RESEARCH ARTICLE

Genetic and Sex-Specific Transgenerational

Effects of a High Fat Diet in Drosophila
melanogaster
Kelly Dew-Budd1,2, Julie Jarnigan1, Laura K. Reed1*
1 Department of Biological Sciences, University of Alabama; Tuscaloosa, AL, United States of America,
2 School of Plant Sciences, University of Arizona; Tucson, AZ, United States of America

* lreed1@[Link]

a11111
Abstract
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 deteriorat-
ing health and making the study of the longer term impacts of a changing lifestyle a priority.
OPEN ACCESS The dietary habits of ancestors have been shown to affect phenotype in several organisms,
Citation: Dew-Budd K, Jarnigan J, Reed LK (2016) including humans, mice, and the fruit fly. Whether the ancestral dietary effect is purely envi-
Genetic and Sex-Specific Transgenerational Effects ronmental or if there is a genetic interaction with the environment passed down for multiple
of a High Fat Diet in Drosophila melanogaster. PLoS generations, has not been determined previously. Here we used the fruit fly, Drosophila
ONE 11(8): e0160857. doi:10.1371/journal.
pone.0160857
melanogaster, to investigate the genetic, sex-specific, and environmental effects of a high
fat diet for three generations’ on pupal body weights across ten genotypes. We also tested
Editor: Gregg Roman, University of Mississippi,
UNITED STATES
for genotype-specific transgenerational effects on metabolic pools and egg size across
three genotypes. We showed that there were substantial differences in transgenerational
Received: February 26, 2016
responses to ancestral diet between genotypes and sexes through both first and second
Accepted: July 26, 2016 descendant generations. Additionally, there were differences in phenotypes between
Published: August 12, 2016 maternally and paternally inherited dietary effects. We also found a treated organism’s reac-
Copyright: © 2016 Dew-Budd et al. This is an open tion to a high fat diet was not a consistent predictor of its untreated descendants’ phenotype.
access article distributed under the terms of the The implication of these results is that, given our interest in understanding and preventing
Creative Commons Attribution License, which permits metabolic diseases like obesity, we need to consider the contribution of ancestral environ-
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
mental experiences. However, we need to be cautious when drawing population-level gen-
credited. eralization from small studies because transgenerational effects are likely to exhibit
Data Availability Statement: All relevant data are
substantial sex and genotype specificity.
within the paper and its Supporting Information files.

Funding: This work was funded by grant

R01GM098856 from National Institutes of General
Medical Sciences, [[Link]]. The funders
Introduction
had no role in study design, data collection and
analysis, decision to publish, or preparation of the The effect of environment on an organism’s phenotype has been well documented, however,
manuscript. the effect on descedants has only recently been examined and shows that an ancestor's behavior
Competing Interests: The authors have declared and environment can affect the health of future generations. Research on human subjects has
that no competing interests exist. revealed sex-specific and non-sex specific effects of food resources on multiple generations’

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 1 / 20

 Transgenerational Dietary Effects

mortality, BMI, and risk of death from diabetes[1,2]. Furthermore, model organisms, such as
mice, play a large role in the advancement of this field with studies on the effects on descendant
phenotypes of diet[3,4], chemicals [5,6], and trauma [1,2,7,8]. Drosophila melanogaster specifi-
cally, has been useful in elucidating the transgenerational effects of diet [3,4,9–16], temperature
[1,2,5,6,17,18], toxins [3,4,19,20], and immunity [5,6,21,22].
In addition to traditional overall direct transgenerational effects of the environment, we
should also consider the role of genotype-by-environment interactions. An extensive geno-
type-by-diet interaction has been revealed in fruit flies [7,8,23,24] and their offspring [9–
13,15,16,25], where some genotypes are highly sensitive to their diets while others are robust to
dietary perturbation. The transgenerational aspect of this interaction could indicate either a
genotype-specific transmission of dietary information or direct influence of the ancestral phe-
notypic variation. We would expect to see phenotypic variation among genotypes due to the
ancestor’s diet if there is genotype-specific transmission, while a consistent correlation between
offspring and ancestral phenotype across genotypes is expected if the effect were strictly due to
parental phenotype (e.g. maternal effect). Maternal effects could be transmitted through the
provisioning of the egg by the mother or by maternal loading of transcripts into the egg [26].
The effect of diet on subsequent generations can be quickly and easily modeled in Drosoph-
ila, 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 [27]. In mammals, studies of epigenetic inheritance must be carried out to the F3 genera-
tion because the F1's germline is derived in utero, thus potentially modifying F2 phenotype
through grandmaternal (non-epigenetic) effects. However, in Drosophila, studies of genotype-
by-diet variation in transgenerational effects need only to be carried out to the F2 generation at
minimum because Drosophila development occurs outside of the mother, thus maternal effects
on an offspring’s germline should be minimal[28,29]. We also have prior evidence that mater-
nal and paternal effects on descendants can vary and should, therefore, be studied separately
[29].
Some of the transgenerational dietary studies on Drosophila have revealed the effects of pro-
tein deficient [11], protein and sugar deficient[9,10], and sugar-enriched food [13,30] on vari-
ous metabolic phenotypes, such as offspring size, development time, survival, weight, and
metabolic pools. However, these studies have not been able to distinguish between transgenera-
tional effects that are merely a result of a parental phenotype (maternal effect) or are the result
of epigenetic inheritance. The distinction between maternal effects or transgenerational epige-
netic inheritance is important in determining the mechanism of transmission [31,32]. Maternal
effects are a change in offspring phenotype caused by the non-genetic maternal contributions
to offspring development, such as egg nutritional content [32]. In contrast, transgenerational
epigenetic inheritance indicates effects on offspring phenotype through the non-Mendelian
transmission of environmental effects via the gametes leading to alteration of gene expression
[31,32].
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
[33,34]. We know from previous work that weight exhibits a substantial genotype-by-diet
interaction [23,24] and we were interested in whether larger variation in reaction norms can be
transmitted across generations, as such effects could influence the long-term health of a popu-
lation. We then replicated these patterns in a subset of four genetic lines.
We also analyzed three homozygous genetic lines derived from an outcrossed population
[35], to determine if patterns we observed in weight also applied to larval triglyceride, trehalose,
and protein levels, as well as egg size, of the organisms experiencing either a high fat or normal

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 2 / 20

 Transgenerational Dietary Effects

diet and their descendants. Larval triglyceride (fat storage), trehalose levels (sugar concentra-
tion), and protein reflect the metabolic condition of the larva. Assaying these metabolic traits
(fly surrogates for metabolic syndrome) can help identify how diet may transmit fitness effects
across generations.
In this study, we asked three primary questions. What is the overall effect of genotype and
sex of descendants on transgenerational effects (i.e. do all genotypes react similarly in extent
and direction)? Do sex-specific effects occur in the transgenerational impacts on phenotype?
And finally, is phenotypic plasticity in the parental generation in response to diet required for
future generations to demonstrate transgenerational effects?

Materials and Methods

Drosophila stocks
To survey transgenerational weight phenotypes, ten genetic lines, here after referred to as the
"10 line study" were randomly chosen from the Drosophila Genetic Reference Panel (DGRP);
153, 440, 748, 787, 801, 802, 805, 900, 907, 911, abbreviated as DGRP# [33,34], and from those,
a subset of four genetic lines (153, 440, 748, and 911) were selected for further replication, the
"4 line study", "Fall" referring to the first replicate and "Spring" for the second replicate. For the
additional metabolic phenotypes, three stocks were randomly selected (here after referred to as
the "3 line study") from the Drosophila Synthetic Population Resource (DSPR); 11291, 12062,
22271, abbreviated here as Line A, Line B, and Line C respectively [35,36].

Experimental design
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 [23,24,37]. The high fat diet was identical to the normal diet except for the addition of
3% coconut oil by weight before distributing 10 mL of food per vial [23,24]. Prior to experi-
mentation, stocks were maintained on normal food in the controlled environment for more
than two generations.
No specific effort was made to control for gut microbiota. Flies acquire their gut microbes
from their diet, which is seeded by other flies (e.g. adults from the previous generation) and
environmental sources [38–40]. Lab strains of Drosophila show lower levels of microbial diver-
sity than wild caught flies [41] presumably due, in part, to the combined effects of a sheltered
environment, standardized diet, and antimicrobial agents in the food. These genetic lines were
maintained in the lab environment and on a diet containing the antimicrobial agents of Tego-
sept and propionic acid for many generations prior to these experiments. Thus, we expect most
of the remaining differences among the genetic lines due to the microbiota are likely to be ulti-
mately under the influence of the genotype of the host and how that genotype filters the small
amount of available microbes in lab diet environment.
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 (Fig
1) was implemented to allow for the distinction of sex-specific transgenerational effects of diet
and resulted in three treatment groups, Maternal Ancestor (MA), Paternal Ancestor, (PA) and
Control, for two generations (F1 and F2). The MA treatment group consisted of P females
reared on a high fat diet mated to P males reared on a normal diet, while the PA treatment
group was the reverse (P males reared on a high fat diet mated to P females reared on a normal

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 3 / 20

 Transgenerational Dietary Effects

Fig 1. Crossing schematic. 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.
doi:10.1371/[Link].0160857.g001

diet). Mating between P males and females reared on the normal diet founded the Control
lineage.
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 Fig 1 for two days on normal food before being placed in laying chambers. The
F1 generation was allowed to pupate, eclose, and mate for two days within their original food
vial, before being placed in laying chambers. The resulting F2 larvae were treated identically to
the F1 larvae. To measure the weight phenotype, at each generation five additional vials were
seeded with 50 1st instar larvae and allowed to mature to pupae for collection. For the 3 line
study, where egg size was measured, several additional vials of F2’s were allowed to mature to
adults and mate for two days before being moved to laying chambers for egg collection.
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.

Phenotypic measurements
All pupae in a vial for the weight phenotypes, known to be quite stable throughout the pupal
stage [42], were collected for each generation when they were within 12 hours of eclosion (when
they can be sexed), placed in Ringer’s solution, and stored at -20°C. Pupae were cleaned, sexed,
and weighed after being patted-dry in groups of three on a high precision balance (Mettler Toledo
XS105). In the 3 line study, the pupae were weighed individually. We have found that wet weights
are much more reproducible than dry weights for the same sample, because for these small mas-
ses humidity in the ambient air rapidly rehydrates dried samples skewing measurements.
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 phe-
notype. 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.

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 4 / 20

 Transgenerational Dietary Effects

Larval trehalose levels were measured using homogenized larvae and the Sigma Glucose
Determination Kit after overnight treatment with trehalase [23,43]. Total larval triglyceride
levels were determined in homogenized larvae using the Sigma Triglyceride Determination Kit
[23,44,45]. Protein concentrations were determined for the trehalose and triglyceride samples
using the AMRESCO Bradford Method Protein Assay Kit [46].
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 Moticam1 2000. The Motic1 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, 16 pa2 b where a is the width and b is the length.

Statistical analysis
Egg size and pupal weight data were normally distributed under a Shapiro-Wilk test. The tre-
halose 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 normal-
ized phenotypes were analyzed by analysis of variance (ANOVA) according to the model:
y ¼ m þ Gi þ Tj þ Gi  Tj þ Eijk

where G and T are the main effects of genotype and treatment respectively, G × T is the geno-
type-by-treatment interaction, and E is the error term for the ith genetic line, jth treatment and
kth individual. Phenotypes were compared between the two treatment groups (High Fat and
Control) for the P generation and the three treatment groups (MA, PA, and Control) for the F1
and F2 generations. We also conducted additional analyses with more effects such as genera-
tion and sex of pupa, and their interactions with genotype and treatment using the same type
of ANOVA model. Correlation between the Spring and Fall replicated four genetic lines was
calculated on both the average weights within each genotype, treatment, generation, and sex
from each replicate, and the differences between treatment (MA or PA) and control averages
for each sex, genotype, and generation.
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, treat-
ments, and, in the case of the 4 line study, replicate, to test for spurious structure in the treat-
ment effects. Each permuted dataset was analyzed by ANOVA and the p-values for all the
tested effects were saved. The real p-values were then compared to the distributions of the 1024
permuted p-values for each ANOVA effect.
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 Con-
trol 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 JMP1 (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.

Results
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 (Fig 1). By measuring the following two generations (F1

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 5 / 20

 Transgenerational Dietary Effects

and F2), reared on a normal diet, we were able to observe genetic and sex-specific transgenera-
tional effects, as well as, genotype-by-diet interaction effects.

Weight phenotypes
10 Line Study. 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 (Fig 1). Genotype and geno-
type-by-treatment effects were highly significant for all generations and sexes tested (Fig 2 and
Fig A in S1 Information, Table 1 and Table A in S1 Information). The patterns of variance
explained by diet and genotype in the parental generation were very similar to what has been
found in previous studies [23,24] where diet alone explained only a very small portion of the
total variance but the interaction between diet and genotype explained a substantial portion of
the population's phenotypic variance (Table A in S1 Information).
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 (Table 1 and Table A in S1 Informa-
tion). The total variance contributed by the sex-by-treatment interaction and those interactions
with genotype and generation in the F1 and F2 generations was 4% (Table 1). There was no sig-
nificant contribution of generation alone to weight, but there were highly significant interac-
tions between generation and treatment, genotype, and sex in the F1 and F2 generations
(Table 1). When considering the main treatment effect and all of its interactions with other fac-
tors in the F1 and F2 generations, those effects were responsible for 18%-19% of the variance in
phenotype within each sex (Fig 3, S1 File). We tested for the robustness of the strong interac-
tion effects found in the ANOVA by performing permutation tests that randomized across
treatments and generations within sex and genotype for the F1 and F2 generations. All p-values
for the interaction effects fell well beyond the distribution of randomly generated p-values
showing the analysis to be robust to random factors (Fig 4A–4H and Fig B in S1 Information).

Fig 2. Weight 10 Line Study Data. 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.
doi:10.1371/[Link].0160857.g002

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 6 / 20

 Transgenerational Dietary Effects

Table 1. Descendants (F1 and F2 generation) variance partition for pupal weight.
Effect1 10 line study variance explained NLP2 4 line study variance explained NLP
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 ns 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 na na 0.0119 28.80
1
ANOVA model effect
2
negative log p-value
ns, non-significant
na, not-applicable

doi:10.1371/[Link].0160857.t001

4 Line Study. We replicated the effects of ancestral diet on pupal weight in four genetic
lines selected from the original 10 line study. We found the overall patterns to be very similar
with larger contributions of genotype and sex to the total variation in pupal weight, and small
contributions of treatment interaction effects (Fig C in S1 Information, Table 1 and Table A in
S1 Information, S1 File). The total contribution of treatment and its interaction effects to varia-
tion in weight within the sexes accounted for more than 20% of the total variance for females
and 14% for males in the Fall replicate but it was smaller in the spring replicate at about 5%
and 10%of the total variance for females and males respectively (Table 1 and Table A in S1
Information, Fig 3, and S1 File).
The main effect of treatment remained consistent across all studies in the males, as did the rel-
ative contribution of treatment-by-genotype-by-generation (Fig 3D). For females, there were
more substantial differences in the relative effect sizes of treatment and treatment's interaction
effects across the three studies (Fig 3D). While the relative contribution of treatment-by-genotype
and treatment-by-genotype-by-generation were consistent in the 10 line study and the subset of
the genetic lines in 4 line study fall replicate, those factors were much less substantial in the
spring replicate of the 4 line study for females (Fig 3C). The main effect of treatment however,
did remain consistent across the Fall and Spring replicates of the 4 line study.
The overall correlation between the Fall and Spring replicates was greater than 80% (Fig
5A). When the major effects of genetic line and sex were controlled for (by taking the difference
between the control and treated sample averages for each sex/genotype/generation) the positive
correlation between the two replicates remained significant (p = 0.002) with an R2 value of
0.155 (Fig 5B). In addition, when permutation tests were performed randomizing across the
spring and fall replicates, treatment, and generation within genotype and sex, the actual p-val-
ues fell to the extreme right tail of the null p-value distribution indicating that the sex and
genotype specific treatment effects are robust across study replicates.

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 7 / 20

 Transgenerational Dietary Effects

Fig 3. Substantial variance due to treatment effects in F1 and F2 generations in the partition of variance effects for
pupal weight phenotypes. The total explained variance is graphed for females (A) and males (B). The 10 line study is the
left most column in each graph while the fall and spring replicates of the 4 line study are the middle and right columns
respectively. Variance components are indicated as genotype (periwinkle), generation (green), and generation-by-genotype
interaction (fuchsia), while the treatment effects (treatment and all its interactions) are indicated with brackets. The
treatment-related effects are enlarged in C (females) and D (males). Variance components of treatment-related effects are
indicated as main treatment (aqua), treatment-by-genotype (red), treatment-by-generation (yellow), and treatment-by-
genotype-by-generation (blue). The main effect of treatment and its interactions with genotype and generation explain
between 5 and 20% of the total variance in the weight phenotype. Relative effect sizes of treatment and treatment-by-
genotype-by-generation were consistent across studies in the males but showed more variation in females. The spring
replicate of the 4 line study showed reduced total treatment variance effects than observed in the 10 line study, and the fall
replicate of the 4 line study, a subset of the 10 line study.
doi:10.1371/[Link].0160857.g003

F2 generation reaction norms. We are especially interested in how the diet of the grand-
parents (P generation) affects the phenotypes of the grandchildren (F2 generation). The reac-
tion norms across the control, MA, and PA treatments varied dramatically across the ten
genetic lines first tested (Figs 2 and 6), and the total variation due to genotype-by-treatment
interactions in the F2 generation explained 22% of the variance in female weight (NLP = 46.6)
and 17% of the variance in male weight (NLP = 46.7) (Fig 3). The large effect of the P genera-
tion treatment was specific to the sex of the P ancestor experiencing the treatment for some
genetic lines (e.g. 911 females, Figs 2 and 6, Table 2) and for other genotypes the treatment
effect was independent of the sex of the ancestor (e.g. 153 females, Figs 2 and 6, Table 2). In
addition, the magnitude and direction of the effect of the ancestral diet varied by genotype and
by sex of the weighed pupae (e.g. 802 males and females, Fig 2). The sex-by-treatment-by-geno-
type interaction effect was highly significant and explained over 6% of the total variance in the
F2 generation (NLP = 52.4, S1 File).
In the four genetic lines that were retested, their F2 reaction norms followed a fairly similar
pattern (Fig 6B and 6D). The correlation between the differences of the control and treated
samples in the F2 generation was positive and substantial with an R2 of 0.517 (p = 0.0442, Fig
5C). Permutation tests were performed on the 4 line study weight data where sample

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 8 / 20

 Transgenerational Dietary Effects

Fig 4. F1 and F2 generations' distribution of p-values for ANOVA effects with support significance of
treatment effects. Arrows indicate actual p-values. Distributions based on 1024 permutations across
treatments and generations (and replicate for the 4 line study) within genotype and sex to randomize
treatment effects. Genotype and sex distributions not shown since they remain highly significant under the
permutation model as expected. A-H are derived from the 10 line study while I-P are derived from the Fall and
Spring replicates of the 4 line study. Note that all real p-values fall in or beyond the extreme tail of the
permutation distribution. Additional ANOVA effect permutations can be found in Figs A and B in S1
Information.
doi:10.1371/[Link].0160857.g004
measurements were randomized across replicates, generations, and treatments within each
genetic line and sex for the F1 and F2 generations. All p-values for the ANOVA effects for the
real data fell in or beyond the extreme tail of the p-values calculated from permuted data indi-
cating that the highly significant effects of treatment interacting with genotype, sex, and gener-
ation are not due to random chance (Fig 4I–4P and Fig B in S1 Information).
Predictive power of ancestral treatment phenotype. We also tested the predictive power
of the ancestral phenotypic difference to the MA or PA treatment on future generations
(Table 3). Correlations were calculated between reactions to dietary treatment rather than raw
weight to control for experimental variation between generations for the 10 line study, the Fall
replicate of the 4 line study (subset of the 10 line study data), and the Spring replicate of the 4
line study (Table 3). Any differences in correlation between the 10 line study and the Fall repli-
cate of the 4 line study demonstrate how a subset of a population may differ from the whole,
while differences between the Fall and Spring replicates of the 4 line study demonstrate how
correlations can breakdown across time in the same genetic sample.
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 (Table 3). In the F2
generation, there was no consistent pattern of the magnitude of the parental treatment effect,
but females showed more positive correlations with the F1 treatment effects and males showed
more negative correlations. However, the patterns of correlation across generations was highly
variable across studies, while ten of the 16 tested correlations replicated in at least two of the

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 9 / 20

 Transgenerational Dietary Effects

Fig 5. Significant correlation between the Fall and Spring replicates of the 4 line study. The overall
correlation of the actual measurements for all three generations across four genetic lines and three

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 10 / 20

 Transgenerational Dietary Effects

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).
doi:10.1371/[Link].0160857.g005

studies, only two were consistently negatively correlated across all three studies, one each in the
F1 and F2, while four of the correlations that replicated in two studies were substantially contra-
dicted in the third (Table 3).

3 Line Study metabolic phenotypes

We were interested in whether the patterns observed in weight were also reflected in other phe-
notypes 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 ran-
domly selected genetic lines from the DSPR.
Metabolic pools. Genetic diversity contributed significantly to variation in protein con-
centration for all three generations, to trehalose in the F1 and F2 generations, and to triglycer-
ides in the P and F1 generations (Table 4). Treatment group contributed to protein levels for
the P and F2 generation, and triglycerides in the P generation (Table 4). Protein levels also
showed a significant genotype-by-treatment interaction in the F1 generation (Table 4). The
genotype-by-treatment effect in the F2 generation explained 2.8% of the variation in triglycer-
ides, 6.0% of the variation in trehalose, and 6.2% of the variance in protein (S1 File). For Line

Fig 6. Reaction norms for pupal weight in the F2 generation. 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.
doi:10.1371/[Link].0160857.g006

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 11 / 20

 Transgenerational Dietary Effects

Table 2. Significant independent contrasts in F2 generation.

10 line study 4 line studya
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
4 Line Study from Spring Replicates
* indicates significance at p< 0.05

doi:10.1371/[Link].0160857.t002

A, larvae exposed to a high fat diet had higher trehalose levels (p = 0.0284), higher protein lev-
els (p<0.0001), and lower triglyceride levels (p = 0.0003) than those on the control diet (Fig D
in S1 Information, Table 5). Additionally, Line A F1:MA had higher protein levels than F1:PA
(Fig D in S1 Information, p = 0.0329) and F2:MA had lower trehalose levels than F2:Control
(Fig D in S1 Information, Table 5, p = 0.0497). Line C showed an increase in protein in the MA

Table 3. Predictive power of ancestral phenotype on descendant pupal weight.

Generation1 Sex2 Treatment3 Ancestor 10 Line 4 Line 4 Line Correlation 10 Line P- 4 Line P- 4 Line P-value Repeatability11
Effect4 Correlation5 Correlation Fall6 Spring7 value8 value Fall9 Spring10
F1 F MA P_HF_diff F -0.59 -0.48 -0.20 1.28E-19 2.31E-06 3.38E-02 *
F1 F PA P_HF_diff M 0.19 0.27 -0.51 1.07E-02 1.44E-02 1.18E-08 x
F1 M MA P_HF_diff F -0.02 -0.49 -0.28 ns 2.21E-06 3.28E-03
F1 M PA P_HF_diff M 0.28 0.54 -0.49 3.14E-04 1.90E-07 3.08E-07 x
F2 F MA P_HF_diff F -0.05 0.06 0.04 ns ns ns
F2 F PA P_HF_diff M 0.25 0.16 0.22 1.36E-04 ns 2.31E-02
F2 M MA P_HF_diff F -0.27 0.58 -0.06 5.90E-04 4.21E-06 ns
F2 M PA P_HF_diff M -0.23 -0.22 -0.26 7.12E-04 2.68E-02 8.70E-03 *
F2 F MA F1_MA_diff F 0.02 -0.05 -0.36 ns ns 1.24E-04
F2 F MA F1_MA_diff M 0.01 -0.60 -0.11 ns 6.78E-07 ns
F2 F PA F1_PA_diff F 0.46 0.41 -0.42 9.17E-13 1.18E-05 6.46E-06 x
F2 F PA F1_PA_diff M 0.40 0.30 0.03 1.05E-09 1.50E-03 ns
F2 M MA F1_MA_diff F 0.24 -0.05 -0.10 1.68E-03 ns ns
F2 M MA F1_MA_diff M -0.12 -0.01 0.16 ns ns ns
F2 M PA F1_PA_diff F 0.02 -0.48 0.24 ns 5.41E-07 1.71E-02
F2 M PA F1_PA_diff M 0.21 -0.43 -0.25 2.23E-03 8.33E-06 1.39E-02 x

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 10 line study
6
correlation between ancestor and progeny phenotype in 4 line study subset of ten genetic lines
7
correlation between ancestor and progeny phenotype in 4 line study replicated
8
P-value of 10 line study correlation
9
P-value of 4 line study subset of ten genetic lines
10
P-value of 4 line study replicate.
11
repeatability across populations and time points (* correlation repeats in all three studies, x one of the studies contradicts the pattern repeated in the other
two studies), ns—non-significant, bold indicates correlation repeated in at least two studies

doi:10.1371/[Link].0160857.t003

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 12 / 20

 Transgenerational Dietary Effects

Table 4. P-values of ANOVA effects for each phenotype separated by generation. Significant p-values are bolded.
Trehalose Levels Triglyceride Levels Female Pupal Weight
Comparison P F1 F2 P F1 F2 P F1 F2
Genotype ns 3.71 3.27 3.33 3.17 ns 2.47 ns 7.89
Treatment ns ns ns 2.37 ns ns ns 1.48 1.41
Genotype * Treatment ns ns ns ns ns ns ns ns ns
Protein Egg Size Male Pupal Weight
P F1 F2 P F1 F2 P F1 F2
Genotype 5.97 2.47 1.42 3.22 ns 11.21 4.22 ns 2.29
Treatment 3.17 ns 1.39 1.40 ns ns ns 3.45 ns
Genotype * Treatment ns 2.21 ns ns ns 1.76 ns 3.22 ns
doi:10.1371/[Link].0160857.t004

and PA treatments in the F1 generation (p = 0.0193 and p = 0.0051 respectively) and a decrease
in protein in the MA treatment relative to both the PA (p = 0.0037) and control (p = 0.0347)
treatments in the F2 generation (Fig E in S1 Information). Line B did not show significant dif-
ferences between treatments for triglyceride, trehalose, or protein levels. Our study pooled
male and female larvae for the metabolite measurements, but future studies of transgenera-
tional effects on larval metabolite pools may want to consider whether the sex of the larvae
plays a role.
Male and female pupal weight. Male and female pupal weight both had genotype effects
for the P and F2 generations (Table 4). Ancestral diet (treatment) caused significant effects in
the F1 and F2 generations for female weight (Table 4), and these effects were driven by Line A
and Line C (Fig F in S1 Information, Table 5).
Egg size. The P and the F2 generations had a large genotypic effect on egg size (Table 4).
As well, egg size was affected by treatment in the P generation and genotype-by-treatment
interaction explained 4.2% of the variation in the F2 generation (Table 4). Interestingly, the
genotype-by-treatment effect is caused by Line A and Line C’s opposing reactions to the treat-
ments (Fig 7 and Fig G in S1 Information). Line B eggs laid by a female who was reared on a
high fat diet (P:MA) were 8.3% larger than the Control (Fig 7 and Fig G in S1 Information,
Table 5, p = 0.0206). Line B eggs laid by a female with a mother reared on a high fat diet (F1:
MA) were 7.2% larger than the F1:PA eggs (Fig 7 and Fig G in S1 Information, Table 5,
p = 0.0054). Line A F2:PA had 4.1% larger eggs than the F2:MA (Fig 7 and Fig G in S1

Table 5. Significant differences between treatment groups for each phenotype/generation/genotype.

Trehalose Levels b Triglyceride Levels b Female Pupal Weight b
a
Comparison P F1 F2 P F1 F2 P F1 F2
MA vs. Control A* - A* A*** - - A* - A* C *
PA vs. Control - - - - A** C**
MA vs. PA - - - - A** -
Protein b Egg Size Male Pupal Weight b
P F1 F2 P F1 F2 P F1 F2
MA vs. Control A*** C* C* B* - - - - -
PA vs. Control C** - - - C** - -
MA vs. PA A* C** - B** A* C** - -
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; p < 0.05 *, p < 0.01 **, p < 0.001 ***)
b
The only comparison made in the P generation was High Fat vs. Control.

doi:10.1371/[Link].0160857.t005

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 13 / 20

 Transgenerational Dietary Effects

Fig 7. Difference in egg size of Maternal Ancestor (MA) and Paternal Ancestor (PA) compared to the Control in the 3 line study. Substantial
differences by sex, genotype, and generation were observed for the influence of the high fat treatment. Significance values indicated as p < 0.05 *,
p < 0.01 **.
doi:10.1371/[Link].0160857.g007

Information, Table 5, p = 0.0423). Opposingly, Line C’s F2:PA had smaller eggs than F2:MA
(7.1%, p = 0.0013) and the F2:Control (Fig 7 and Fig G in S1 Information, Table 5, 5.9%,
p = 0.0067).
Egg-body size correlation. We also looked at the correlation between male and female
pupal weight and egg size (Table B in S1 Information). Overall, treatment groups with large
male pupae laid larger eggs (p = 0.0004). This relationship was driven by genotype-specific rela-
tionships and Line B’s positive correlation between pupal size and offspring’s egg size
(p = 0.0194). Line B’s female pupal weight was positively correlated with the size of the eggs
they laid (p = 0.0113). In contrast, Line C’s female pupae had a negative correlation with laid
egg size (p = 0.0311). Interestingly, there was a negative correlation between Line B’s egg size
and the future male and female pupal weights (p = 0.0262 and 0.0469, respectively).

Discussion
In this study we examined the sex-specific effects of a high fat diet on two subsequent genera-
tions in the model organism, D. melanogaster. We have demonstrated three important patterns
about transgenerational epigenetic effects of a diet: 1) differences in ancestral diet exhibit a sig-
nificant interaction with genotype and sex in descendants' phenotype, 2) epigenetic impacts on
phenotype vary according to the sex of the affected ancestor, 3) ancestral reaction to a differ-
ence in diet is not a consistent predictor of a descendants' weight.

Substantial genotype and sex-dependent transgenerational effects

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 [13]. We found that the genotype-by-treatment interaction effect in
both the F1 and F2 generations for weight was highly significant and explained 1–22% of the
total variation in phenotype within a given generation and sex. To put this into perspective,

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 14 / 20

 Transgenerational Dietary Effects

effect sizes for quantitative trait loci deemed important for influencing the genetic component
of human phenotypes and disease through GWAS are routinely on the order of 1% [47]. Thus,
the influence of ancestral diet interacting with genotype appears to be as important, and most
cases much more important, than the effect of individual genetic loci. Further study with many
more replicates will be needed to make a robust estimate of the absolute effect of the interaction
between ancestral diet and genotype, but clearly is a non-negligible effect.
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 10 line study, and 1.0 and 3.1% of the variation respectively in the 4
line study. Overall, when considering F1 and F2 in combination, 5–10% of the variance in
weight was due to the parent generation's diet (treatment) and its interactions with the descen-
dants' genotype, sex, and generation (Table 1). The importance of treatment interactions with
genotype and generation are even more apparent when the sexes are analyzed separately, with
the main effect of treatment and all of its interactions explaining between 5 and 21% of the phe-
notypic variance (Fig 3).
We observed similar magnitudes of treatment effects in the phenotypes measured in the 3
line study, suggesting that the transgenerational impact of ancestral diet can influence multiple
traits, including ones closely tied to fitness such as egg size. Xia and Belle [25] also recently
found that adult diet can effect longevity and reproduction out as far as the F3 generation in a
single genotype of Drosophila. Our findings of treatment effects across generations is consis-
tent with previous research that has found that the dry weight of F1 adults whose parents were
fed a high sugar [13] or low protein diet [11] were heavier than the controls. It should be noted
that the variance explained by the main effect of ancestral diet is not directly additive to the
interaction of the treatment with other factors such as sex or genotype, thus it is possible that
the treatment can have an average directional impact across all samples, but also have a distinct
effect on one sex or genotype.

Sex-specific transmission
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 (Table 2). For example, in the 10 line study, the MA treatment in the F2
generation had no significant impact of female pupal weight but was associated with a substan-
tial decrease in weight in the male pupae. Conversely, the PA treatment was associated with a
decrease in the weight of female F2 pupae but not in male F2 pupae. Overall, the MA and PA
treatments differed from each other in F2 pupal weights within sex for five of the ten genetic
lines each for females and males (Table 2). For seven out of the eight contrasts between MA
and PA in the F2 generation in the 4 line study, the pattern replicated (Table 2). Similar pat-
terns were observed for the other phenotypes in the 3 line study. Thus, the sex of the grandpar-
ent experiencing a distinct dietary treatment influences the nature of the transgenerational
impact on the grand-offspring.
In humans, Kaati et al. [1] found that transgenerational dietary influences occurred only
through the paternal line in their human cohort, where the father and paternal grandmother’s
exposure to famine during their slow growth period was associated with lower cardiovascular
causes of death in the present generation. The paternal influence may be transmitted via DNA
methylation and small non-coding RNA expression in the spermatozoa of obese males [48].
Our data does not show clear indications of strictly paternal (PA) inheritance, and instead
there are effects through both the paternal and maternal lineage.

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 15 / 20

 Transgenerational Dietary Effects

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 genera-
tions [30]. In our experiment, one of the three genetic lines tested for changes in metabolic
pools showed MA high fat treatment effects on trehalose levels in the F2 generation (Table 5).
Interestingly, paternal low protein diet did not affect female body size in a previous study of
Drosophila [11], but paternal high fat diet did affect female size in a mouse model [49]. Tem-
perature effects on body size in Drosophila have also been observed to transmit in a sex-specific
manner, where mothers who developed at higher temperatures had offspring with a decreased
body mass, while the fathers that develop at higher temperatures produced offspring with a
longer wingspan [17]. Taken as a whole, these results show that sex-specific multi-generation
effects can occur, but will vary by genotype.

Parental reaction an inconsistent predictor of descendants’

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 pur-
pose 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 dif-
ference 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 differ-
entiate 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 mecha-
nisms 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 cor-
relations 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 con-
trol for the one tested genotype [30]. This indicates a parental reaction to diet is not required
for descendant phenotypic effects, which may be an example of direct induction in Drosophila
[50]. We saw a number of genetic lines where the treatment in the parental generation had little
or no effect on the phenotypes of the parents, but the F1 and F2 descendants showed a substan-
tial impact from the parental diet (Fig 2).
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 10
line study, but the subset of four genetic lines assayed in the Fall showed a more substantial
main effect of treatment that replicated in the spring as well (Fig 3C). Thus, the shift in the
genetic population analyzed changed the overall variance due directly to the ancestral genera-
tion's diet. Overall, the predictability of ancestral diet is largely genotype-specific and future

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 16 / 20

 Transgenerational Dietary Effects

studies should utilize large genotypic sample sizes to accurately characterize a population's gen-
eral transgenerational phenotypic effects and avoid attributing a genotypic idiosyncrasy to
population-level patterns.
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 epi-
genetic 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, categori-
cally, 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 inter-
acting with genetic variation [51].

Conclusions
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 indi-
cator of effects in other related traits. For example, female pupae were affected in some genetic
lines while males were not affected (Fig 2).
Sex-specific treatment and phenotyping across generations has shown its usefulness in Dro-
sophila, 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 transgenera-
tional 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 (e.g.
high fat treated maternal grandfather) and phenotyping males and females separately. Addi-
tionally, the additive effect of sex should be considered in organisms whose ancestors of both
sexes were raised in a high fat background, as in Valtonen et al. [11]. These additional tests will
help determine the origin, and thus mechanism, for the transgenerational transmission of die-
tary information.
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 geno-
types 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 popula-
tion. Awareness of genetic variation for the impact of ancestral environment could provide
important insights into the varied causes of the current obesity epidemic.

Supporting Information
S1 File. Supplemental Data Tables. Tables of data means and standard errors, raw data, and
variance partitions under different models are given.
(XLSX)

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 17 / 20

 Transgenerational Dietary Effects

S1 Information. Supplemental Figs and Tables. 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 4 line study, while D- G depict geno-
type-, generation-, and treatment-specific means and standard errors for measured pheno-
types. Table A gives ANOVA variance partition for the parental generation. Table B gives the
correlation between body weight and egg size.
(PDF)

Acknowledgments
We thank the Reed Lab members for technical assistance with experiments, as well as, exten-
sive constructive criticism on manuscript and data presentation.

Author Contributions
Conceptualization: KDB LKR.
Formal analysis: KDB LKR.
Funding acquisition: LKR.
Investigation: KDB JJ LKR.
Methodology: KDB LKR.
Project administration: LKR.
Resources: LKR.
Supervision: LKR.
Validation: LKR JJ.
Visualization: KDB LKR.
Writing - original draft: KDB LKR.
Writing - review & editing: KDB JJ LKR.

References
1. Kaati G, Bygren LO, Edvinsson S. Cardiovascular and diabetes mortality determined by nutrition during
parents”and grandparents” slow growth period. European Journal of Human Genetics. 2002; 10: 682–
688. doi: 10.1038/[Link].5200859 PMID: 12404098
2. Pembrey ME, Bygren LO, Kaati G, Edvinsson S, Northstone K, Sjöström M, et al. Sex-specific, male-
line transgenerational responses in humans. European Journal of Human Genetics. 2006; 14: 159–
166. doi: 10.1038/[Link].5201538 PMID: 16391557
3. McPherson NO, Fullston T, Bakos HW, Setchell BP, Lane M. Obese father“s metabolic state, adiposity,
and reproductive capacity indicate son”s reproductive health. Fertil Steril. 2014; 101: 865–873. doi: 10.
1016/[Link].2013.12.007 PMID: 24424359
4. Wei Y, Schatten H, Sun Q-Y. Environmental epigenetic inheritance through gametes and implications
for human reproduction. Hum Reprod Update. 2015; 21: 194–208. doi: 10.1093/humupd/dmu061
PMID: 25416302
5. Paoloni-Giacobino A. Epigenetic effects of methoxychlor and vinclozolin on male gametes. Vitam
Horm. 2014; 94: 211–227. doi: 10.1016/B978-0-12-800095-3.00008–0 PMID: 24388192
6. Zhu J, Lee KP, Spencer TJ, Biederman J, Bhide PG. Transgenerational transmission of hyperactivity in
a mouse model of ADHD. J Neurosci. 2014; 34: 2768–2773. doi: 10.1523/JNEUROSCI.4402-13.2014
PMID: 24553919

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 18 / 20

 Transgenerational Dietary Effects

7. Dias BG, Ressler KJ. Parental olfactory experience influences behavior and neural structure in subse-
quent generations. Nat Neurosci. 2014; 17: 89–96. doi: 10.1038/nn.3594 PMID: 24292232
8. Gapp K, Jawaid A, Sarkies P, Bohacek J, Pelczar P, Prados J, et al. Implication of sperm RNAs in
transgenerational inheritance of the effects of early trauma in mice. Nat Neurosci. 2014; 17: 667–669.
doi: 10.1038/nn.3695 PMID: 24728267
9. Prasad NG, Shakarad M, Rajamani M, Joshi A. Interaction between the effects of maternal and larval
levels of nutrition on pre-adult survival in. Evolutionary Ecology Research. 2003; 5: 903–911.
10. Vijendravarma RK, Narasimha S, Kawecki TJ. Effects of parental larval diet on egg size and offspring
traits in Drosophila. Biol Lett. 2010; 6: 238–241. doi: 10.1098/rsbl.2009.0754 PMID: 19875510
11. Valtonen TM, Kangassalo K, Pölkki M, Rantala MJ. Transgenerational effects of parental larval diet on
offspring development time, adult body size and pathogen resistance in Drosophila melanogaster.
PLoS ONE. 2012; 7: e31611. doi: 10.1371/[Link].0031611 PMID: 22359607
12. Somer RA, Thummel CS. Epigenetic inheritance of metabolic state. Current Opinion in Genetics &
Development. 2014; 27: 43–47. doi: 10.1016/[Link].2014.03.008
13. Matzkin LM, Johnson S, Paight C, Markow TA. Preadult parental diet affects offspring development
and metabolism in Drosophila melanogaster. PLoS ONE. 2013; 8: e59530. doi: 10.1371/[Link].
0059530 PMID: 23555695
14. Xia B, de Belle JS. Transgenerational programming of longevity and reproduction by post-eclosion die-
tary manipulation in Drosophila. Aging (Albany NY). 2016; 8: 1115–1134. doi: 10.18632/aging.100932
15. Brookheart RT, Duncan JG. Drosophila melanogaster: An emerging model of transgenerational effects
of maternal obesity. Mol Cell Endocrinol. 2015. doi: 10.1016/[Link].2015.12.003
16. Aldrich JC, Maggert KA. Transgenerational inheritance of diet-induced genome rearrangements in Dro-
sophila. PLoS Genet. 2015; 11: e1005148. doi: 10.1371/[Link].1005148 PMID: 25885886
17. Crill WD, Huey RB, Gilchrist GW. Within- and Between-Generation Effects of Temperature on the Mor-
phology and Physiology of Drosophila melanogaster. Evolution. [Society for the Study of Evolution,
Wiley]; 1996; 50: 1205–1218.
18. Schiffer M, Hangartner S, Hoffmann AA. Assessing the relative importance of environmental effects,
carry-over effects and species differences in thermal stress resistance: a comparison of Drosophilids
across field and laboratory generations. Journal of Experimental Biology. 2013; 216: 3790–3798. doi:
10.1242/jeb.085126 PMID: 23821714
19. Branco AT, Lemos B. High intake of dietary sugar enhances bisphenol A (BPA) disruption and reveals
ribosome-mediated pathways of toxicity. Genetics. 2014; 197: 147–157. doi: 10.1534/genetics.114.
163170 PMID: 24614930
20. Stern S, Snir O, Mizrachi E, Galili M, Zaltsman I, Soen Y. Reduction in maternal Polycomb levels con-
tributes to transgenerational inheritance of a response to toxic stress in flies. J Physiol (Lond). 2014;
592: 2343–2355. doi: 10.1113/jphysiol.2014.271445
21. Linder JE, Promislow DEL. Cross-generational fitness effects of infection in Drosophila melanogaster.
Fly (Austin). 2009; 3: 143–150.
22. Nystrand M, Dowling DK. Transgenerational interactions involving parental age and immune status
affect female reproductive success in Drosophila melanogaster. Proceedings of the Royal Society of
London Series B-Biological Sciences. 2014; 281: 20141242. doi: 10.1098/rspb.2014.1242
23. Reed LK, Williams S, Springston M, Brown J, Freeman K, DesRoches CE, et al. Genotype-by-diet inter-
actions drive metabolic phenotype variation in Drosophila melanogaster. Genetics. 2010; 185: 1009–
1019. doi: 10.1534/genetics.109.113571 PMID: 20385784
24. Reed LK, Lee K, Zhang Z, Rashid L, Poe A, Hsieh B, et al. Systems Genomics of Metabolic Phenotypes
in Wild-Type Drosophila melanogaster. Genetics. 2014. doi: 10.1534/genetics.114.163857
25. Xia B, de Belle JS. Transgenerational programming of longevity and reproduction by post-eclosion die-
tary manipulation in Drosophila. Aging (Albany NY). 2016; 8: 1115–1134. doi: 10.18632/aging.100932
26. Tadros W, Lipshitz HD. Setting the stage for development: mRNA translation and stability during oocyte
maturation and egg activation in Drosophila. Dev Dyn. 2005; 232: 593–608. doi: 10.1002/dvdy.20297
PMID: 15704150
27. Baker KD, Thummel CS. Diabetic larvae and obese flies-emerging studies of metabolism in Drosophila.
Cell Metab. 2007; 6: 257–266. doi: 10.1016/[Link].2007.09.002 PMID: 17908555
28. Skinner MK. What is an epigenetic transgenerational phenotype? F3 or F2. Reprod Toxicol. 2008; 25:
2–6. doi: 10.1016/[Link].2007.09.001 PMID: 17949945
29. Lecomte V, Youngson NA, Maloney CA, Morris MJ. Parental programming: how can we improve study
design to discern the molecular mechanisms? Bioessays. 2013; 35: 787–793. doi: 10.1002/bies.
201300051 PMID: 23881837

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 19 / 20

 Transgenerational Dietary Effects

30. Buescher JL, Musselman LP, Wilson CA, Lang T, Keleher M, Baranski TJ, et al. Evidence for transge-
nerational metabolic programming in Drosophila. Dis Model Mech. 2013; 6: 1123–1132. doi: 10.1242/
dmm.011924 PMID: 23649823
31. Youngson NA, Whitelaw E. Transgenerational epigenetic effects. Annual Review of Genomics and
Human Genetics. 2008; 9: 233–257. doi: 10.1146/[Link].9.081307.164445 PMID: 18767965
32. Ho DH, Burggren WW. Epigenetics and transgenerational transfer: a physiological perspective. J Exp
Biol. 2010; 213: 3–16. doi: 10.1242/jeb.019752 PMID: 20008356
33. Mackay TFCT, Richards SS, Stone EAE, Barbadilla AA, Ayroles JFJ, Zhu DD, et al. The Drosophila
melanogaster Genetic Reference Panel. Nature. 2012; 482: 173–178. doi: 10.1038/nature10811
PMID: 22318601
34. Huang W, Massouras A, Inoue Y, Peiffer J, Ràmia M, Tarone AM, et al. Natural variation in genome
architecture among 205 Drosophila melanogaster Genetic Reference Panel lines. Genome Research.
2014; 24: 1193–1208. doi: 10.1101/gr.171546.113 PMID: 24714809
35. King EG, Macdonald SJ, Long AD. Properties and Power of the Drosophila Synthetic Population
Resource for the Routine Dissection of Complex Traits. Genetics. 2012; 191: 935–949. doi: 10.1534/
genetics.112.138537 PMID: 22505626
36. Macdonald SJ, Long AD. Joint estimates of quantitative trait locus effect and frequency using synthetic
recombinant populations of Drosophila melanogaster. Genetics. 2007; 176: 1261–1281. doi: 10.1534/
genetics.106.069641 PMID: 17435224
37. Williams S, Dew-Budd K, Davis K, Anderson J, Bishop R, Freeman K, et al. Metabolomic and Gene
Expression Profiles Exhibit Modular Genetic and Dietary Structure Linking Metabolic Syndrome Pheno-
types in Drosophila. G3 (Bethesda). 2015; 5: 2817–2829. doi: 10.1534/g3.115.023564
38. Broderick NA, Lemaitre B. Gut-associated microbes of Drosophila melanogaster. Gut Microbes. 2012;
3: 307–321. doi: 10.4161/gmic.19896 PMID: 22572876
39. Buchon N, Broderick NA, Lemaitre B. Gut homeostasis in a microbial world: insights from Drosophila
melanogaster. Nat Rev Microbiol. 2013; 11: 615–626. doi: 10.1038/nrmicro3074 PMID: 23893105
40. Charroux B, Royet J. Gut-microbiota interactions in non-mammals: what can we learn from Drosophila?
Semin Immunol. 2012; 24: 17–24. doi: 10.1016/[Link].2011.11.003 PMID: 22284578
41. Staubach F, Baines JF, Künzel S, Bik EM, Petrov DA. Host species and environmental effects on bac-
terial communities associated with Drosophila in the laboratory and in the natural environment. PLoS
ONE. 2013; 8: e70749. doi: 10.1371/[Link].0070749 PMID: 23967097
42. Church RB, Fw R. A Biochemical Study of Growth of Drosophila Melanogaster. Journal of Experimental
Zoology Part a-Comparative Experimental Biology. 1966; 162: 337–&.
43. Rulifson EJ, Kim SK, Nusse R. Ablation of insulin-producing neurons in flies: growth and diabetic phe-
notypes. Science. 2002; 296: 1118–1120. doi: 10.1126/science.1070058 PMID: 12004130
44. Clark AG, Keith LE. Variation among Extracted Lines of Drosophila-Melanogaster in Triacylglycerol
and Carbohydrate Storage. Genetics. 1988; 119: 595–607. PMID: 3136052
45. De Luca M, Yi NJ, Allison DB, Leips J, Ruden DM. Mapping quantitative trait loci affecting variation in
Drosophila triacylglycerol storage. Obesity Research. 2005; 13: 1596–1605. PMID: 16222063
46. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utiliz-
ing the principle of protein-dye binding. Analytical Biochemistry. 1976; 72: 248–254. doi: 10.1016/0003-
2697(76)90527-3 PMID: 942051
47. Rockman MV. The QTN program and the alleles that matter for evolution: all that's gold does not glitter.
Evolution. 2012; 66: 1–17. doi: 10.1111/j.1558-5646.2011.01486.x PMID: 22220860
48. Donkin I, Versteyhe S, Ingerslev LR, Qian K, Mechta M, Nordkap L, et al. Obesity and Bariatric Surgery
Drive Epigenetic Variation of Spermatozoa in Humans. Cell Metab. 2016; 23: 369–378. doi: 10.1016/j.
cmet.2015.11.004 PMID: 26669700
49. Fullston T, Ohlsson Teague EMC, Palmer NO, DeBlasio MJ, Mitchell M, Corbett M, et al. Paternal obe-
sity initiates metabolic disturbances in two generations of mice with incomplete penetrance to the F2
generation and alters the transcriptional profile of testis and sperm microRNA content. FASEB J. 2013;
27: 4226–4243. doi: 10.1096/fj.12-224048 PMID: 23845863
50. Jablonka E, Raz G. Transgenerational epigenetic inheritance: prevalence, mechanisms, and implica-
tions for the study of heredity and evolution. Q Rev Biol. 2009; 84: 131–176. PMID: 19606595
51. Azevedo RB, French V, Partridge L. Life-history consequences of egg size in Drosophila melanogaster.
Am Nat. 1997; 150: 250–282. doi: 10.1086/286065 PMID: 18811284

PLOS ONE | DOI:10.1371/[Link].0160857 August 12, 2016 20 / 20