Biogerontology (2022) 23:129–144

https://doi.org/10.1007/s10522-022-09953-2 (0123456789().,-volV)
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RESEARCH ARTICLE

Mapping sex differences in the effects of protein

and carbohydrates on lifespan and reproduction
in Drosophila melanogaster: is measuring nutrient intake
essential?
Matthew R. Carey . C. Ruth Archer . James Rapkin . Meaghan Castledine .
Kim Jensen . Clarissa M. House . David J. Hosken . John Hunt

Received: 11 October 2021 / Accepted: 21 January 2022 / Published online: 5 February 2022
Ó The Author(s) 2022

Abstract Understanding how diet affects reproduc- nutrient composition of forty agar-based diets, gener-
tion and survival is a central aim in evolutionary ating broad coverage of nutrient space. We find that
biology. Although this relationship is likely to differ male and female lifespan was maximised on low
between the sexes, we lack data relating diet to male protein, high carbohydrate blends (* 1P:15.9C). This
reproductive traits. One exception to this general nutrient ratio also maximised male reproductive rates,
pattern is Drosophila melanogaster, where male but females required more protein to maximise daily
dietary intake was quantified using the CApillary fecundity (1P:1.22C). These results are consistent with
FEeder (CAFE) method. However, CAFE feeding CAFE assay outcomes. However, the approach
reduces D. melanogaster survival and reproduction, so employed here improved female fitness relative to
may distort diet-fitness outcomes. Here, we use the CAFE assays, while effects of agar versus CAFE
Geometric Framework of Nutrition to create nutrient feeding on male fitness traits depended on the nutrient
landscapes that map sex-specific relationships composition of experimental diets. We suggest that
between protein, carbohydrate, lifespan and reproduc- informative nutrient landscapes can be made without
tion in D. melanogaster. Rather than creating land- measuring individual nutrient intake and that in many
scapes with consumption data, we map traits onto the cases, this may be preferable to using the CAFE
approach. The most appropriate method will depend
on the question and species being studied, but the
Supplementary Information The online version contains approach adopted here has the advantage of creating
supplementary material available at https://doi.org/10.1007/
s10522-022-09953-2.

M. R. Carey K. Jensen
Department of Metabolism, Digestion and Reproduction, Department of Animal Science - ANIS Nutrition, Aarhus
Imperial College London, London, UK University, Tjele, Denmark

M. R. Carey
C. R. Archer
J. Rapkin
C. M. House
J. Hunt (&)

M. Castledine
D. J. Hosken School of Science, Western Sydney University,
Centre for Ecology and Conservation, University of Hawkesbury Campus, Richmond, NSW, Australia
Exeter, Cornwall Campus, Cornwall, UK e-mail: [email protected]

C. R. Archer
Institute of Evolutionary Ecology and Conservation
Genomics, University of Ulm, Albert-Einstein Allee 11,
89069 Ulm, Germany

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nutritional landscapes when dietary intake is hard to typically require more protein for reproduction and
quantify. therefore a single diet cannot maximise both lifespan
and reproduction, resulting in a nutrient-based trade-
Keywords Caloric restriction
Drosophila off between these traits (Lee et al. 2008; Maklakov
melanogaster
Geometric framework of nutrition
et al. 2008; Harrison et al. 2014; Archer et al. 2015;
Longevity
Reproduction
CAFE assays Rapkin et al. 2017). It is not clear if males face a
similar trade-off because existing data suggest that in
many insects, males can consume a single nutrient
ratio to promote both lifespan and reproduction
Introduction (Maklakov et al. 2008; Harrison et al. 2014; Jensen
et al. 2015; Rapkin et al. 2017). However, our
In many species, dietary restriction (where individuals understanding of how nutrition affects male repro-
are fed less food but without causing malnutrition) duction lags far behind our understanding of effects in
extends lifespan but reduces fertility (Masoro 2005; females across all taxa, making it difficult to determine
Speakman and Mitchell 2011; Nakagawa et al. 2012; if this apparent sex-difference represents a general
Simons et al. 2013). Longer lives in response to dietary pattern. In particular, most research using the Geo-
restriction are taxonomically widespread—although metric Framework of Nutrition has used insect mod-
most frequently observed in lab models (Nakagawa els, and while accumulating research in other taxa has
et al. 2012). Accordingly, dietary restriction has been shown that nutrient blend as well as total nutrient
described as a paradigm in aging biology (Anderson intake affect reproductive effort and survival (e.g.
and Weindruch 2012) and understanding it is a major mice Solon-Biet et al. 2015; e.g. sticklebacks Moatt
aim of aging research (Jensen et al. 2015; Moatt et al. et al. 2019), sex-differences in the relationship
2020). A long-standing evolutionary explanation for between nutrients and life-history are even less well
greater lifespan under dietary restriction is that understood outside of insects.
resources are reallocated away from reproduction It has been suggested that failure to robustly
towards somatic maintenance (Shanley and Kirkwood characterise how diet affects male reproductive
2000). The rationale being that there is little gain to investment could have skewed our understanding of
reproducing when food is limited and offspring sex-differences in the relationship between nutrition
survival prospects are poor. Instead, it is better to and reproductive success (Moatt et al. 2016). While
invest in surviving to reproduce when environmental studies frequently test for effects of dietary restriction
conditions improve. This idea probably oversimplifies on lifespan in both sexes (see e.g. data used in meta-
the relationship between nutrition, lifespan and repro- analysis by Nakagawa et al. 2012), far fewer studies
duction (Adler and Bonduriansky 2014), and is test how dietary restriction affects reproduction
unlikely to apply to all species e.g. long-lived species (Moatt et al. 2016). Of those linking nutrition and
that reproduce over multiple seasons (Shanley and reproduction, few assay males, and those that do
Kirkwood 2006). However, trade-offs involving seldom capture a significant proportion of male
reproduction may play a role in explaining why in reproductive costs (Moatt et al. 2016). That is, to
some species, individuals live longer when they eat reproduce males must attract mates and fertilise their
less (Moatt et al. 2020; Zanco et al. 2021). ova, often against a backdrop of intense male–male
The relationship between food, sex and death is not competition both before and after mating (Hosken and
just about how much food individuals consume. The House 2011; Archer and Hosken 2021). Few dietary
Geometric Framework of Nutrition has shown that restriction studies assay how nutrition affects this full
both the amount of food consumed and its nutrient spectrum of male reproductive behaviours, and as a
ratio interact to affect lifespan. For example a general result, apparent sex-differences in the magnitude of
trend in insects is that high protein, low carbohydrate dietary restriction impacts on reproduction may be an
intake reduces lifespan in both sexes (Lee et al. 2008; artefact of experimental design rather than a genuine
Maklakov et al. 2008; Fanson et al. 2009; Bruce et al. biological signal (Moatt et al. 2016).
2013; Harrison et al. 2014; Jensen et al. 2015; Malod In the Geometric Framework of Nutrition literature,
et al. 2017; Rapkin et al. 2017). However, females there have been tests of how nutrients interact to affect

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male reproductive traits including the number of (Simpson and Raubenheimer 1995, 2007, 2012). How-
offspring sired (Reddiex et al. 2013; Jensen et al. ever, while the CAFE method enables accurate
2015), sexual signalling (Maklakov et al. 2008; South quantification of nutrient intake (Deshpande et al.
et al. 2011; Harrison et al. 2014; Archer et al. 2015; 2014), this approach reduces lifespan and reproduc-
Bunning et al. 2016; Rapkin et al. 2017; Moatt et al. tion in Drosophila (Lee et al. 2008; Jensen et al. 2015).
2019) and sperm number and viability (Bunning et al. Worryingly, sex-differences in ethanol preferences
2015; Morimoto and Wigby 2016). However, few not seen in CAFE assays emerge when solid foods are
studies have tested how nutrition affects lifespan and provided and intake measured using tracer methods
reproduction in both sexes concurrently (see however (Park et al. 2018). If the CAFE approach (or similar
Lee et al. 2008; Harrison et al. 2014; Archer et al. methods e.g. feeding using pipette tips) can alter sex-
2015; Rapkin et al. 2017; Moatt et al. 2019). Once differences in responses to a particular dietary treat-
more, existing studies typically measure female ment, it is possible that the widespread use of the
reproduction directly (i.e. fecundity in mated females), CAFE approach in small invertebrates (e.g. Lee et al.
but use proxy measures of male reproductive effort. 2008; Fanson et al. 2009; Reddiex et al. 2013; Jensen
For example in field crickets calling effort is fre- et al. 2015; Malod et al. 2017) has skewed our
quently used as a measure of male reproduction understanding of the sex-specific relationship between
(Archer and Hunt 2015). Such proxies only provide a nutrition and phenotype.
snapshot of how diet affects male reproductive With this in mind, we tested how consumption of
investment and do not assess copulation and ejacula- protein and carbohydrates affected lifespan and
tion costs, which can represent a major portion of male reproduction in D. melanogaster, using broad mea-
reproductive effort (Martin and Hosken 2004; Brown sures that capture most of the costs of female and male
et al. 2009). There are exceptions to this however, with reproduction. Additionally, we tested whether it was
studies showing that protein and carbohydrate affect possible to create informative nutrient landscapes
both female fecundity and male competitive siring without collecting consumption data. That is, can we
success in Drosophila melanogaster (Reddiex et al. avoid the costs of CAFE (reduced fitness) without
2013; Jensen et al. 2015). However, these Drosophila losing the insights offered by the accurate quantifica-
studies come with some caveats. For example, Red- tion of food consumption? Here, we dispense with
diex et al. (2013) used yeast as a protein source; yeast creating nutrient landscapes using individual con-
contains more nutrients than just protein and so diets sumption data, and plot trait values onto the protein
varying in their yeast content inevitably vary in more and carbohydrate content of experimental diets. To
than just dietary protein. This makes it more compli- generate broad coverage of the nutrient space, we
cated to determine which specific nutrients affected utilise more diets than is common in Geometric
male phenotypes compared to work using fully Framework studies: 800 flies of each sex were fed one
chemically defined (i.e. holidic) diets. Additionally, of 40 fully chemically defined (i.e. holidic) diets that
Reddiex et al. (2013) only tested for effects on early- varied in their ratio and amount of protein and
life reproduction and did not test for lifespan impacts. carbohydrate.
Finally, both Jensen et al. (2015) and Reddiex et al.
(2013) used the CApillary FEeder (CAFE) method (Ja
et al. 2007) to measure the nutrient intake of flies. Methods and materials
By providing liquid diets in a micro-capillary, the
CAFE method enables the quantification of food Fly stock and maintenance
consumption in small invertebrates such as Droso-
phila (Ja et al. 2007), and so its use can reveal how Dahomey Drosophila melanogaster stocks (supplied
intake may vary with dietary dilution (e.g. detect by Nick Priest, University of Bath) and Krüppel
compensatory feeding). Accurately quantifying nutri- mutation stocks (Bloomington Stock Centre, received
ent intake is central to creating nutrient landscapes September 2015) were maintained in each of two large
using the Geometric Framework of Nutrition to population cages (1m3) with overlapping generations
determine how each nutrient independently, and via at 25 °C under a 12:12 light:dark cycle. Stocks were
interactions with other nutrients, affects phenotype maintained at * 2000 individuals allowed to mate

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freely, and fed ‘Jazz mix’ diet (Fisher Scientific, light:dark regime as stock populations. Lifespan and
Loughborough, UK), provided in wide-neck 1000 ml reproduction were measured in the same experimental
jars. Stock cultures were maintained using this proto- animals: lifespan by monitoring survival daily from
col for ca. 9 months prior to use. Experimental adult eclosion and reproductive effort by counting
animals were cultivated using small vials (25 mm 9 eggs (females) or offspring sired in a competitive
95 mm). To collect experimental animals, multiple mating assay (males). We commenced measuring
small vials were put into each of the two population female fecundity at day five post hatching, by pairing
cages for a maximum of 6 h. These vials were then each focal female at random with a 5-day-old virgin
incubated while flies developed. Virgin flies of both male mating partner for 12-h. Mating partners were
sexes were collected from both of the Dahomey and collected and housed in the same manner as experi-
Krüppel population cages within 4 h of eclosion, and mental individuals until mating. This mating regime
then allocated at random to one of 40 dietary was continued for the duration of an experimental
treatments. individual’s lifetime (i.e. females were paired with
mates every 5 days, for 12-h, beginning at
Artificial diets 18:00–19:00 shortly before lights went out in exper-
imental incubators) and each time a new 5-day-old
In total, 40 artificial, holidic (i.e. fully chemically mating partner was used. This regime was designed to
defined) diets that varied in both protein (P) to ensure that females did not suffer reduced fecundity
carbohydrate (C) ratio (i.e. P:C) and total nutritional due to sperm limitation, or reduced longevity due to
content (i.e. P ? C), were created using the estab- the direct costs of mating and male harassment
lished protocol outlined in Simpson and Abisgold (rationale in Taylor et al. 2008). All experimental
(1985). Diets varied along 10 different P:C ratios (3:1, flies had their food caps changed 24 h prior to mating.
2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:5, 1:8, 1:16) and were The food caps were also changed 6 h after mating
diluted with indigestible cellulose such that they partner removal, at which time female egg production
contained 12, 36, 60 or 80% total nutrition (i.e. was counted under a binocular microscope. This
P ? C) (for detail please see Fig. S1 and Table S1). regime is summarised in Fig. S2 in the online
Powdered forms of each diet were created using the supplement.
methods outlined in South et al. (2011) and then Male reproductive effort, also measured every 5
combined with water, agar and Nipagin (ratio— days, was quantified using similar methods to those
10:10:1:0.1 diet, water, agar and Nipagin). To achieve outlined in Jensen et al. (2015). In brief, we counted
this, agar was added to water, which was boiled and the number offspring produced by a focal male when
then left to cool to \ 60 °C, after which Nipagin and in competition with a 5-day-old virgin male with the
each powered diet was added. Black food colouring Krüppel dominant eye mutation with a wild-type
was added to foods to increase the contrast between female. This allowed for offspring paternity to be
eggs and food to make egg counting easier. These easily assigned and provided us with a biologically
agar-based diets were provided in ‘vial caps’ (1.6 cm relevant measure of male fitness that we could
diameter, 1.6 cm deep) that could be securely fitted to compare to previous work (i.e. siring success). After
the vials in which experimental individuals were the 12-h mating period, the competing Krüppel male
housed. Caps were changed every 5 days both before and the wild-type tester female were removed, and the
and after mating. The experimental diets provided female established on 7 ml of standard ‘Jazz mix’ diet
food, moisture and oviposition substrate for experi- for 14 days. On day 14, vials were frozen and wild-
mental females. type and Krüppel counted. This regime is summarised
in Fig. S3 in the online supplement.
Experimental protocol Reproductive effort was assessed every 5 days
across an individual’s lifetime. Total reproductive
A total of 20 virgin flies of each sex were assigned to effort equates to the sum of these measures (females—
each of 40 artificial diets at random (N = 1600) on the total eggs laid, males—total offspring sired) and daily
day of hatching and housed individually in a vial reproductive effort is the average reproductive output
containing experimental diet and kept under the same per mating opportunity (females—the average number

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of eggs produced per mating opportunity, males—the et al. (2015) (Fig. S4). Second, the divergence between
average number of offspring sired per mating oppor- the global nutritional maxima (calculated from 95%
tunity). Daily reproductive effort was calculated as the CR of the nutritional landscape) was estimated using
average of each count because each reproductive assay the Euclidean distance (d) and corresponding 95% CIs
captured around 1 days’ worth of reproductive activity using the CRcompare function in the ‘OptimaRegion’
i.e. up to 18 h of egg laying for females, and one package in R (Fig. S4). See Rapkin et al. (2018) for a
competitive mating trial for males. Flies that died full overview and justification of this analysis.
before their first mating or escaped during the
experiment were replaced, resulting in a sample size
of 1600 individuals (800 males and 800 females). Results

Statistical analysis Nutrient effects on lifespan and reproduction

in the sexes
A multivariate response-surface approach outlined in
South et al. (2011) was used to estimate the linear and In both sexes, increased intake of dietary protein
nonlinear (i.e. quadratic and correlational) effects of reduced lifespan but dietary carbohydrates increased it
protein (hereafter, P) and carbohydrate (C) intake on (Table 1, Fig. 1a, b). Both nutrients also had signif-
lifespan, daily reproductive effort and total reproduc- icant quadratic effects on lifespan in the sexes. For
tive effort within each sex. To visualise the multivari- dietary carbohydrates the quadratic coefficient was
ate nutritional landscapes for each trait, non- negative for both sexes, meaning that there was a peak
parametric thin-plate splines were constructed in R in lifespan on the nutritional landscape for this nutrient
using the Tps function in the Fields package (Nychka (Table 1; Fig. 1a, b). For dietary protein, the quadratic
et al. 2015) of R (R Core Development Team, version coefficient was positive for both sexes, reflecting a
3.1.2, Vienna, Austria, www.r-project.org). The minimum lifespan at medium to high values of this
locations of the nutritional optima and their 95% nutrient on the nutritional landscape (due to the optima
confidence regions (CRs) were optimised using the in lifespan being at low levels of dietary protein;
OptRegionTps function in the ‘OptimaRegion’ pack- Fig. 1a, b). There was also a significant negative
age (del Castillo et al. 2020). Full details of this correlational effect between dietary protein and car-
approach is provided in Rapkin et al. (2018). bohydrate on lifespan in both sexes, further highlight-
A sequential model-building approach (Draper and ing the increase in lifespan on diets low in protein and
John 1988) was used to determine whether the linear high in carbohydrate (Table 1, Fig. 1a, b). Indeed,
and nonlinear (quadratic and correlational) effects of formal analysis revealed that lifespan was maximised
nutrient intake differed across our response variables at a P:C ratio of 1P:15.93C in males (Fig. 2a) and
(lifespan, daily reproductive effort and total repro- 1P:15.88C in females (Fig. 2b).
ductive effort). Full details of this approach are Statistical comparison of nutritional landscapes for
outlined in Text S1. The sequential model building lifespan across the sexes revealed significant sex
approach can quantify differences in linear and differences in the linear, quadratic and correlational
nonlinear gradients for different response variables effects of dietary protein and carbohydrates (Table 2).
but cannot quantify the direction of this difference in The significant sex difference in linear effects of
nutritional space (Rapkin et al. 2015; Bunning et al. dietary protein and carbohydrates occurred because
2016). It is possible for response variables to show lifespan decreased more steeply with increased dietary
differences in the magnitude of linear and nonlinear protein and increased more steeply with carbohydrates
gradients, but at the same time occupy a similar in males than in females (Table 2). The significant sex
location in nutritional space. Two additional measures difference in quadratic effects was driven exclusively
were therefore calculated to quantify any difference in by the protein content of the diet and occurred because
the location of nutritional optima. First, the angle (h) the coefficient was more positive in males than in
and 95% confidence interval (CI) between nutritional females (Table 2). The significant sex difference in the
vectors for the two response variables of interest were correlational effects existed because the coefficient
calculated using the procedure outlined in Bunning was more negative in males than in females (Table 2).

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Table 1 The linear (P) and carbohydrate (C), quadratic (P 9 P; (DRE) and total reproductive effort (TRE) for male and female
C 9 C) and correlational (P 9 C) effects of protein (P) and Drosophila melanogaster
carbohydrates (C) on lifespan (LS), daily reproductive effort
Response variables Linear effects Nonlinear effects
P C P9P C9C P9C

(A) Males
LS
Coefficient ± SE - 0.31 ± 0.03 0.38 ± 0.03 0.27 ± 0.03 - 0.31 ± 0.03 - 0.32 ± 0.05
t799 9.94 12.14 9.99 10.20 7.00
P value 0.0001 0.0001 0.0001 0.0001 0.0001
DRE
Coefficient ± SE - 0.15 ± 0.04 0.15 ± 0.04 0.04 ± 0.03 - 0.08 ± 0.04 - 0.17 ± 0.06
t799 4.30 4.20 1.29 2.17 2.91
P value 0.0001 0.0001 0.19 0.03 0.004
TRE
Coefficient ± SE - 0.30 ± 0.03 0.30 ± 0.03 0.20 ± 0.03 - 0.25 ± 0.03 - 0.30 ± 0.05
t799 9.34 9.38 6.68 7.62 5.94
P value 0.0001 0.0001 0.0001 0.0001 0.0001
(B) Females
LS
Coefficient ± SE - 0.17 ± 0.03 0.25 ± 0.03 0.18 ± 0.03 - 0.26 ± 0.04 - 0.12 ± 0.05
t799 5.13 7.39 5.55 7.45 2.15
P value 0.0001 0.0001 0.0001 0.0001 0.032
DRE
Coefficient ± SE 0.27 ± 0.03 0.17 ± 0.03 - 0.27 ± 0.03 - 0.21 ± 0.03 0.04 ± 0.05
t799 7.92 4.95 8.56 6.20 0.79
P value 0.0001 0.0001 0.0001 0.0001 0.43
TRE
Coefficient ± SE 0.09 ± 0.03 0.33 ± 0.03 - 0.09 ± 0.03 - 0.35 ± 0.03 - 0.07 ± 0.05
t799 2.63 9.81 2.80 10.20 1.32
P value 0.009 0.0001 0.005 0.0001 0.19
SE standard error and t799 the test of the coefficient with 799 degrees of freedom

However, despite the nutrient effects on lifespan being quadratic effect of dietary protein for total reproduc-
consistently greater in males than in females, the small tive effort (but this was not significant for daily
angle between the linear nutritional vectors reproductive effort) and a significant negative quad-
(h = 12.50°, 95% CIs: 3.27°, 20.76°) and the small ratic effect of dietary carbohydrates on both traits
Euclidean distance between the optima (d = 18.64, (Table 1a; Fig. 1c, e). Likewise, there was also a
95% CIs: 15.67, 21.06), indicates that the optima for significant negative correlational effect between diet-
lifespan occupied similar regions on the nutritional ary protein and carbohydrates on daily and total
landscape for the sexes (Figs. 1a, b, 2a, b). reproductive effort (Table 1a; Fig. 1c, e). Formal
Male daily and total reproductive effort increased analysis revealed that daily reproductive effort was
with dietary carbohydrates and decreased with maximised at a P:C ratio of 1P:12.78C (Fig. 2c) and
increased dietary protein (Table 1a; Fig. 1c, e). As total reproductive effort at 1P:15.92C (Fig. 2e).
for male lifespan, there was a significant positive

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Fig. 1 Nutrient landscapes Males Females

relating dietary protein and days days
carbohydrate to sex-specific a b
lifespan (LS) and
reproductive traits.
Nonparametric thin-plate
spline contour visualizations
of the responses surfaces
describing the effects of
protein and carbohydrate
intake on a male LS,
b female LS, c male
offspring production rate,
d female egg production
rate, e total offspring
offspring eggs
production in males, and c d
f total egg production in
females in Drosophila
melanogaster
Percentage carbohydrate (%)

offspring eggs
e f

Percentage protein (%)

In females, daily reproductive effort and total Statistical comparison of the nutritional landscapes
reproductive effort both increased with dietary protein for daily and total reproductive effort across the sexes
and dietary carbohydrates (Table 1b, Fig. 1d, f). There revealed significant sex differences in the linear,
were also significant negative quadratic coefficients quadratic and correlational effects of dietary protein
for dietary protein and carbohydrates indicating a and carbohydrates (Table 2). For both daily reproduc-
well-defined peak for daily (Fig. 1d) and total repro- tive effort and total reproductive effort, the significant
ductive effort (Fig. 1f). The correlational effect sex difference in the linear effects of nutrients was
between dietary protein and carbohydrates, however, driven exclusively by dietary protein and the fact that
was not significant for either daily or total reproduc- daily reproductive effort and total reproductive effort
tive effort (Table 1b). Formal analysis revealed that increased with dietary protein in females but
daily reproductive effort was maximised at a P:C ratio decreased with this nutrient in males (Table 2). The
of 1P:1.16C (Fig. 2d) and total reproductive effort at significant sex difference in the quadratic effects of
1P:1.85C in females (Fig. 2f). nutrients was due to a positive quadratic coefficient for

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Fig. 2 Confidence regions Male Female

surrounding dietary optima.
Nutritional optima are given s a s b
as X,Y coordinates: a male
lifespan = 4.25,67.70,
b female
lifespan = 3.53,56.07,
c male daily reproductive
effort = 5.19, 66.32,
d female daily reproductive
effort = 36.50, 42.22,
e male total reproductive
effort = 4.08,64.96,
f female total reproductive
effort = 25.43,47.10.
Percentage carbohydrate (%)

Nutritional optima are

shown as black points and c d
the grey regions surrounding
these are 95% confidence
intervals

e f

Percentage protein (%)

dietary protein in males but a negative coefficient in between the linear nutritional vectors (daily reproduc-
females and the quadratic coefficient for dietary tive effort: h = 33.26°, 95% CIs 22.27°, 44.31°; total
carbohydrates being more negative for females than reproductive effort: h = 44.35°, 95% CIs 35.58°,
males (indicative of a more pronounced peak) 53.73°) and the large Euclidean distance between the
(Table 2). The significant sex difference in the nutritional optima (daily reproductive effort:
correlational effects existed because the coefficient d = 43.01, 95% CI 42.12, 43.63; total reproductive
was negative in males but not significant in females effort: d = 34.53, 95% CIs 33.30, 35.14) for the sexes.
(Table 2). These pronounced sex differences in the
magnitude and sign of nutritional effects resulted in The trade-off between lifespan and reproduction
the optima for daily and total reproductive effort being within the sexes
located in different regions on the nutritional land-
scape for males (Figs. 1c, e, 2c, e) and females Sequential model testing revealed that there were
(Figs. 1d, f, 2d, f), as evidenced by the large angles significant differences in the linear and nonlinear

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Table 2 Sequential model building analysis that contrasts the reproductive effort (TRE), both between the sexes, and
linear and nonlinear effects of protein (P) and carbohydrate between traits within the sexes
(C) on lifespan (LS), daily reproductive effort (DRE) and total
SSR SSC DF1 DF2 F P

Males vs. females

LS
Linear 1356.44 1343.05 2 1594 7.95 0.0004A
Quadratic 1169.55 1164.35 2 1590 3.55 0.03B
Correlational 1137.26 1131.30 1 1588 8.37 0.004
DRE
Linear 1552.56 1483.24 2 1594 37.25 0.0001C
Quadratic 1433.03 1375.95 2 1590 32.98 0.0001D
Correlational 1373.69 1367.47 1 1588 7.22 0.007
TRE
Linear 1425.72 1365.03 2 1594 35.44 0.0001E
Quadratic 1254.97 1207.66 2 1590 31.14 0.0001F
Correlational 1188.58 1181.13 1 1588 10.02 0.002
Male
LS vs. DRE
Linear 1411.59 1380.95 2 1594 17.68 0.0001G
Quadratic 1297.67 1262.59 2 1590 22.09 0.0001H
Correlational 1228.69 1225.36 1 1588 4.31 0.038
LS vs. TRE
Linear 1276.97 1274.67 2 1594 1.43 0.24
Quadratic 1097.12 1094.11 2 1590 2.19 0.11
Correlational 1039.76 1039.68 1 1588 0.12 0.74
DRE vs. TRE
Linear 1442.96 1424.74 2 1594 10.19 0.0001I
Quadratic 1369.81 1352.18 2 1590 10.37 0.0001J
Correlational 1321.39 1318.99 1 1588 2.89 0.09
Female
LS vs. DRE
Linear 1524.23 1445.34 2 1594 43.50 0.0001K
Quadratic 1364.34 1277.71 2 1590 53.90 0.0001L
Correlational 1276.93 1273.41 1 1588 4.39 0.04
LS vs. TRE
Linear 1463.56 1433.41 2 1594 16.76 0.0001M
Quadratic 1312.80 1277.90 2 1590 21.71 0.0001N
Correlational 1273.06 1272.75 1 1588 0.40 0.53
DRE vs. TRE
Linear 1445.75 1423.52 2 1594 12.45 0.0001O
Quadratic 1249.83 1231.44 2 1590 11.87 0.0001P
Correlational 1231.33 1229.61 1 1588 2.23 0.14
A B
Univariate test: P: F1,1594 = 8.73, P = 0.003, C: F1,1594 = 7.74, P = 0.005; P 9 P: F1,1590 = 7.08, P = 0.008, C 9 C:
F1,1590 = 0.20, P = 0.66; CP: F1,1594 = 73.96, P = 0.0001, C: F1,1594 = 0.18, P = 0.67; DP 9 P: F1,1590 = 51.60, P = 0.0001, C 9 C:
F1,1590 = 9.10, P = 0.003; EP: F1,1594 = 70.16, P = 0.0001, C: F1,1594 = 0.29, P = 0.59; FP 9 P: F1,1590 = 50.91, P = 0.0001, C 9 C:
F1,1590 = 6.80, P = 0.009; GP: F1,1594 = 11.79, P = 0.001, C: F1,1594 = 24.78, P = 0.0001; HP 9 P: F1,1590 = 30.19, P = 0.0001, C 9
C: F1,1590 = 18.45, P = 0.0001; IP: F1,1594 = 10.23, P = 0.001, C: F1,1594 = 10.90, P = 0.001; JP 9 P: F1,1590 = 13.39, P = 0.0001, C
9 C: F1,1590 = 9.48, P = 0.002; KP: F1,1594 = 85.00, P = 0.0001, C: F1,1594 = 3.08, P = 0.08; LP 9 P: F1,1590 = 107.66, P = 0.0001,
C 9 C: F1,1590 = 0.51, P = 0.47; MP: F1,1594 = 30.19, P = 0.0001, C: F1,1594 = 2.63, P = 0.11; NP 9 P: F1,1590 = 37.14, P = 0.0001,
C 9 C: F1,1590 = 3.44, P = 0.06; OP: F1,1594 = 14.26, P = 0.0001, C: F1,1594 = 11.53, P = 0.001; PP 9 P: F1,1590 = 19.02,
P = 0.0001, C 9 C: F1,1590 = 6.86, P = 0.009

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138 Biogerontology (2022) 23:129–144

effects of nutrients on lifespan and daily reproductive nutritional optima for lifespan and daily reproductive
effort in males (Table 2). The significant difference in effort suggests that any nutrient-based trade-off
the linear effects occurred because the negative between these traits is likely to be weak in males.
impacts of dietary protein and the positive effects of In females, there were significant differences in the
dietary carbohydrates were stronger for lifespan than linear, quadratic and the correlational effects of
daily reproductive effort (Table 2). Likewise, the dietary protein and carbohydrates on lifespan and
significant difference in quadratic effects was due to daily reproductive effort (Table 2). The significant
the quadratic coefficients for dietary protein being difference in the linear effects occurred because daily
more positive, but the coefficient for dietary carbohy- reproductive effort increased with dietary protein but
drates being more negative, for lifespan than daily lifespan decreased with this nutrient (Table 2). The
reproductive effort (Table 2). The significant differ- significant difference in the quadratic effects was due
ence in the correlational effects was due to the to the quadratic coefficient for dietary protein being
correlational coefficient being more negative for positive for lifespan but negative for daily reproduc-
lifespan than daily reproductive effort. In contrast, tive effort (Table 2). Similar differences in the linear
the linear and nonlinear effects of dietary protein and and quadratic (but not correlational) effects of dietary
carbohydrates did not differ significantly between protein and carbohydrates were found for lifespan and
lifespan and total reproductive effort (Table 2). How- total reproductive effort (Table 2). Again, significant
ever, the linear and quadratic (but not the correla- differences in the linear effects occurred because total
tional) effects of these nutrients differed significantly reproductive effort increased with dietary protein but
between daily reproductive effort and total reproduc- lifespan decreased with this nutrient. The significant
tive effort (Table 2). The difference in linear effects differences in the quadratic effects were due to the
reflects that the negative effects of dietary protein and quadratic coefficient for dietary protein being positive
the positive effects of dietary carbohydrates were for lifespan but negative for total reproductive effort
stronger for total reproductive effort than for daily (Table 2). There were significant differences in the
reproductive effort (Table 2). The significant differ- linear, quadratic but not the correlational effects of
ence in quadratic effects was due to the quadratic dietary protein and carbohydrates on daily reproduc-
coefficients for dietary protein being more positive, tive effort and total reproductive effort (Table 2).
and the coefficient for dietary carbohydrates being Significant difference in linear effects occurred
more negative, for total reproductive effort than for because there was a stronger positive effect of dietary
daily reproductive effort (Table 2). protein on daily reproductive effort than on total
Collectively, the differences observed in our reproductive effort, while the opposite pattern was true
sequential models for males were all due to changes for dietary carbohydrates (Table 2). The significant
in the magnitude of nutrient effects on lifespan, daily difference in the quadratic effects was the result of the
reproductive effort and total reproductive effort, rather quadratic coefficient for dietary protein being more
than changes in the sign or significance of these negative for daily reproductive effort than for total
effects. This suggests that the optima for lifespan, reproductive effort but again the opposite pattern was
daily reproductive effort and total reproductive effort true for dietary carbohydrates (Table 2).
in males are located in similar regions on the Collectively, the differences observed in our
nutritional landscape (Figs. 1a, c, e, 2a, c, e). sequential models for females were due to changes
Accordingly, the angle between the linear nutritional in the sign and magnitude of nutrient effects on
vectors and the Euclidean distances between the lifespan, daily reproductive effort and total reproduc-
nutritional optima for lifespan and daily reproductive tive effort, suggesting that optima for the traits are
effort (h = 6.73°, 95% CIs 0.00°,15.52°, d = 12.81, located in different regions of the nutritional landscape
95% CIs 11.08, 14.35), lifespan and total reproductive (Figs. 1b, d, f, 2b, d, f). In agreement with this view,
effort (h = 22.90°, 95% CIs 13.68°, 31.36°; d = 13.88, both the angle between the linear nutritional vectors
95% CIs 12.26, 15.55) and daily reproductive effort and the Euclidean distance between the nutritional
and total reproductive effort (h = 28.61°, 95% CIs optima for lifespan and daily reproductive effort
16.89°,40.88°; d = 16.00, 95% CIs 14.47, 17.67) were (h = 26.48, 95% CIs 17.97°, 34.45°; d = 40.06 95%
all modest. In particular, the small angle between the CIs 39.51, 40.58), were larger in females than males.

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Biogerontology (2022) 23:129–144 139

This suggests that nutrient-based trade-offs between consumed, rather than on energy alone (Mair et al.
lifespan and daily reproductive effort are likely to be 2005; Lee et al. 2008; Maklakov et al. 2008; Fanson
more substantial in females than males. However, the et al. 2009; Nakagawa et al. 2012; Bruce et al. 2013;
angles and distances between lifespan and total Jensen et al. 2015; Le Couteur et al. 2016; Malod et al.
reproductive effort (h = 9.16, 95% CIs 0.36°, 2017; Piper et al. 2017; Rapkin et al. 2017). In both
16.88°; d = 32.96, 95% CIs 31.92, 33.96) and daily sexes, lifespan was greatest on low protein, high
reproductive effort and total reproductive effort carbohydrate diets (female: 1P:15.88C, male:
(h = 17.41, 95% CIs 9.16°, 25.20°; d = 14.48, 95% 1P:15.93C). Why lifespan is often greatest in individ-
CIs 13.89, 15.13) were modest. uals consuming low protein diets is not fully under-
stood. One possibility is that individuals fed high
protein diets use protein for energy, and in doing so
Discussion incur a metabolic cost. This metabolic penalty could
be associated with transformations and waste produc-
Understanding how diet affects lifespan, reproduction tion associated with protein deamination and preven-
and the trade-off between these traits is a key aim in tion of toxicity (Anderson et al. 2020). What is clear is
life-history biology and fundamental to aging research that this is a general trend, with increased lifespan on
(Moatt et al. 2020; Regan et al. 2020). However, a lack low protein diets being documented in insect species
of data testing how diet affects the full spectrum of including Queensland fruit flies (Bactrocera tryoni)
male reproductive costs (Moatt et al. 2016) means that (Fanson et al. 2009; Fanson and Taylor 2012), the
sex differences in the relationship between food, sex marula fruit fly, Ceratitis cosyra (Malod et al. 2017),
and death remain poorly understood (Moatt et al. honey bees (Apis mellifera) (Archer et al. 2014; Paoli
2020). The limited data available testing how diet et al. 2014), and the crickets Teleogrylls commodus
affects survival and reproduction in both sexes con- (Rapkin et al. 2017) and Gryllus veletis (Harrison et al.
comitantly, largely come from D. melanogaster. 2014). High protein intake also reduces survival in
These data are complicated because flies are typically mice (Solon-Biet et al. 2015) and low intake of
fed via the CAFE method, which has clear and costly animal-derived proteins in middle-aged adults
effects on overall fitness (Lee et al. 2008; Jensen et al. (* 50–65 year olds) has positive effects on health
2015). Here, we used agar-based diets to test how and survival, but detrimental effects in the over 65 s
protein and carbohydrate affect survival and repro- (Levine et al. 2014).
duction in both sexes, creating nutrient landscapes While low dietary protein intake improved lifespan,
without measuring consumption. We find that long- it also reduced female egg production. This finding is
evity was greatest on high carbohydrate, low protein consistent with previous Drosophila research (dis-
diets in both sexes. However, these nutrients had sex- cussed below) and insect work more broadly, where
specific effects on reproduction; male daily siring egg or offspring production is optimised on diets that
success was maximized on low protein, high carbo- are more balanced in their P:C ratio. For example, egg
hydrate diets but female daily fecundity was maxi- production is greatest on a P:C ratio of between 1P:1C
mized in flies fed diets richer in protein. The general (Fanson and Taylor 2012) and 1P:2.3C in Queensland
topography of the nutrient landscapes we created are fruit flies (Fanson et al. 2009), 1P:2.5C in marula fruit
similar to those created using the CAFE approach, but flies (Malod et al. 2017), 1P:1C in the Australian black
the absolute values for lifespan and reproduction we field cricket (Maklakov et al. 2008; Rapkin et al. 2017)
report were generally higher—particularly for and 3P:1C in the field cricket G. veletis (Harrison et al.
females. While quantifying food intake is key to 2014). The positive effects of protein on female
tackling many research questions, our results show reproductive success likely reflects that in many
that it is possible to create high resolution and insects, protein helps stimulate oogenesis and regulate
informative nutritional landscapes, while avoiding vitellogenesis (Wheeler 1996). However, there are
the limitations of CAFE approach. exceptions to this general pattern; in the cockroach
In agreement with a wealth of previous research, Naupheota cinerea female clutch size improves with
lifespan and reproduction depended on both the total carbohydrate consumption and is independent of
amount and specific blend of nutrients that individuals

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140 Biogerontology (2022) 23:129–144

protein consumed during adulthood (Bunning et al. diets and thus avoid using the CAFE approach, which
2016). reduces D. melanogaster fitness (Lee et al. 2008;
Male fertility does not appear to rely so heavily on Jensen et al. 2015). Rather than measuring dietary
protein consumption. In the insects studied to date, intake, the standard method to create nutrient land-
male reproductive effort is typically maximised on scapes (Simpson and Raubenheimer 1995), we created
low protein, high carbohydrate diets (Maklakov et al. landscapes by mapping phenotype (lifespan, daily
2008; Jensen et al. 2015; Rapkin et al. 2017). Although reproductive effort and total reproductive effort) to the
once more there are exceptions to this general trend; protein and carbohydrate content of 40 experimental
protein consumption increases sexual signaling in the diets to achieve a broad, detailed coverage of the
field cricket G. veletis (Harrison et al. 2014), courtship nutrient space. We find that dietary optima for the
activity in male sticklebacks (Gasterosteus aculeatus) traits assessed here were in similar regions of the
(Moatt et al. 2019) and testes mass in mice (Solon-Biet nutrient landscape to previous work using the CAFE
et al. 2015). In the current study, males conformed to method. For example, the dietary optima for lifespan
the more general insect trend, maximizing their here (1P:15.93C in males, 1P:15.88C in females) are
lifespan and reproductive output when fed the same almost identical to those identified by Jensen et al.
low protein, high carbohydrate nutrient blend. Accord- (2015) (1P:16C), despite the two studies using different
ingly, we find that females experienced a nutrient- nutrient ratios and concentrations. The dietary optima
based trade-off between lifespan and daily reproduc- identified here are even similar to estimates from
tive investment (different nutrient blends increase studies using different diet formulations (e.g. where
expression of each trait) but males did not. yeast is used a protein source), although there is some
Males might not always manage to avoid a dietary sex-specific variation between some studies (Lee et al.
mediated trade-off between survival and reproductive 2008; Bruce et al. 2013; Kim et al. 2020; Skorupa et al.
rates. For males to fertilize ova they often need to 2008).
outcompete their rivals, court females via energeti- Dietary optima for male reproductive traits
cally expensive displays and transfer functional sperm observed here are similar to those identified by Jensen
and seminal fluid (Hosken et al. 2019; Archer and et al. (2015) (i.e. daily offspring sired optimised at
Hosken 2021). Each of these reproductive traits may 1P:12.78C versus 1P:16C). Although both estimates
be optimised on different nutritional blends. In differ from the 1P:2C ratio that optimised male
cockroaches for example, high carbohydrate intake reproductive output in work by Reddiex et al. (2013)
allows males to invest heavily in pheromones that and the 1P:9C estimate from Morimoto & Wigby
increase their attractiveness (South et al. 2011), but (2016). These other studies tested how diet affects
high protein intake improves sperm numbers (Bunning male reproduction in the days following adult eclo-
et al. 2015). This means that the nutrient blend that sion, while Jensen et al. (2015) and our present study
increases success in pre-copulatory sexual selection tested these effects over the entire life-course. In fact,
differs from the diet that promotes male success in the nutrient ratio that maximised offspring production
post-copulatory sexual selection. Accordingly in young male flies here (i.e. 5 days post-eclosion)
whether (and how much) dietary optima diverge was * 1P:2C. This suggests that the nutrient blend
between longevity and reproductive effort, depends associated with high male reproductive output in the
on the traits in which males must invest to produce short term may differ from that generating higher
offspring (Moatt et al. 2019). This almost certainly reproductive output over the entire life-course. Once
differs between species and environments that vary in again, understanding nuanced effects of diet on male
the intensity of male-male competition before and reproductive capacity relies on collecting more data
after mating (Kokko and Rankin 2006). It will remain linking nutrition and reproduction in both sexes, in
unclear how often males avoid a lifespan-reproductive more species.
trade-off until we better quantify how reproductive Finally, the dietary optima we identify for female
effort is affected by diet in both sexes in a broad range daily fecundity (1P:1.16C) are similar to the 1P:2C ratio
of animal taxa. identified by Jensen et al. (2015), as well as earlier
A secondary aim of our work was to create nutrient work in D. melanogaster using yeast as a protein
landscapes using fully chemically defined, agar-based source (Lee et al. 2008). Our results diverge from

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previous published estimates when considering total generations of lab adaptation etc.), but this suggests
(rather than daily) reproductive success in females, that there is further room for diet optimisation. In
which we find is greatest at a higher protein intake than particular, male fertility values in the current study are
in previous work. We suspect that this is because low– a possible explanation for this that warrants
particularly pronounced improvements in fecundity in investigation is cytoplasmic incompatibility (Werren
females fed agar based foods (discussed below), drive et al. 2008) given that female fecundity (measured as
dietary optima for total reproductive success towards egg counts) was high, while male fertility (measured
higher protein nutrient blends. as offspring sired in competitive assay) was low and
While the nutrient blends that optimise lifespan and the Wolbachia status of stocks was not known.
reproduction are similar between studies, female Measuring consumption is vital to testing how
fitness was substantially higher in flies fed agar diets. individuals self-regulate their dietary intake and to
For example, females fed the optimal agar diet for detect compensatory feeding. The CAFE method
lifespan lived around 11.67 days longer than females remains the best way to do this on a large scale
fed the optimal diet for lifespan in Jensen et al. (2015). (Deshpande et al. 2014). Accordingly, the conclusion
Egg laying rates are also consistently high in the that despite costly effects on fitness the CAFE
current study. These improvements suggest that agar approach does not appear to skew the qualitative
based diets do not have the costly phenotypic effects in relationship between nutrients and phenotype is reas-
females that are characteristic of the CAFE approach. suring. However, we show that it is possible to create
While males fed the optimal agar diet for lifespan an informative, high resolution nutrient landscape
survived for 8.2 days longer than individuals fed the without measuring food consumption. When might it
optimal liquid diet in Jensen et al. (2015), for some be more appropriate to adopt this alternative
equivalent P:C ratios flies lived longer on the CAFE approach? There are clear scenarios where it is not
diets than on agar based foods. If flies cannot consume appropriate to measure consumption using a technique
CAFE diets as readily (Moatt et al. 2020), it may be that reduces fitness—for example when characterising
easier to overconsume suboptimal nutrient blends how nutrition affects detailed demographic measures
when fed agar diets. This would explain why males fed of aging. More generally, there may be merit in
optimal P:C ratios perform better on agar diets, but investing the time saved from measuring dietary
these benefits are lost as diets become more imbal- intake towards increasing sample sizes for researchers
anced. While this idea is speculative, it raises the interested in phenotypes that require large sample
possibility that use of the CAFE approach may have sizes (e.g. calculating age-dependent mortality rates,
concealed the magnitude of effects of nutritional characterising behaviour). However, the most obvious
imbalances on phenotype. More work is needed to test case where it makes sense not to measure consumption
this idea. Further, male fertility was broadly equiva- is in species that live, excrete on and reproduce in their
lent in the current study and in Jensen et al. (2015). own food. Drosophila are an example of this but so are
This may reflect the influence of the agar based diets. nematodes and numerous pests of stored products (e.g.
Overall, it is hard to reconcile consistently positive flour beetles—House et al. 2016). Perhaps creating
effects on females of agar based diets with more landscapes without measuring consumption will
equivocal impacts on males. Moreover, while we see enable researchers to expand the taxonomic scope of
pronounced improvements in fitness traits in the research using the Geometric Framework? Although if
current work relative to flies fed via the CAFE individual nutrient intake is not assayed then it is vital
approach, our fitness trait values are still on the low that a larger number of experimental diets are used to
side—virgin females from the Genetic Reference cover nutrient space with sufficient resolution to
Panel lines (DGRP) live for 55.28 days (Ivanov produce a meaningful landscape. Ultimately, the
et al. 2015), which is around 20 days longer than the advent of tools that allow us to better measure dietary
average lifespan of flies fed that diets best for survival intake of agar-based foods may mean we do not have
here. There may be many reasons for this (e.g. our flies to choose between the costs and benefits of either
were mated and mating can be costly in D. approach. In the interim, it is reassuring that the
melanogaster, we assayed flies on novel experimental relationship between food and phenotype is strikingly
diets rather than the foods they have adapted to over

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Publisher’s Note Springer Nature remains neutral with
nutritional bases of aging. Gerontol Ser A 62:707–713.
regard to jurisdictional claims in published maps and
https://doi.org/10.1093/gerona/62.7.707
institutional affiliations.
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