the neonate nutrition hypothesis: early feeding affects the body stoichiometry of daphnia offspring

The neonate nutrition hypothesis: early feeding affects thebody stoichiometry of Daphnia offspring

MARCUS LUKAS*, PAUL C. FROST† AND ALEXANDER WACKER*

*Department of Ecology and Ecosystem Modelling, Institute of Biochemistry and Biology, University of Potsdam, Potsdam, Germany†Department of Biology, Trent University, Peterborough, ON, Canada

SUMMARY

1. Aquatic herbivores consume variable quantities and qualities of food. In freshwater systems,

where phosphorus (P) is often a primary limiting element, inadequate dietary P can slow maternal

growth and reduce body P content. There remains uncertainty about whether and how dietary effects

on mothers are transferred to offspring by way of egg provisioning.

2. Using the keystone herbivore Daphnia, we tested a novel explanation (the ‘neonate nutrition

hypothesis’) to determine whether the early nutrition of newborns affects their elemental composition

and whether the indications of differences in maternal P nutrition found previously might be overes-

timated.

3. We thus examined the P content of mothers and their eggs from deposition through development

to the birth of neonates. We examined further whether very short periods of ingestion (3 h) by the

offspring alter the overall P content of juvenile Daphnia.

4. We showed that strong dietary P effects on mothers were not directly transferred to their eggs.

Irrespective of the supply of P in the maternal diet, the P content of eggs in different developmental

stages and in (unfed) neonates did not differ. This indicates that Daphnia mothers do not reduce the

quality (in terms of P) of newly produced offspring after intermittent periods (i.e. several days) of

poor nutrition. In contrast, the P content of neonates reflected that of their food after brief periods of

feeding, indicating that even temporary exposure to nutrient poor food immediately after birth may

strongly affect the elemental composition of neonates.

5. Our results thus support the neonate nutrition hypothesis, which, like differential maternal provi-

sioning, is a possible explanation for the variable elemental quality of young Daphnia.

Keywords: ecological stoichiometry, food quality, maternal effects, nutrient limitation, zooplankton

Introduction

Animals have the ability to alter their life history in

response to changing environmental conditions by vary-

ing the allocation of resources (Stearns, 1992, 2000). For

example, some Daphnia species produce larger eggs

when food quantity is low, probably because neonates

derived from larger eggs survive longer without food

(Gliwicz & Guisande, 1992; Guisande & Gliwicz, 1992).

Studies of life history traits like somatic growth, devel-

opment time, age and size at maturation and reproduc-

tive effort bridge the gap between physiology at the

individual level and demography at the population level

(Urabe & Sterner, 2001) and are at the heart of theoreti-

cal predictions of population dynamics of consumers

and their prey (Gurney et al., 1990; McCauley et al.,

1990). Despite this importance, there remains much to

be learned about how resource allocation from mother

to offspring changes in response to poor food quality.

Uncertainty about resource allocation remains, even

for the relatively well-studied aquatic herbivore,

Daphnia. Past investigations of Daphnia have largely

focussed on understanding how resources are allocated

between somatic tissues and reproduction. While it is

clear that the number or mass of eggs produced

decreases with declining quality of the diet (e.g. Sterner

Correspondence: Marcus Lukas, Department of Ecology and Ecosystem Modelling, Institute of Biochemistry and Biology, University of

Potsdam, Am Neuen Palais 10, Potsdam D 14469, Germany. E-mail: [email protected]

© 2013 John Wiley & Sons Ltd 2333

Freshwater Biology (2013) 58, 2333–2344 doi:10.1111/fwb.12213

& Hessen, 1994; DeMott, 1998), how phosphorus (P)

flows into reproductive tissue and the production of

eggs is less certain. In contrast, changes in embryo

carbon (C) content during the ontogenesis of eggs are

well described (Urabe & Watanabe, 1990; Glazier, 1991;

Boersma, 1995). Data on the P content of Daphnia eggs

are largely absent, particularly for mothers consuming

diets varying in P content (see Faerøvig & Hessen,

2003). The few studies of the response of eggs and neo-

nates to differences in P content of the maternal diet

have reported quite variable results. While some have

found a constant P content in eggs (although usually

only a single egg development stage has been studied)

with decreasing P supply (e.g. Faerøvig & Hessen,

2003; Becker & Boersma, 2005), others have observed a

reduced P content in the recently released offspring of

P-stressed Daphnia (DeMott, Gulati & Siewertsen, 1998;

Boersma & Kreutzer, 2002; Frost et al., 2010). Here, we

examine an additional explanation (the ‘neonate nutri-

tion hypothesis’) for the variable P content of neonate

Daphnia. Our objective was to determine the effects of

early nutrition on the body P content of newborns

before and just after release.

Because offspring receive nutrients directly from their

mother during egg production (Rossiter, 1996; Mitchell

& Read, 2005), differences in the P content of neonates

have been assumed to result from maternal nutrition

(e.g. DeMott et al., 1998; Frost et al., 2010). However, the

strength of those effects (i.e. the P content of neonates

being determined by maternal nutrition) could also

reflect the nutritional environment of neonates immedi-

ately after birth. If so, the effect of maternal nutrition

might be less than previously reported (e.g. Frost et al.,

2010) or not always manifested by different P allocation

in deposited eggs. Therefore, we tested three compo-

nents of our ‘neonate nutrition hypothesis’ (NNH) : (i)

over short time periods (3 days), the P supply in the

maternal diet would affect the mother’s own somatic

tissue but not that of her eggs, (ii) egg P content would

not change during egg development, but egg C content

would. This would lead to recently described differences

between the P : C ratio of eggs and neonates [compare

e.g. Faerøvig & Hessen (2003) and Becker & Boersma

(2005) with DeMott et al. (1998) and Boersma & Kreutzer

(2002)], and (iii) fresh neonates exposed to different die-

tary P supply would have different body P content. This

last component would not apply to neonates hatched

under starved conditions because they would not ingest

any food that would alter their body chemistry. We

suggest our hypothesis as a means for resolving, at least

partially, the differences between direct measurements

on eggs and these on newborns. Therefore, we examined

whether Daphnia magna alters P provisioning to the

different tissues (somatic or egg tissue) when the dietary

P supply decreases over a 3-day period. More specifi-

cally, we analysed the body P content and P : C ratio of

eggs (in the first egg stage) and somatic tissues of

Daphnia fed different amounts of dietary P for 3 days.

Further, we investigated the carbon and phosphorus

content of eggs from development until birth and,

finally, examined whether the very short periods of

feeding (3 h) can influence the overall body P content of

the newly released animals.

Methods

Organisms

The stock culture of Daphnia magna was raised in

filtered lake water (0.2-lm pore-sized membrane filter)

with saturating amounts of the P-replete cultured green

alga Scenedesmus obliquus (SAG 276-3a, culture collec-

tion Goettingen, Germany) provided for food. For the

growth experiment, the highly ingestible and non-toxic

cyanobacterium, Synechococcus elongatus (SAG 89.79)

(von Elert, Martin-Creuzburg & Le Coz, 2003), was

used as food for D. magna. Synechococcus elongatus

(SYN) was cultured in aerated 2-L flasks containing

WC medium with vitamins (Guillard, 1975) and

diluted daily (dilution rate 0.2 day�1) to ensure nutri-

ent and phosphorus (P) repletion. SYN was maintained

at an illumination of 40 lmol m�2 s�1 using a 16-h/8-h

light/dark cycle. For P-limited S. elongatus (SYN P-), an

aliquot of previously P-sufficient cultured SYN was

transferred to P-free WC medium and cultivated until

molar P : C ratios reached at least 1 mmol mol�1. KCl

(100 lmol L�1) was added to P-free WC medium in

order to prevent a limitation by potassium (K) due to

the omission of the K and P containing stock solution

(50 lmol L�1 K2HPO4). Only SYN P- was used in the

experiments and pulsed with P, according to Roth-

haupt (1995) and Boersma (2000), shortly before prepa-

ration of the different food P levels to prevent

potential indirect P effects. Such could be changes in

fatty acid composition or different digestibility, caused

by different culture conditions (Boersma, 2000; Ravet &

Brett, 2006) which occur especially in green algae. The

effect of direct P limitation could then be much lower

than that of indirect P limitation (Ravet & Brett, 2006).

All organisms were raised at 20 °C.

To classify eggs at different development stages, we

used a system developed by Threlkeld (1979). This

© 2013 John Wiley & Sons Ltd, Freshwater Biology, 58, 2333–2344

2334 M. Lukas et al.

system classifies five different stages of Daphnia egg

development in which the embryos develop from a

spherical stage I, without any morphological differentia-

tion, to stage II when they lose their egg membrane and

build up their antennae. After the embryos develop two

red eyes in stage III, these eyes change colour to brown

in stage IV and merge into a single eye when they are

completely developed neonates in stage V.

Experimental procedure

We used third-clutch neonates for our experiments, fed

on cyanobacteria with different P content. The cyanobac-

teria lacked sterols and polyunsaturated fatty acids

(PUFA) and, to ensure growth and reproduction of

Daphnia (Sperfeld & Wacker, 2011), were enriched with

liposomes containing cholesterol (final concentration

14.2 lg cholesterol per mg C) and eicosapentaenoic acid

(EPA, final concentration 6.4 lg EPA per mg C). The

liposomes were prepared according to Wacker &

Martin-Creuzburg (2012). Throughout the experiment,

Daphnia were transferred daily into jars with renewed

food suspensions.

To analyse the P content of maternal somatic tissue

and eggs separately, we dissected eggs from the brood

pouch using a gentle stream of water from an extended

Pasteur pipette that was carefully inserted into the

brood pouch of the animals (Wacker & Martin-

Creuzburg, 2007), which were not injured by the proce-

dure. The eggs of all mothers of each replicate (a repli-

cate consisted of a 300-mL jar with three mothers each)

and the mothers themselves were transferred separately

into pre-weighed aluminium boats. After drying for

48 h at 50 °C, Daphnia and eggs were weighed on an

electronic balance (�1 lg; CP2P, Sartorius, Goettingen,

Germany) and the dry mass determined. After weighing,

the aluminium boats of mothers and eggs of each

replicate were used for the analysis of P content.

Preparation of food

For the experiments on tissue P content and the effects

of short-term ingestion by adults, we used filtered lake

water containing little dissolved P, which was further

depleted by adding (and later removing) a P-limited

culture of green algae to the water (Lukas, Sperfeld &

Wacker, 2011). For the experiment on egg provision

and neonate nutrition, we used P-free artificial Daphnia

medium (ADaM, Kluettgen et al., 1994). Before the daily

preparation of food suspensions, SYN P- (P : C <

1 mmol mol�1) was incubated for 40 min with

P (75 lmol L�1 K2HPO4). An incubation time of 40 min

was sufficient for SYN P- to obtain constant high P : C

ratios (Lukas, Sperfeld & Wacker, 2011) resulting in a

change from P-deficient into P-sufficient SYN (hereafter

designated as SYN P*). After P incubation, P concentra-

tions of SYN P- and SYN P* were subsequently deter-

mined daily on duplicate saved samples. Target P : C

ratios were achieved by mixing SYN P- and SYN P*

(P : C ratios in Table 1) in calculated proportions using

the previously determined P concentrations. Thereby,

the daily mixed P : C ratios varied by no more than 5%

among daily preparations and did not differ from the

target P : C ratios, which we tested twice during the

experiment. To integrate the dietary P concentration

over the whole experiment, we calculated the mean of

the daily mixed P : C ratios.

The daily prepared food suspensions started with an

equal food concentration (2 mg cyanobacterial C L�1),

which was estimated from photometric light extinction

(800 nm) using previously determined carbon-extinction

equations. Before preparation of food suspensions, sam-

ples of SYN P- and SYN P* were filtered onto pre-

combusted glass fibre filters (25 mm, GF/F, Whatman)

and dried for later analysis of particulate organic carbon

using a carbon analyser (HighTOC+N, Elementar, Ha-

nau, Germany) to calculate actual P : C ratios.

Tissue P content

Daphnia used for the experiment were raised in lake

water with S. obliquus for 6 days from birth. When they

released eggs of their first clutch into the brood pouch,

we transferred these animals into six dietary P levels of

SYN for 3 days. Because their resources had recently

been allocated to reproduction alone when animals had

very recently released their new eggs (Tessier &

Goulden, 1982), and the almost empty ovaries could be

investigated visually under a stereomicroscope, we

Table 1 P:C ratios (mean � 1 SD) of P-limited cultured SYN (SYN

P-) and with P incubated SYN P- (SYN P*) in all three experiments

P : C SYN P-

(mmol mol�1)

P : C SYN P*(mmol mol�1)

Tissue P contents

(duration n = 9 days)

0.71 � 0.05 7.46 � 0.58

Egg provision and

neonate nutrition


0.91 � 0.06 6.40 � 0.29

Short-term (3-h) ingestion


0.70 � 0.03 7.16 � 0.37


Neonate nutrition hypothesis 2335

assumed that the second clutch (used for our analysis)

had largely been based on our experimental diets. The

three lower P treatments were replicated four times, the

higher P treatments three times. Each replicate started

with three animals per separate jar containing 250 mL of

food suspension. The experiment was terminated after

3 days, when the neonates of the first clutch were

released and mothers released the second clutch into the

brood pouch. These eggs of the second clutch, which

were all in the spherical egg stage I (Threlkeld, 1979),

and their mothers were then analysed for P content as

described below.

Egg provision and neonate nutrition

Daphnia used for this experiment were raised in lake

water with S. obliquus from birth for 6 days until they

released the eggs of the first clutch into the brood

pouch. The animals released their first clutch between

zero and 12 h; therefore, we checked Daphnia twice a

day, initially, when the first animals approached first

clutch release and again when all animals had released

their eggs. Subsequently, we transferred the mothers to

three different P levels of the diet (P- c. P : C

1.5 mmol mol�1, P med c. P : C 2.8 mmol mol�1, P+ c.

P : C 6.4 mmol mol�1) for 3 days. Each P treatment was

replicated 19 times, starting with three animals in each

jar containing 250 mL of food suspension. We used four

replicates each for every egg stage tested (I, III/IV, V

and neonates). Three further replicates of each P treat-

ment were used to test the effect of the short-term inges-

tion by neonates. When mothers released the second

clutch into the brood pouch (the neonates of the first

clutch were released previously), we sampled the first

four replicates of each P treatment and collected the

eggs in egg stage I and the respective mothers (see

Experimental procedure for further information). The

mothers in the remaining jars were kept at their respec-

tive P supplies until we collected eggs in egg stage III/

IV and egg stage V over the following days. To deter-

mine the effect of the short-term feeding by neonates on

their P content, we starved mothers carrying the last egg

stage (three replicates of each P treatment). Conse-

quently, neonates that hatched between time zero and

6 h could not have ingested any food for the previous

3 h, on average. At the same time, mothers in four repli-

cates of each P treatment were allowed to release their

neonates with (maternal) food present; hence, these neo-

nates were allowed to consume food for an average of

3 h each. We then analysed the P and C contents of the

eggs and the neonates as described below.


In this experiment, we pre-conditioned Daphnia on food

with five different P treatments from birth for 9 days.

Each dietary P treatment of SYN was replicated eight

times, each replicate with nine animals in a jar contain-

ing a 300 mL food suspension. To quantify the influence

of short-term ingestion on the overall P (and C) contents

of the animal, we separated the animals within each die-

tary P availability into two subsamples (n = 4 for each)

after the pre-conditioning phase: (i) animals directly col-

lected from their treatment food and (ii) transferred into

P-deficient food (2 mg C L�1 SYN, P : C c. 1 mmol

mol�1) for 3 h prior to collection. Within these 3 h, the

animals of the second subsample ingested P-deficient

food rather than their treatment food (i.e. different diets

with different P : C ratios). Therefore, we determined

the ingestion rates of the animals by measuring the

change in the optical density (720 nm, UVmini-1240,

Shimadzu, China) of the food suspension within the 3 h.

Assuming that the initial carbon concentration of

2 mg C L�1of SYN corresponded to the measured opti-

cal density at the beginning of the 3 h, we calculated the

final carbon concentration by knowing the optical den-

sity after 3 h. Ingestion rate (IR, lg C ind.�1 h�1) was

calculated using eqn (1) of Helgen (1987):

IR ¼ Cstart � Cend

Dt� V

Nð1Þ

where Cstart is the initial carbon concentration (in

mg C L�1), Cend is the final carbon concentration after

3 h, t is the time (h), V is the culture volume (mL), and

N is the number of animals. All animals collected were

weighed for dry mass and analysed for body P.

To estimate the amount of carbon ingested, we used

the difference in Daphnia P : C ratios between the two

subsamples (animals with treatment food vs. animals

with P-deficient food for 3 h) at each P level in the food.

Using this difference, and the associated C content of

the animals, we calculated the amount of P ingested.

This amount of P and the respective food P : C ratio

were used to calculate the amount of C ingested for each

dietary P treatment (see Appendix S1 in Supporting

Information for further information).

Analysis

We analysed the P concentration of aliquots of SYN

P- and SYN P* filtered onto membrane filters (Tuffryn,

25 mm, 0.45 lm, Pall Corporation, NY, U.S.A.) and sam-

ples of dried Daphnia. Phosphorus concentration was

determined using the molybdate blue reduction method



(Murphy & Riley, 1962) after dissolving tissues with sul-

phuric acid and an oxidative hydrolysation using

K2S2O8 at 120 °C and 120 kPA by autoclaving.

The carbon content of animals and eggs was calcu-

lated using carbon dry mass ratios determined prior to

the experiment (0.41 and 0.52 lg C lg�1 dry mass,

respectively) or was directly measured using a carbon

analyser (Elemental Analyser Euro EA, HEKAtech, Weg-

berg, Germany).

All statistical analyses including one-way and two-

way ANOVAs, two-way ANCOVAs, Tukey’s HSD post hoc

tests and two-tailed t-tests were performed using the

statistical software package R v.2.5.1., which is under

general public licence (R Development Core Team,

2007). We log-transformed the data of the dietary P sup-

ply for the two-way ANCOVA for the experiment on

effects of short-term ingestion, where we tested whether

there was a difference between the P : C ratios of ani-

mals that ingested P-deficient food for 3 h and those

that were collected and measured directly from their

treatments.

For a better visualisation, we correlated tissue P : C

ratio with the food P : C ratio and fitted the data by a

modified Monod model (Monod, 1950; Wacker & von

Elert, 2001) using eqn (2):

P : C ¼ P : Cmaxc� S0

c� S0 þ KSð2Þ

where P:Cmax is the fitted maximum tissue P : C ratio

(mmol mol�1), c is the resource concentration (food

P : C ratio, mmol mol�1), S0 is the threshold concentra-

tion for a (hypothetical) tissue P : C ratio of zero (mmol

mol�1), and KS is the half-saturation constant (mmol

mol�1).

Results

Tissue P content

P : C ratios of eggs and soma from Daphnia consuming

different dietary P content differed strongly. Both tissue

types generally lay above the 1 : 1 line between tissue and

food P content (Fig. 1). While the P content of Daphnia

mothers (soma without eggs) decreased with decreasing

dietary P, egg P content showed almost no response to

different P diets and remained at 6.3 � 0.4 mmol mol�1

(mean � SD, n = 15) across nearly all the P : C ratios of

the diets provided. We found that the P content of the

eggs was reduced only at the very lowest dietary P avail-

ability (two-sample t-test: t16 = 3.6, P = 0.002). In contrast,

the P : C ratio of the somatic tissue of the animals was

substantially higher than those of eggs when food P : C

ratio was 2.0 mmol mol�1 or higher.

Egg provision at different egg stages

The P : C ratio of mothers was strongly influenced by

the P content of the food (Fig. 2; two-way ANOVA: P

supply: F2,26 = 219.0, P < 0.001) but this was variable

Food P:C ratio (mmol mol–1)(molar C:P ratio)

0 1(1000)

2(500)

3(333)

4(250)

5(200)

6(167)

7(143)

Tis

sue

P:C

rat

io (

mm

ol m

ol –

1 )

0

5

6

7

8

9

10

* * * *

1:1

Fig. 1 P : C ratios (mean � SD) of Daphnia magna mothers

(squares) cultivated in food differing in P supply and their eggs

(triangles) in the first egg stage. Regression lines indicate nonlinear

relationships (see results in Table S1) using eqn (2). ‘*’ means sig-

nificantly differences between mothers and their eggs (P < 0.01,

two-sample t-test). The dotted line represents the 1 : 1 ratio

between tissue and food P contents.

P– P med P+

Mot

her

P:C

rat

io (

mm

ol m

ol–1

)

0

2

4

6

8

10

aba

b

aa aa

bbc

1. egg stage 3./4. egg stage 5. egg stage

Fig. 2 P : C ratios (mean � SD, n = 4) of Daphnia magna mothers

(without eggs) measured at the different egg stages. The animals

were cultivated in food differing in P supply (P- c. P : C

1.5 mmol mol�1, P med c. P : C 2.8 mmol mol�1, P+ c. P : C

6.4 mmol mol�1). Different letters indicate significant differences

among bars within a P treatment (P < 0.05, Tukey’s post hoc test).



among the times that we collected the different egg

stages (egg development stage: F2,26 = 8.5, P = 0.001). We

analysed maternal tissues only until the eggs reached the

fifth egg stage to avoid fluctuations associated with sub-

sequent egg production. In addition to the decrease in

maternal P content when the P content in their food

decreased, body P content decreased further longer they

were fed on P-deficient food. In contrast, the P content of

mothers fed ‘P med’ and P-sufficient food remained

more or less stable (see post hoc tests in Fig. 2). These

differences in body P content of mothers with contrasting

diets were evident from the significant interaction in the

two-way ANOVA (P supply 9 egg development stage:

F4,26 = 5.8, P = 0.002).

Contrary to the mothers, the P : C ratio of eggs did

not respond to P availability in the food, but increased

during egg development (Fig. 3a; two-way ANOVA,

P supply: F2,32 = 0.7, P = 0.50; egg development stage:

F3,32 = 65.2, P < 0.001; egg development stage 9 P

supply: F6,32 = 3.2, P = 0.012). The significant interaction

in this ANOVA indicates that the increase in the P : C ratio

of the eggs varied with P content of the diet. In the fol-

lowing, we used the ‘C or P per egg’ unit, which

allowed us to distinguish the separate effects on P and

C contents. In other words, we were interested in the

decline in carbon due to respiration or other carbon con-

sumptive processes. The P content per egg did not vary

throughout egg development or among different dietary

P treatments (Fig. 3b; two-way ANOVA, egg development

stage: F3,32 = 1.5, P = 0.23; P supply: F2,32 = 0.1, P = 0.87;

egg development stage 9 P supply: F6,32 = 1.8, P = 0.13).

Therefore, the reason for the changes in the P : C ratio

of eggs was based on changes in C content. This

decreased from 3.9 (�0.33) to 2.6 (�0.40) lg C per egg

(mean � SD, N = 12, respectively N = 9) during devel-

opment from egg stage one to neonate (two-way ANOVA,

egg development stage: F3,32 = 46.8, P < 0.001; P supply:

F2,32 = 0.1, P = 0.94; egg development stage 9 P supply:

F6,32 = 1.1, P = 0.40). Since in both two-way ANOVAs (for

P and C contents of the eggs), the effect of P supply as

well as the interaction between egg development stage

and P supply was not significant, we conclude that there

was no influence of dietary P supply on the P : C ratio

of eggs in any of the different egg stages.

Neonate nutrition

To determine the potential effect of ingested food on

neonate body P content, we starved a first subsample of

mothers carrying the last egg stage, while a second

subsample of mothers released their neonates while

provided with food. Consistent with the results from

previous egg measurements, the P : C ratio of starved

neonates did not vary under changing P supply (Fig. 4).

However, the P : C ratio of neonates hatching during an

exposure time of 3 h (on average) to the food of their

mothers increased with greater P availability in the food

(Fig. 4; Table 2). This indicates that relatively short peri-

ods (3 h on average) of feeding are sufficient to alter the

body P content of newborn Daphnia. This effect was

strongest under P-limited conditions, because food P : C

ratio differed most strongly from the P : C ratio of the

neonates (compared with almost equal P content of neo-

nates and food under P-saturated conditions).

Changes in neonate P : C ratio after feeding across the

dietary P gradient were derived from changes in both

the P and C contents per neonate (Fig. 5). First, provid-

ing food affected neonate P content, but only when food

was P-rich (increase by 31–42% compared with starved

animals, one extreme of 80%). Second, supplying food

increased the C content per neonate, both in animals fed

Egg

P:C

rat

io (

mm

ol m

ol–1

)

0

2

4

6

8

10

12E

gg P

con

tent

(ng

P p

er e

gg)

Egg

C c

onte

nt (

µg C

per

egg

)

0

20

40

60

80

P– P med P+0

2

4

6

8

a

b b

a

aab

b

b

aa

bc

c

a

bb

a aa

bb

baba

a

a a aa

a a a

b

aab

ab

(a)

(b)

1. egg stage 3./4. egg stage 5. egg stage Neonate

Fig. 3 Elemental composition (mean � SD) of Daphnia offspring

when their mothers were cultivated in food differing in P supply

(P- c. P : C 1.5 mmol mol�1, P med c. P : C 2.8 mmol mol�1, P+ c.

P : C 6.4 mmol mol�1). (a) P : C ratios and (b) P (uniform bars)

and C contents (striped bars) of eggs at different egg stages (n = 4)

and of neonates (hatched within 3 h on average in jars with no

food, n = 3). Different letters indicate significant differences among

bars within a P treatment (P < 0.05, Tukey’s post hoc test). Note, in

no case did maternal P affect P : C ratios, P and C contents of

developmental stages (see Results).



P-rich (increase between 29 and 60% compared with

starved neonates) and P-deficient food (increase between

8 and 49% compared with starved neonates), but not in

animals grown under medium P conditions.


We measured lower P : C ratios in adult animals which

had been fed P-deficient food for 3 h prior to collection

and compared these to animals that had been collected

directly from the different P treatments of 9 days pre-

conditioning (Fig. 6). The positive relationship between

body P content of D. magna with increasing dietary P

supply was still obvious but had diminished, indicating

that a 3-h period of ingestion could influence the P con-

tent of the body. We found further that when more die-

tary P was available, there was a greater difference

between the P : C ratios of animals which had ingested

P-deficient food for 3 h and those collected directly from

the treatment food they had been pre-conditioned on

(i.e. without further opportunity to ingest food) (two-

way ANCOVA with log-transformed food P : C ratios, pre-

conditioning P supply: F1,36 = 210.8, P < 0.001; animals

directly collected vs. animals collected after additionally

feeding on P-deficient food: F1,36 = 31.8, P < 0.001; inter-

action: F1,36 = 4.8, P = 0.035).

For each P treatment, we used the difference in P : C

ratios between animals collected from the treatment food

they had been pre-conditioned on and those which had

ingested P-deficient food for 3 h prior to collection to

calculate the amount of carbon ingested within 3 h. The

calculated amount of C ingested was not different from

the measured amount ingested within 3 h (see Table S3,

two-way ANCOVA, P supply: F1,35 = 0.6, P = 0.45; calcu-

lated vs. measured C: F1,35 = 1.7, P = 0.20; P sup-

ply 9 calculated vs. measured C: F1,35 = 2.6, P = 0.11).

Furthermore, we found that the proportion of the

amount of C ingested to the overall carbon content of

the whole animal was 15.4 � 3.1% and did not vary

with changing P supply (see Fig. S1).

Discussion

We found that the effects of phosphorus (P) deficient

food on mothers were not transferred to their offspring

by way of reduced P allocation to eggs. Eggs and fresh

neonates, for the most part, did not vary in P content in

Table 2 Results of the linear regressions in Fig. 4

Starved neonates Neonates in food

r² <0.01 0.42

n 9 12

Slope � SE 0.004 � 0.115 0.261 � 0.098

95% CI (�0.269–0.277) (0.043–0.478)t-value 0.1 2.7

P-value 0.98 0.024

Intercept � SE 8.56 � 0.48 6.93 � 0.40

95% CI (7.43–9.69) (6.03–7.83)

t-value 18.0 17.2

P-value <0.001 <0.001

Neo

nate

P c

onte

nt (

ng P

per

neo

nate

)

Neo

nate

C c

onte

nt (

µg C

per

neo

nate

)

0

20

40

60

80

P– P med P+0

2

4

6

8

a

b

b

b

a

a

a

a

a

aa

a

Neonate CNeonate P

Withoutfood

Withfood

Fig. 5 P (solid or open bars) and C contents (hatched bars) of neo-

nates hatched within 3 h on average in jars without food (=neonatestarvation) and neonates hatched within the same time in mothers

food (=neonate food). Different letters indicate significant differences

among bars within a P treatment (P < 0.05, Tukey’s post hoc test).

Food P:C ratio (mmol mol–1)

0 1 2 3 4 5 6 7

Neo

nate

P:C

rat

io (

mm

ol m

ol–1

)

0

6

7

8

9

10

Fig. 4 P : C ratios of neonates hatched within 3 h on average in jars

without food (open circles, dashed line) and neonates hatched

within the same time in mothers food (filled circles, solid line). Each

data point represents all of the offspring from one replicate

(between 15 and 26 neonates). Data for neonates hatched without

food are the same as shown as mean � SD in Fig. 3. Regression

lines indicate linear regressions (results in Table 2). The food P : C

ratio was calculated as the mean P : C ratio of five experiment days.



relation to the supply of P in the maternal diet. On the

other hand, we found that the P : C ratio of Daphnia

eggs increased during embryonic development because

the carbon content decreased within this time. Interest-

ingly, P : C ratio of the newborn offspring varied

strongly with varying P supply of their own food, indi-

cating that ingestion, and probably also assimilation, by

the neonates is probably the primary cause of the vari-

able neonate body P content observed here.

There was evidence supporting the first component of

our hypothesis that maternal feeding for 3 days on a

P-poor diet changed mothers’ own somatic tissue, but

not the P content of their eggs. In our experiments

examining the tissue P content of mothers, P content of

the diet affected maternal P : C ratio strongly, which is

consistent with other studies on cladocerans (DeMott

et al., 1998; Acharya, Kyle & Elser, 2004; DeMott & Pape,

2005). We suggest that below a certain dietary P : C

threshold, close to that previously found (P : C ratio of

3.7 mmol mol�1, Lukas et al., 2011), maternal tissue

P : C ratio decreases with a declining supply of P in the

diet. Moreover, this threshold P : C ratio is in the range

of threshold elemental ratios for growth reported previ-

ously (Sterner & Hessen, 1994; Anderson & Hessen,

2005).

In contrast to the variable maternal P : C ratio, the

P : C ratio of eggs in the first stage showed almost no

response to dietary P and thus is consistent with reports

of a constant P content of eggs (in stage one) with

decreasing P supply (Faerøvig & Hessen, 2003; Becker &

Boersma, 2005). This largely corroborates the first part of

our hypothesis that there is no effect of different P sup-

ply in the maternal diet on egg P content. These results

are for mothers experiencing acute P starvation (i.e. with

a P-deficient diet in the 3 d before egg deposition),

which differs from studies of mothers grown under

chronic P starvation (e.g. DeMott et al., 1998; Frost et al.,

2010), where dietary P was limited from birth. Hence,

whether the P content of deposited eggs varies with

maternal diet may depend on the duration of P-stress

experienced by the mother. Moreover, rapid changes in

P depletion in dynamically mixing water columns (e.g.

within 48 h in Einarsson et al., 2004) may determine the

duration of nutritional stress on zooplankton across

short timescales. Hence, speed of appearance, duration

and speed of disappearance of P limitation appear to

interact with its severity and should be studied further.

We found the P content of embryos to remain stable

during development through the five egg stages. This

corroborates the second component of our hypothesis

that there would be no differences in egg P content dur-

ing egg maturation. However, the P : C ratio of newly

deposited eggs (5.9 � 0.4 mmol mol�1, mean � SD,

n = 12) was quite low compared with neonates

(8.6 � 0.7 mmol mol�1 mean � SD, n = 9) (present

study, see also Faerøvig & Hessen, 2003). This pattern of

an increasing P : C ratio during egg development is

likely to have resulted from the respiratory loss of

carbon within the embryo (Urabe & Watanabe, 1990;

Glazier, 1991; Boersma, 1995). Assuming a respiratory

quotient of 1.16 (Lampert & Bohrer, 1984; Bohrer &

Lampert, 1988), we converted our measured carbon loss

during egg development until birth into a respiration

rate, following Urabe & Watanabe (1990). This estimate

of respiration was 6.4 lL O2 mg C�1 h�1 during this

time period. This is slightly less than the respiration rate

of D. galeata embryos found by Urabe & Watanabe

(1990) and a little above that found for D. magna

embryos by Glazier (1991) and for D. galeata by Boersma

(1995). A higher estimate of respiration may be due to

the inclusion of non-respiratory losses, such as the shed-

ding of egg membranes (Glazier, 1991), and possibly an

increased carbon loss during starvation of neonates. As

eggs receive no further resources from the mother after

deposition, our results indicate that P, but not C, is

retained efficiently in the developing embryo.

Once neonates start to ingest food, ambient food qual-

ity should reassert itself as a determinant of neonate

body stoichiometry. If so, the longer the newly hatched

Food P:C ratio (mmol mol–1)

0 2 3 4 5 6 7

Som

atic

tiss

ue P

:C r

atio

(m

mol

mol

–1)

0

6

7

8

9

10

1:1

Fig. 6 P : C ratios (mean � SD, n = 4) of adult Daphnia magna

pre-conditioned for 9 d on food with different P:C ratios. After

pre-conditioning, we allowed a subsample of the animals to ingest

P-deficient food (P : C c. 1 mmol mol�1) for 3 h; animals directly

collected from their treatment food (filled squares), animals with

P-deficient food for 3 h (open squares). Regression lines indicate

nonlinear regressions (results in Table S2) using eqn (2). The dotted

line represents the 1 : 1 ratio between tissue and food P contents.



individuals are exposed to the environmental food

source, the more the body P content of the neonates at

the time of sampling should diverge from their original

body P content. This difference would originate from

the uptake of food of contrasting P content into the

small-sized body of a Daphnia neonate. Neonates

released when food is high in P might increase their

P : C ratio rapidly, whereas the P : C ratio of neonates

taking P-deficient food might decrease. We assume that

young Daphnia can quickly assimilate P from the food

ingested because: (i) Daphnia has a high P assimilation

efficiency under P limitation (e.g. DeMott et al., 1998)

and (ii) the amount of unassimilated food in the gut is a

negligible fraction of that ingested over 3 h (Lampert,

1977a,b). Our study showed that as long as neonates do

not experience the maternal food with a different P con-

tent, eggs and neonates of D. magna have the same P

content per capita, independently of their egg develop-

ment stage and the maternal dietary P supply. In con-

trast, the P : C ratio of neonates exposed to the maternal

food supply declined with reduced P availability in that

food. This resulted because of an increased body C con-

tent but no change in body P content. However, since

both P and C increased in equal proportion when neo-

nates were fed P-rich food, we found no difference

between the P : C ratio of starved neonates and those

which ingested food. Consequently, our results indicate

that differentially nourished mothers can provide eggs

with the same amount of P, and the differences

observed recently in the P content of newborn offspring

of Daphnia (DeMott et al., 1998; Boersma & Kreutzer,

2002; Frost et al., 2010) may partly be caused by the

ingestion of the maternal food differing in P content

rather than solely by maternal (provisioning) effects. In

these latter studies, neonates were allowed to ingest the

maternal food for a period of between 3 and 24 h.

Both the neonate nutrition experiment (Figs 4 & 5)

and the short-term (3 h) ingestion experiment with

adults (Fig. 6) suggest that food ingested within a short

period after birth contributes to the total P and C con-

tents of neonates. While Daphnia P : C ratio was strongly

affected by P-limited food in the neonate nutrition

experiment, we found the strongest effect on Daphnia

P : C for P-saturated food in the short-term ingestion

experiment. This result is likely to reflect differences in

methods between the two experiments. In the neonate

nutrition experiment, neonates ingested food (with

different P supply) for the first time in their lives.

Because neonates actually were P saturated (see P con-

tent of eggs), their P content was affected more strongly

by ingesting P-deficient food rather than P-rich food

(which was similar to their own body P content). In con-

trast, in our short-term ingestion experiment, adult

Daphnia were pre-conditioned on different dietary P

availabilities and then were transferred to P-deficient

food for 3 h. Here, the P content of new food matched

the P content of Daphnia fed previously on P-limited

food. Hence, we found no effect for the P-limited cul-

tured animals. Rather, the effect was high when animals

had been fed on P-saturated food during pre-condition-

ing (which was largely different to the new P-limited

food). Consequently, both experiments clearly show the

strong effect of short periods of ingestion on the elemen-

tal composition of small animals such as Daphnia.

The differences between the P content of adult ani-

mals that had fed on P-deficient food for 3 h and those

which were collected directly from their treatment food

(see short-term ingestion experiment) were used to esti-

mate the carbon ingested as a proportion of the overall

carbon body content. This yielded an estimate of about

15% of total C of an adult Daphnia that was ingested

within 3 h. We cannot state whether all of the carbon

ingested was also assimilated, but the assimilation

efficiency of S. elongatus is quite high (Arnold, 1971;

Lampert, 1977a,b). Assuming this high assimilation effi-

ciency, and abundant food, a Daphnia would typically

ingest about 100% of its body mass per day (e.g. DeMott

et al., 1998; Darchambeau, Faerøvig & Hessen, 2003).

Hence, 3 h of feeding should equal ingestion of about

15% of body mass of food, which agrees with our

results.

We used a two-member mixing model, following

Frost, Hillebrand & Kahlert (2005), to see whether we

can transfer our result derived from adult D. magna to

neonates (see Appendix S2 in Supporting Information).

We conclude that at least 20%, and up to 30%, of the

mass of a neonate could be derived from ingestion dur-

ing the first 3 h. This was corroborated by the results of

our neonate nutrition experiment in which the carbon

content of a neonate increased by a minimum of 8% to a

maximum of 60% (32 � 19%, mean � SD, N = 12),

when the neonates fed for a mean time of 3 h. From our

estimation of ingestion rates (30% and 15% of body mass

within 3 h for neonates and adults, respectively), we

expect that the early nutrition of neonates might have an

even greater influence on body P content.

Our study provides good evidence that Daphnia does

not change P allocation to individual eggs, even as a

maternal P content is depleted within a 3-d period. This

time period is shorter than used in other studies, for

example, in DeMott et al. (1998) and Frost et al. (2010),

which makes direct comparison difficult. Nevertheless,



our results reveal that very short periods of ingestion

(3 h after birth) affect the offspring P content, which

was non-variable during development before birth.

While DeMott et al. (1998) concluded that a female diet

strongly influenced the specific P content of the off-

spring and Frost et al. (2010) assumed maternal effects

to be the primary reason for lower P content in the off-

spring of P-stressed Daphnia, we suggest that early neo-

nate nutrition could contribute substantially to the

overall effect of maternal food P content on neonates.

However, the extent to which feeding after birth or

direct maternal effects themselves lead to the subsequent

effects on growth and reproduction shown by Frost et al.

(2010) remains unclear and should be examined by

future work.

In conclusion, our results show the importance of two

issues. First, any measurements of substances in the

body (here P, but also possibly other components of the

food, such as other minerals, fatty acids and sterols)

might be altered by the ingestion of food which differs

from the experimental diet. This effect could be espe-

cially significant for relatively small animals and hence

should need to be accounted for in future studies. How-

ever, the ratio of carbon to the compound measured

would determine the strength of overestimation (i.e. a

low dietary P : C ratio would lead to lower biased mea-

surements compared with food with higher P : C ratios).

Second, acute periods of P limitation are usually not suf-

ficient to reduce the P content of deposited eggs of

Daphnia and may not produce the maternal effects previ-

ously reported.

Thus, it appears that the early nutritional environment

(immediately after birth) of neonates can strongly influ-

ence their elemental composition (and perhaps their

growth rate) and we conclude that apparent differences

in P content of Daphnia offspring may have resulted, at

least to some extent, from variable neonate nutrition.

This result indicates that the location of neonate release

by mothers within the water column could have very

strong effects on neonate performance (i.e. if food P : C

ratios vary spatially). Such spatial heterogeneity of

phytoplankton in lakes occurs within short periods of

time, forced by internal and environmental driving

factors (Serra et al., 2007; Caron et al., 2008; Alexander &

Imberger, 2013). Assuming that spatial heterogeneity in

phytoplankton abundance and species composition

could be correlated with the spatial distribution of nutri-

ents (Dickman, Stewart & Servantvildary, 1993), different

P : C ratios in Daphnia food are very likely. Future work

should examine fine-scale patterns in food P : C ratios

in lakes and whether Daphnia mothers can vary the loca-

tion of neonate release. Moreover, our results indicate

that Daphnia mothers do not reduce quality of the eggs

after a 3-d period of poor nutrition. Acute P starvation

can thus be mediated by female Daphnia by: (i) reducing

the number of offspring in response to nutritional con-

straints and/or (ii) depleting their own body P to the

benefit of their offspring. These strategies may be eco-

logically worthwhile by postponing negative effects of

low P supply onto the next generation when P supply

remains poor. Instead, when P supply recovers quickly,

the strategies are even more promising to sufficiently

compensate the gap in P supply.

Acknowledgments

We thank S. Heim and E. Sperfeld for technical assis-

tance and advice. We also thank A. Hildrew and two

anonymous reviewers for valuable comments on this

manuscript. This study was supported by the German

Research Foundation (DFG, WA 2445/4-1).

References

Acharya K., Kyle M. & Elser J.J. (2004) Biological stoichiom-

etry of Daphnia growth: an ecophysiological test of the

growth rate hypothesis. Limnology & Oceanography, 49,

656–665.

Alexander R. & Imberger J. (2013) Phytoplankton patchiness

in Winam Gulf, Lake Victoria: a study using principal

component analysis of in situ fluorescent excitation spec-

tra. Freshwater Biology, 58, 275–291.

Anderson T.R. & Hessen D.O. (2005) Threshold elemental

ratios for carbon versus phosphorus limitation in Daph-

nia. Freshwater Biology, 50, 2063–2075.

Arnold D.E. (1971) Ingestion, assimilation, survival, and

reproduction by Daphnia pulex fed seven species of blue-

green algae. Limnology and Oceanography, 16, 906–920.

Becker C. & Boersma M. (2005) Differential effects of phos-

phorus and fatty acids on Daphnia magna growth and

reproduction. Limnology and Oceanography, 50, 388–397.

BoersmaM. (1995) The allocation of resources to reproduction

inDaphnia galeata - against the odds. Ecology, 76, 1251–1261.

Boersma M. (2000) The nutritional quality of P-limited algae

for Daphnia. Limnology & Oceanography, 45, 1157–1161.

Boersma M. & Kreutzer C. (2002) Life at the edge: is food

quality really of minor importance at low quantities?

Ecology, 83, 2552–2561.

Bohrer R.N. & Lampert W. (1988) Simultaneous measure-

ment of the effect of food concentration on assimilation

and respiration in Daphnia magna Straus. Functional Ecol-

ogy, 2, 463–471.

Caron D.A., Stauffer B., Moorthi S., Singh A., Batalin M.,

Graham E.A. et al. (2008) Macro- to fine-scale spatial and



temporal distributions and dynamics of phytoplankton

and their environmental driving forces in a small mon-

tane lake in southern California, U.S.A.. Limnology and

Oceanography, 53, 2333–2349.

Darchambeau F., Faerøvig P.J. & Hessen D.O. (2003) How

Daphnia copes with excess carbon in its food. Oecologia,

136, 336–346.

DeMott W.R. (1998) Utilization of a cyanobacterium and a

phosphorus-deficient green alga as complementary

resources by daphnids. Ecology, 79, 2463–2481.

DeMott W.R., Gulati R.D. & Siewertsen K. (1998) Effects of

phosphorus-deficient diets on the carbon and phosphorus

balance of Daphnia magna. Limnology & Oceanography, 43,

1147–1161.

DeMott W.R. & Pape B.J. (2005) Stoichiometry in an ecologi-

cal context: testing for links between Daphnia P-content,

growth rate and habitat preference. Oecologia, 142, 20–27.

Dickman M., Stewart K. & Servantvildary M. (1993) Spatial

heterogeneity of summer phytoplankton and water chem-

istry in a large volcanic spring-fed lake in Northern

Iceland. Arctic and Alpine Research, 25, 228–239.

Einarsson A., Stefansdottir G., Johannesson H., Olafsson

J.S., Gislason G.M., Wakana I. et al. (2004) The ecology of

Lake Myvatn and the River Laxa: variation in space and

time. Aquatic Ecology, 38, 317–348.

von Elert E., Martin-Creuzburg D. & Le Coz J.R. (2003)

Absence of sterols constrains carbon transfer between cy-

anobacteria and a freshwater herbivore (Daphnia galeata).

Proceedings of the Royal Society of London Series B-Biological

Sciences, 270, 1209–1214.

Faerøvig P.J. & Hessen D.O. (2003) Allocation strategies in

crustacean stoichiometry: the potential role of phospho-

rus in the limitation of reproduction. Freshwater Biology,

48, 1782–1792.

Frost P.C., Ebert D., Larson J.H., Marcus M.A., Wagner

N.D. & Zalewski A. (2010) Transgenerational effects of

poor elemental food quality on Daphnia magna. Oecologia,

162, 865–872.

Frost P.C., Hillebrand H. & Kahlert M. (2005) Low algal

carbon content and its effect on the C: P stoichiometry of

periphyton. Freshwater Biology, 50, 1800–1807.

Glazier D.S. (1991) Separating the respiration rates of

embryos and brooding females of Daphnia magna - impli-

cations for the cost of brooding and the allometry of met-

abolic-rate. Limnology and Oceanography, 36, 354–362.

Gliwicz Z.M. & Guisande C. (1992) Family-planning in Daph-

nia - resistance to starvation in offspring born to mothers

grown at different food levels. Oecologia, 91, 463–467.

Guillard R.R.L. (1975) Cultures of phytoplankton for feed-

ing of marine invertebrates. In: Culture of Marine Inverte-

brate Animals, pp. 29–60. (Eds W.L. Smith & M.H.

Chanley), Plenum, New York.

Guisande C. & Gliwicz Z.M. (1992) Egg size and clutch size

in 2 Daphnia species grown at different food levels. Jour-

nal of Plankton Research, 14, 997–1007.

Gurney W.S.C., McCauley E., Nisbet R.M. & Murdoch

W.W. (1990) The physiological ecology of Daphnia - a

dynamic model of growth and reproduction. Ecology, 71,

716–732.

Helgen J.C. (1987) Feeding rate inhibition in crowded

Daphnia pulex. Hydrobiologia, 154, 113–119.

Kluettgen B., Duelmer U., Engels M. & Ratte H.T. (1994)

ADaM, an artificial freshwater for the culture of

zooplankton. Water Research, 28, 743–746.

Lampert W. (1977a) Studies on the carbon balance of Daph-

nia pulex as related to environmental conditions. I. Meth-

odological problems of the use of 14C for the

measurement of carbon assimilation. Archiv f€ur Hydrobiol-

ogie Supplement, 48, 287–309.

Lampert W. (1977b) Studies on the carbon balance of Daph-

nia pulex as related to environmental conditions. II. The

dependence of carbon assimilation on animal size, tem-

perature, food concentration and diet species. Archiv f€ur

Hydrobiologie Supplement, 48, 310–335.

Lampert W. & Bohrer R. (1984) Effect of food availability

on the respiratory quotient of Daphnia magna. Comparative

Biochemistry and Physiology A-Physiology, 78, 221–223.

Lukas M., Sperfeld E. & Wacker A. (2011) Growth Rate

Hypothesis does not apply across colimiting conditions:

cholesterol limitation affects phosphorus homoeostasis of

an aquatic herbivore. Functional Ecology, 25, 1206–1214.

McCauley E., Murdoch W.W., Nisbet R.M. & Gurney

W.S.C. (1990) The physiological ecology of Daphnia -

development of a model of growth and reproduction.

Ecology, 71, 703–715.

Mitchell S.E. & Read A.F. (2005) Poor maternal environment

enhances offspring disease resistance in an invertebrate.

Proceedings of the Royal Society B-Biological Sciences, 272,

2601–2607.

Monod J. (1950) La technique de culture continue. Theorie

et applications. Annales de l’Institut Pasteur, 79, 390–410.

Murphy J. & Riley J.P. (1962) A modified single solution

method for determination of phosphate in natural waters.

Analytica Chimica Acta, 26, 31–36.

R Development Core Team. (2007) R: A Language and Envi-

ronment for Statistical Computing. R Foundation for Statisti-

cal Computing, Version 2.5.1. R Development Core Team,

Vienna, Austria.

Ravet J.L. & Brett M.T. (2006) Phytoplankton essential fatty

acid and phosphorus content constraints on Daphnia

somatic growth and reproduction. Limnology and Oceanog-

raphy, 51, 2438–2452.

Rossiter M.C. (1996) Incidence and consequences of inher-

ited environmental effects. Annual Review of Ecology and

Systematics, 27, 451–476.

Rothhaupt K.O. (1995) Algal nutrient limitation affects

rotifer growth rate but not ingestion rate. Limnology and


Serra T., Vidal J., Casamitjana X., Soler M. & Colomer J.

(2007) The role of surface vertical mixing in phytoplank-



ton distribution in a stratified reservoir. Limnology and


Sperfeld E. & Wacker A. (2011) Temperature- and

cholesterol-induced changes in eicosapentaenoic acid

limitation of Daphnia magna determined by a promising

method to estimate growth saturation thresholds.

Limnology and Oceanography, 56, 1273–1284.

Stearns S.C. (1992) The Evolution of Life Histories. Oxford

University Press, Oxford, U.K.

Stearns S.C. (2000) Life history evolution: successes, limita-

tions, and prospects. Naturwissenschaften, 87, 476–486.

Sterner R.W. & Hessen D.O. (1994) Algal nutrient limitation

and the nutrition of aquatic herbivores. Annual Review of

Ecology and Systematics, 25, 1–29.

Tessier A.J. & Goulden C.E. (1982) Estimating food limita-

tion in cladoceran populations. Limnology and Oceanogra-

phy, 27, 707–717.

Threlkeld S.T. (1979) Estimating cladoceran birth rates -

Importance of egg mortality and the egg age distribution.

Limnology and Oceanography, 24, 601–612.

Urabe J. & Sterner R.W. (2001) Contrasting effects of differ-

ent types of resource depletion on life-history traits in

Daphnia. Functional Ecology, 15, 165–174.

Urabe J. & Watanabe Y. (1990) Influence of food density on

respiration rate of 2 crustacean plankters, Daphnia galeata

and Bosmina longirostris. Oecologia, 82, 362–368.

Wacker A. & Martin-Creuzburg D. (2007) Allocation of

essential lipids in Daphnia magna during exposure to poor

food quality. Functional Ecology, 21, 738–747.

Wacker A. & Martin-Creuzburg D. (2012) Biochemical

nutrient requirements of the rotifer Brachionus calyciflorus:

co-limitation by sterols and amino acids. Functional Ecol-

ogy, 26, 1135–1143.

Wacker A. & von Elert E. (2001) Polyunsaturated fatty

acids: evidence for non-substitutable biochemical

resources in Daphnia galeata. Ecology, 82, 2507–2520.

Supporting Information

Additional Supporting Information may be found in the

online version of this article:

Figure S1. Relationship of the amount of carbon ingested

to the whole carbon content of the animal.

Table S1. Results of the non-linear regressions of the

tissue P : C ratios of mothers and eggs shown in Fig. 1.

Table S2. Results of the non-linear regressions of the

P : C ratios of adult D. magna shown in Fig. 6.

Table S3. Calculated amount of C ingested and the mea-

sured amount of C ingested per individual within 3 h at

different food P : C ratios.

Appendix S1. Calculation method to estimate the

amount of C ingested based on the differences in Daphnia

P : C ratios.

Appendix S2. Two-member mixing model to quantify

the influence of short-term ingestion on the P and C

content of a neonate.

(Manuscript accepted 8 July 2013)



Supporting Information Appendix S1:

The following calculations were conducted for all five dietary P levels.

We calculated the difference in Daphnia P:C ratios between the animals with treatment food

and those animals with P-deficient food for three hours (Equation 3).

: ( ) = : − : (3)

This balance contains information about the amount of P and C ingested within three hours,

because a decrease in P:C ratio is caused either by losing P or gaining C (both by ingesting P-

poor food). We could not depict the amount of C ingested with this method, but since we

measured the overall C content of the animals after the three hours of ingestion we can

translate the P:Cbalance into the amount of P ingested (Equation 4).

( ) = : × ( ) × 31 (4)

This amount of P ingested can be converted into amount of C ingested by using the respective

food P:C ratio for each dietary P level (Equation 5).

( ) = ( ) : ( ) (5)

Supporting Information Appendix S2: We used a two-member mixing model (Equation 6)

following Frost et al. (2005) in order to quantify the influence of short-term ingestion (3 h) on

the overall phosphorus and carbon content of a neonate. We therefore assumed a neonate

carbon content (neonate C) without any additionally ingested food after birth as 100%. The C

content of the neonate was used to calculate the phosphorus content of the neonate (neonate

P) assuming a P:C ratio of 8.7 mmol mol-1, which was the mean of all (starved) neonates in

our neonate nutrition experiment. Furthermore our model included terms for the amount of

additionally ingested phosphorus (Pi) and the amount of additionally ingested carbon (Ci).

Thereby Ci was assumed as percentage value between 0 (starvation) and 30 % of neonate’s C

content. Pi was calculated via Ci and the respective P:C ratio of the different diets between 1.5

and 6.7 mmol mol-1.

The model of neonate P:C ratio influenced by different amounts of additionally ingested

food predicts that the P:C ratio of the neonates would remain constant if they would starve

(Fig. A). This is in accordance to our laboratory data showing no effect of dietary P on

neonates P:C ratio when they starve. But if food with different P content was present, the

model predicts a decrease of neonates’ P:C ratios with lower dietary P. This decrease would

be intensified within an increasing amount of Ci, and Pi respectively. This complies with our

laboratory data showing explicitly lower neonate P:C ratios when they fed P-deficient food.

We conclude that Daphnia neonates could ingest at least 20%, in fact even up to 30%, of their

body C content within 3 h.

i

i

CCneonatePPneonate

CPneonatewhole++

=: (6)

Two-member mixing model where neonate P is the phosphorus content of a neonate

and neonate C is the carbon content of a neonate. Pi is the amount of additionally

ingested phosphorus and Ci is the amount of additionally ingested carbon.

Fig. A Two-member mixing model of neonate P:C ratios influenced by short-term ingestion

(three hours) of different proportions of food differing in dietary P. We tested for ingestion

amounts between 0% (= without food) and 30% relative to the body C mass. The model

predictions are plotted as diamonds. Additionally the mean of neonates’ P:C ratios measured

in the lab (see Fig. 4) are displayed as circles

Reference

Frost P.C., Hillebrand H. & Kahlert M. (2005) Low algal carbon content and its effect on the

C : P stoichiometry of periphyton. Freshwater Biology, 50, 1800-1807.

Food P:C ratio (mmol mol-1)1 2 3 4 5 6 7

Neo

nate

P:C

ratio

(mm

ol m

ol-1

)

0

6

7

8

9

10

model 0% model 15% model 20% model 30% lab without foodlab with food

Supporting Information Figure S1: Relationship of the amount of carbon ingested to the

whole carbon content of the animal (%). Linear regression (n =18): slope (± SE) 0.11 ± 0.70

mmol mol-1; t16 = 0.2; P = 0.875, intercept (± SE) 15.4 ± 3.1 %; t16 = 5.1; P < 0.001.

Food P:C ratio (mmol mol-1)

1 2 3 4 5 6 7

A

mon

ut o

f car

bon

inge

sted

____

____

____

____

____

____

____

____

____

____

__ (

%)

Car

bon

cont

ent o

f the

who

le a

nim

al

0

5

10

15

20

25

30

Supporting Information Table S1: Results of the non-linear regressions (Equation 2) of the

tissue P:C ratios (mothers, n = 20; eggs, n = 18) shown in Fig. 1. All parameters given in

(mmol P mol C-1).

Mothers Parameter ± SD t17-value P-value

S0 0.19 ± 0.33 0.6 0.58

Ks 0.54 ± 0.25 2.1 0.048

P:Cmax 10.12 ± 0.52 19.6 < 0.001

Eggs Parameter ± SD t15-value P-value

S0 0.96 ± 0.12 7.9 < 0.001

Ks 0.01 ± 0.02 0.4 0.67

P:Cmax 6.36 ± 0.15 41.5 < 0.001

Supporting Information Table S2: Results of the non-linear regressions (Equation 2) of the

P:C ratios of adult D. magna (each n = 20) shown in Fig. 6. All parameters given in (mmol P

mol C-1).

Treatment food Parameter ± SD t17-value P-value

S0 1.04 ± 0.27 3.8 0.001

Ks 0.54 ± 0.19 2.9 0.009

P:Cmax 10.15 ± 0.38 27.0 < 0.001

P-deficient food Parameter ± SD t17-value P-value

S0 1.11 ± 0.29 3.9 0.001

Ks 0.37 ± 0.14 2.6 0.019

P:Cmax 8.84 ± 0.28 31.5 < 0.001

Supporting Information Table S3: Calculated amount of C ingested and the measured

amount of C ingested per individual within 3 h (both mean ± SD, n =4) at different food P:C

ratios.

Food P:C ratio

(mmol mol-1)

Calculated C ingestion

(µgC)

Measured C ingestion within 3 h

(µgC individual-1)

2.02 11.9 ± 8.6 10.4 ± 0.7

2.53 8.4 ± 5.0 10.0 ± 1.1

3.38 7.8 ± 2.4 9.5 ± 0.8

4.50 9.6 ± 1.5 10.2 ± 0.3

6.75 6.9 ± 2.6 11.4 ± 0.9

the neonate nutrition hypothesis: early feeding affects the body stoichiometry of daphnia offspring

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