coconut oil) on the phenotype of future generations by rearing a parental generation (P) on either a normal or high fat diet and then mating them to partners reared on a normal diet (
We wanted to explore the genotype-by-ancestral diet interaction effects in a population sample so we measured male and female weights for ten genetic lines from the wild-derived DGRP using the crossing scheme described above (
Polka dots indicate treatment is significantly different from Control. Black border indicates treatments are significantly different from each other. Multiple testing was corrected using false discovery rate of 0.05.
sex | 0.3917 | 312 | 0.4492 | 312 |
genotype | 0.1906 | 312 | 0.2855 | 312 |
treatment | 0.0055 | 13.25 | 0.0078 | 18.33 |
generation | 0.0001 | 0.0013 | 3.91 | |
genotype*sex | 0.012 | 23.02 | 0.0050 | 11.02 |
treatment*sex | 0.0014 | 3.52 | 0.0010 | 2.38 |
treatment*genotype | 0.0361 | 67.03 | 0.0054 | 10.03 |
generation*sex | 0.0067 | 16.96 | 0.0043 | 11.18 |
generation*genotype | 0.0145 | 28.55 | 0.0051 | 11.20 |
generation*treatment | 0.0024 | 5.74 | 0.0032 | 7.60 |
treatment*genotype*sex | 0.019 | 32.83 | 0.0078 | 15.39 |
generation*genotype*sex | 0.0077 | 13.7 | 0.0018 | 3.85 |
generation*treatment*sex | 0.0017 | 4.2 | 0.0031 | 7.45 |
generation*treatment*genotype | 0.0157 | 26.09 | 0.0069 | 13.50 |
generation*treatment*genotype*sex | 0.0207 | 36.27 | 0.0100 | 13.40 |
time replicate | 0.0119 | 28.80 |
1 ANOVA model effect
2 negative log p-value
When the weight measurements were pooled across the sexes the majority of the variance in weight was explained by sex, genotype, and genotype-by-treatment, with slight contributions from genotype-by-sex and genotype-by-treatment-by-sex (
The total explained variance is graphed for females (A) and males (B). The
Arrows indicate actual p-values. Distributions based on 1024 permutations across treatments and generations (and replicate for the
We replicated the effects of ancestral diet on pupal weight in four genetic lines selected from the original
The main effect of treatment remained consistent across all studies in the males, as did the relative contribution of treatment-by-genotype-by-generation (
The overall correlation between the Fall and Spring replicates was greater than 80% (
The overall correlation of the actual measurements for all three generations across four genetic lines and three treatments and two sexes showed robust correlation (A). The difference between the average control and treated measurements, across replicates within genotype/sex/generation combinations, also shows a significant positive correlation. (B). When the F2 data points are isolated from the total set in (B), the correlation is stronger and significant (C).
We are especially interested in how the diet of the grandparents (P generation) affects the phenotypes of the grandchildren (F2 generation). The reaction norms across the control, MA, and PA treatments varied dramatically across the ten genetic lines first tested (Figs
The left panels indicate the 10 reaction norms across the three treatments for female pupal weight (A) and male pupal weight (C). The right panels indicates the subset of four genetic lines from the original 10 in the Fall and their Spring replicates for female (B) and male (D) pupal weight, bold indicates the reaction norm from the Spring replicate. For most genetic lines, the reaction norms across the two time replicates in panels B and D maintained a similar pattern.
153 | 440 | 748 | 787 | 801 | 802 | 805 | 900 | 907 | 911 | 153 | 440 | 748 | 911 | ||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
female | Control vs PA | ||||||||||||||
female | Control vs MA | ||||||||||||||
female | MA vs PA | ||||||||||||||
male | Control vs PA | ||||||||||||||
male | Control vs MA | ||||||||||||||
male | MA vs PA |
a
* indicates significance at p< 0.05
In the four genetic lines that were retested, their F2 reaction norms followed a fairly similar pattern (
We also tested the predictive power of the ancestral phenotypic difference to the MA or PA treatment on future generations (
Generation |
Sex |
Treatment |
Ancestor Effect |
Repeatability |
||||||
---|---|---|---|---|---|---|---|---|---|---|
F1 | F | MA | P_HF_diff F | * | ||||||
F1 | F | PA | P_HF_diff M | -0.51 | 1.18E-08 | |||||
F1 | M | MA | P_HF_diff F | -0.02 | ||||||
F1 | M | PA | P_HF_diff M | -0.49 | 3.08E-07 | |||||
F2 | F | MA | P_HF_diff F | -0.05 | 0.06 | 0.04 | ||||
F2 | F | PA | P_HF_diff M | 0.16 | ||||||
F2 | M | MA | P_HF_diff F | -0.27 | 0.58 | -0.06 | 5.90E-04 | 4.21E-06 | ||
F2 | M | PA | P_HF_diff M | * | ||||||
F2 | F | MA | F1_MA_diff F | -0.36 | 1.24E-04 | |||||
F2 | F | MA | F1_MA_diff M | 0.01 | -0.60 | -0.11 | 6.78E-07 | |||
F2 | F | PA | F1_PA_diff F | -0.42 | 6.46E-06 | |||||
F2 | F | PA | F1_PA_diff M | 0.03 | ||||||
F2 | M | MA | F1_MA_diff F | 0.24 | -0.05 | -0.10 | 1.68E-03 | |||
F2 | M | MA | F1_MA_diff M | -0.12 | -0.01 | 0.16 | ||||
F2 | M | PA | F1_PA_diff F | 0.02 | -0.48 | 0.24 | 5.41E-07 | 1.71E-02 | ||
F2 | M | PA | F1_PA_diff M | 0.21 | 2.23E-03 |
1 generation of weighed pupae
2 sex of weighed pupae
3 Treatment—MA (maternal ancestor on high fat), PA (paternal ancestor on high fat)
4 generation (P, F1), treatment (HF, MA, PA), and sex of the difference between control and treated ancestor
5 correlation between ancestor and progeny phenotype in the
6 correlation between ancestor and progeny phenotype
7correlation between ancestor and progeny phenotype in
8 P-value of
9 P-value of
10 P-value of
11 repeatability across populations and time points (* correlation repeats in all three studies,
In general, pupae weight differences in the F1 generation tended to be negatively correlated with the MA treatment effect, but positively correlated in the PA treatment (
We were interested in whether the patterns observed in weight were also reflected in other phenotypes including trehalose, triglyceride, and protein levels, male and female pupae weight, and egg size. These phenotypes were measured in the P, F1 and F2 generations for three randomly selected genetic lines from the DSPR.
Genetic diversity contributed significantly to variation in protein concentration for all three generations, to trehalose in the F1 and F2 generations, and to triglycerides in the P and F1 generations (
Significant
Trehalose Levels | Triglyceride Levels | Female Pupal Weight | |||||||
Comparison | P | F1 | F2 | P | F1 | F2 | P | F1 | F2 |
Genotype | |||||||||
Treatment | |||||||||
Genotype * Treatment | |||||||||
Protein | Egg Size | Male Pupal Weight | |||||||
P | F1 | F2 | P | F1 | F2 | P | F1 | F2 | |
Genotype | |||||||||
Treatment | |||||||||
Genotype * Treatment |
Trehalose Levels |
Triglyceride Levels |
Female Pupal Weight |
|||||||
Comparison |
P | F1 | F2 | P | F1 | F2 | P | F1 | F2 |
MA vs. Control | - | A* | A*** | - | - | A* | - | ||
PA vs. Control | - | - | - | - | A** | ||||
MA vs. PA | - | - | - | - | - | ||||
Protein |
Egg Size | Male Pupal Weight |
|||||||
P | F1 | F2 | P | F1 | F2 | P | F1 | F2 | |
MA vs. Control | C* | - | - | - | - | - | |||
PA vs. Control | - | - | - | C** | - | - | |||
MA vs. PA | C** | - | A* |
- | - |
a Only significant comparisons are listed. A significant difference is represented by the genotype’s letter designation, the level of significance, and the directionality of the difference. A bold entry indicates ‘x’ was a larger value in the comparison ‘x vs. y’. (t-test;
b The only comparison made in the P generation was High Fat vs. Control.
Male and female pupal weight both had genotype effects for the P and F2 generations (
The P and the F2 generations had a large genotypic effect on egg size (
Substantial differences by sex, genotype, and generation were observed for the influence of the high fat treatment. Significance values indicated as
We also looked at the correlation between male and female pupal weight and egg size (Table B in
In this study we examined the sex-specific effects of a high fat diet on two subsequent generations in the model organism,
Our findings were consistent with previous studies that investigated only one generation past treatment (F1) compared to two in our study, in that each genotype showed a unique array of transgenerational effects [
Interactions involving the sex of the descendant and the diet of the ancestor are also highly significant in the F1 and F2 generations, explaining 2.5 and 6.7% of the total variation in weight in each generation in the
We observed similar magnitudes of treatment effects in the phenotypes measured in the
Considering all measured phenotypes, maternal and paternal ancestors did not always affect descendants to the same magnitude. In many of the instances where an ancestral treatment of one sex (MA or PA) varied from Control, the other ancestral sex high fat treatment does not differ from Control (
In humans, Kaati
In addition, a previous study in Drosophila found that a maternal (MA) high sugar diet affected the metabolic pools of both male and females in a specific genetic line for two generations [
The impact of the diet on the parental phenotype may be due to differences in feeding rate between the two diets and/or due to inherent nutritional differences of the diets. For the purpose of the questions posed in this study, which focus primarily on whether the environmental experience of the parent generation impacts the F1 and F2 generations, we do not need to know the underlying mechanism of the diet effect in the parental generation to be able assess how difference in the parental environment impact subsequent generations. However, future studies on how the added fat effects palatability would be interesting since it would allow us to differentiate between the impact of diet composition and total calorie consumption on subsequent generation's phenotypes.
The population showed a high degree of variation to an ancestral high fat diet and this may indicate that the transmission of transgenerational effects is rooted in genotype-specific mechanisms and less in broadly applied maternal effects, thus genetic mapping may be a useful way to identify how genetic variants affect transgenerational inheritance, and thus identify the underlying genetic mechanisms that contribute to these patterns. However, the moderate correlations observed between generations for pupal weight across ten genetic lines implies that there may be some general predictions about transgenerational effects at the population level. Correlations in phenotype across generations in a genetically variable population implies that there is an aspect to the inheritance mechanism(s) that can function independently of the genotypes of the individuals, such as through environmentally determined epigenetic modification.
Similarly, in a previous study, glucose levels in females fed a high sugar diet were not changed but the offspring in the F1 and F2 generations had higher glucose levels than the control for the one tested genotype [
Certainly, explorations of potential mechanisms for transgenerational inheritance within genetically variable populations must be sensitive both to genotype-dependent and genotype-independent factors (such as sex and other environmental factors). For example, for female weight we found that the main effect of the parental treatment was negligible overall in the
In a genotype-specific context, another explanation for the "skipped" generational dietary effects is Mendelian inheritance of high fat exposed alleles. Due to independent assortment of high fat diet exposed alleles in the F2 generation into four possible epigenetic "genotypes," we might have expected to see an increase in variance in phenotypes in F2 generation for the treated relative to control if there was Mendelian inheritance of additive transgenerational epigenetic effects. However, genotypes with phenotypic responses only in later generations did not show a consistent increase in variance for the F2:MA and F2:PA compared to F2:Control, and there was no effect of generation or treatment on the within-genotype variation. Thus, two identical-by-decent alleles from the high fat exposed grandparent did not apparently amplify the phenotypic response in the F2 generation, implying that epigenetic effects are not, categorically, additive.
Finally, we found that correlations between egg size and pupal size were genotype-specific, confirming previous findings of no consistent effects of egg size on adult weight or offspring egg size across a population, which may be due to accumulation of environmental effects interacting with genetic variation [
In this study, we found that there was only a moderate link between parental reaction to a high fat diet and their untreated descendants' phenotype, and the associations exhibited extensive genotype-specific reactions. This indicates that there is a potential for deleterious effects to occur in descendants even when the ancestor did not demonstrate a negative reaction to the environmental stress. Additionally, a transgenerational effect in one sex was not a reliable indicator of effects in other related traits. For example, female pupae were affected in some genetic lines while males were not affected (
Sex-specific treatment and phenotyping across generations has shown its usefulness in Drosophila, humans, and mice, where the two sexes can perform in dramatically different ways. Sex-specific effects may offset each other and prevent researchers from detecting transgenerational environmental impacts if the sexes are not analyzed separately. Future studies should focus on the elucidation of the sex-specific effects by parsing treatment groups further (
Finally, the dramatic differences between all genetic lines show the importance of testing multiple genotypes to assess the genetic diversity of a transgenerational trait, since not all genotypes react to the same extent or direction. Epigenetic effects only evaluated in a single genetic background may lead to inaccurate conclusions because of genetic variation in a real population. Awareness of genetic variation for the impact of ancestral environment could provide important insights into the varied causes of the current obesity epidemic.
Tables of data means and standard errors, raw data, and variance partitions under different models are given.
(XLSX)
Figs A-G and Tables A and B can be found in the S1 Information file. Figs A and B give distribution of P-value permutations, Fig C graphs the differences between control and treated flies for the
(PDF)
We thank the Reed Lab members for technical assistance with experiments, as well as, extensive constructive criticism on manuscript and data presentation.