Making eggs from nectar: the role of life history and dietary carbon

turnover in butterfly reproductive resource allocation

Diane M. O’Brien, Carol L. Boggs and Marilyn L. Fogel

O’Brien, D. M., Boggs, C. L. and Fogel, M. L. 2004. Making eggs from nectar: the roleof life history and dietary carbon turnover in butterfly reproductive resourceallocation. �/ Oikos 105: 279�/291.

The diets of many butterflies and moths change dramatically with development: fromherbivory in the larvae to nectarivory in the adults. These diets are nutritionallydistinct, and thus are likely to contribute differentially to egg manufacture. We examinethe use of dietary resources in egg manufacture by four butterfly species with differentpatterns of oviposition and lifespan; three in the Nymphalidae (Euphydryas chalcedona,Speyeria mormonia and Heliconius charitonia ), and one in the Pieridae (Coliaseurytheme ). Each species was fed two isotopically distinct adult diets based onsucrose, both of which differed from the larval hostplant in 13C content. Egg isotopiccomposition was analyzed to quantify the contribution of carbon from the larval andadult diets to egg manufacture. In all four species, egg 13C content increased to anasymptotic maximum with time, indicating that adult diet is an increasingly importantsource of egg carbon . The 13C increase closely resembled that of a nectar-feedinghawkmoth, and was well-described by a model of carbon flow proposed for thatspecies. This similarity suggests that the turnover from larval to adult dietary supportof egg manufacture is conserved among nectar-feeding Lepidoptera. Species variedwidely in the maximum % egg carbon that derives from the adult diet, from 44% in E.chalcedona to nearly 80% in S. mormonia . These differences were related both to theextent of oocyte provisioning prior to adult emergence, and to egg composition. Aspecies’ lifetime use of larval vs adult resources in egg manufacture reflected both thecarbon turnover of the eggs and the timing of oviposition. Thus, the extent to whichdietary resources are important in egg manufacture in butterflies depends ondevelopment (egg provisioning in teneral adults), behavior (timing of oviposition)and nutritional physiology (nutrient synthesis and turnover).

D. M. O’Brien and C. Boggs, Center for Conservation Biology, Dept of BiologicalSciences, Stanford Univ., Stanford, CA 94305. Present address for DMO: Dept ofBiological Sciences, Wellesley College, 106 Central St., Wellesley, MA 02481, USA(dobrien@wellesley.edu). �/ M. Fogel, Geophysical Laboratory, Carnegie Institution ofWashington, 5251 Broad Branch Rd. NW, Washington DC 20015, USA.

Assumptions about how organisms use nutritional

resources are fundamental to predictions about life

history and evolutionary ecology (Reznick 1985, Stearns

1992, Rose and Bradley 1998). Nutrition is a key

determinant of reproduction (Wheeler 1996); therefore,

reproductive strategies need to be considered in light of

nutrient availability (Boggs 1992, de Jong and van

Noordwijk 1992). Although energy is commonly used

as a currency for reproduction and survival, other

nutrients, such as nitrogen (Slansky and Feeny 1977,

Brodbeck et al. 1993), phosphorous (Elser et al. 1996,

Markow et al. 1999), or essential carbon compounds

(Douglas 1998, O’Brien et al. 2002) may pose greater

constraints upon reproduction and population growth.

The complexities of nutrient requirements, synthesis,

storage, conversion and metabolism are poorly under-

Accepted 25 September 2003

Copyright # OIKOS 2004ISSN 0030-1299

OIKOS 105: 279�/291, 2004

OIKOS 105:2 (2004) 279

stood, especially for free-living animals with mixed or

complex diets. There is a growing appreciation for the

physiological complexity of resource allocation (Rose

and Bradley 1998, Rivero and Casas 1999, Zera and

Harshman 2001, Zhao and Zera 2002), in particular, for

hormonal controls of nutrient use (Sinervo and Svensson

1998, Zera et al. 1998). A similar focus on nutritional

physiology will further help to strengthen the connec-

tions between diet and life history.

Resource allocation in holometabolous insects is

particularly interesting because diets and energetic needs

change between life stages, causing nutrient intake and

nutrient demand to be temporally separated (Boggs

1981, 1986, Rivero et al. 2001). In these insects,

mechanisms for appropriately timed nutrient storage

and release are extremely important (Delobel et al. 1993,

Wheeler and Buck 1995, Snigirevskaya et al. 1997,

Djawdan et al. 1998, Wheeler et al. 2000, Pan and Telfer

2001). In most butterflies and many moths, diets change

dramatically from herbivory in the larvae to nectarivory

in the adults. These diets are nutritionally distinct, and

thus are likely to contribute differently to physiological

demands. Nectar is rich in sugars but relatively depau-

perate in amino acids, fatty acids, and other nutritionally

important compounds (Baker and Baker 1983). The

larval diet is the primary supply of protein, which is

stored for use during metamorphosis and oogenesis

(Wheeler et al. 2000, Pan and Telfer 2001). Butterfly

researchers have long appreciated that both larval and

adult diets are important to reproduction and are both

used in egg manufacture (Norris 1934, Dunlap-Pianka et

al. 1977, Hill 1989, Hill and Pierce 1989, Alm et al. 1990,

Boggs and Ross 1993, Boggs 1997a). Because their life

histories vary, the Lepidoptera have also provided a

model system for comparing reproductive output among

species with varying degrees and types of adult foraging

(Boggs 1986, 1990, 1992, 1997b, 2003).

We use natural variation in 13C to identify egg carbon

deriving from the larval and adult diets in butterflies

with nectivorous adults, but differing lifespans and age-

specific fecundities. 13C content differs between plants

with C3 and C4 photosynthesis, but also can vary among

plants sharing a photosynthetic mode (O’Leary 1981,

1988). Thus, it can be a powerful tracer of carbon flow

from multiple plant sources. Natural abundances of light

stable isotopes are a well-established tracer of nutrient

flow, within both organisms (Metges et al. 1990, Hobson

et al. 1997, Schmidt et al. 1999) and ecosystems

(Rounick and Winterbourn 1986, Peterson and Fry

1987, Ostrom et al. 1997, Wolf and Martınez del Rio

2000). In a previous study, O’Brien and colleagues (2000)

used carbon isotopes to trace the use of larval and adult

diets in reproduction by a hawkmoth. Females were fed

one of two sucrose-based adult diets made from beet

(C3) or cane (C4) sugar, both of which differed from the

natural larval hostplant in 13C content. This approach

enabled them to trace carbon use in eggs with high

accuracy, and they proposed a descriptive carbon turn-

over model to describe how egg nutrients were incorpo-

rated into eggs (O’Brien et al. 2000).

In this study, we apply a similar approach to

investigate the dietary sources of reproductive nutrients

in four nectar-feeding butterfly species, three from the

family Nymphalidae and one from the family Pieridae.

The species selected exhibit a range of variation in

lifespan and age-specific fecundity, and differ in degree

of shared phylogenetic history. We investigate whether

the turnover pattern modeled in hawkmoth eggs is

exhibited in other nectar-feeding Lepidoptera. If so, we

evaluate whether the extent of adult diet use or the

rapidity of larval to adult diet turnover vary according to

differences in life history. We predict that the reliance on

adult dietary carbon in egg manufacture will be higher in

those species whose reproductive effort takes place later

in the adult lifespan.

This study uses sucrose solution as an experimental

proxy for the adult nectar diet, as it elicts a strong

feeding response from butterflies (Romeis and Wackers

2000) and is easy to acquire in different isotopic forms.

This proxy is a nutritional oversimplication, especially as

some butterflies have been shown to detect and slightly

prefer nectars containing amino acids (Alm et al. 1990,

Rusterholz and Erhardt 1998, Mevi-Schutz and Erhardt

2002). We do not suggest that other dilute components

of nectar could not play some nutritional role in

oviposition. This study might slightly underestimate

the importance of the adult diet in species that capitalize

on nectar amino acids. However, as the vast majority of

carbon provided by the adult diet is in the form of sugar

(Baker and Baker 1983), sugar is a reasonable proxy

given our carbon isotope mass-balance approach.

Methods

Study organismsEuphydryas chalcedona

Euphydryas chalcedona (Nymphalidae: Nymphalinae;

Melitaeini) is a univoltine butterfly distributed through-

out western North America. Individuals were field

collected as 5th and 6th instar larvae from Scrophularia

californicus and Mimulus aurantiacus at the Silver Creek

Hills Butterfly Reserve near San Jose, California, USA.

Larvae were placed in mesh bags on potted S. californi-

cus for the remainder of their larval development, and

bags were moved as needed to ensure a constant supply

of fresh leaves. Pupae were stored at 208C until adults

emerged. Average female forewing length was 29.89/0.4

mm. Females mated either the day of emergence or the

day following. Once mated, females were placed in wire

mesh cages (20�/20�/30 cm) and provided fresh S.

californicus leaves for oviposition. Females oviposited

280 OIKOS 105:2 (2004)

daily or every other day in clusters of typically 100�/200

eggs (all other species laid eggs singly). Females were

handfed twice daily. Of the 10 females to successfully

mate and start the experiment, 2 were removed because

they either waited unusually long to oviposit, or didn’t

eat consistently. The remaining 8 butterflies were used in

both isotope analyses and feeding and fecundity mea-

surements.

Speyeria mormonia

Speyeria mormonia (Nymphalidae: Heliconiinae: Heli-

coniini: Argynniti) is a temperate zone univoltine

butterfly that occurs in montane and subalpine habitat

in western North America. Adult females were captured

during August 1999 near Gothic, Gunnison County,

Colorado, USA, and oviposited in captivity. Speyeria

mormonia overwinters as unfed first instar larvae;

therefore, their hatchlings were placed in humid vials

and stored at 48C. The following February (2000), larvae

were warmed to room temperature and placed in mesh

bags tied over leaves of potted violets (Viola soraria ).

Bags were moved as needed to provide larvae with a

constant supply of fresh leaves. Pupae were stored at

208C until adults emerged. Average female forewing

length was 26.99/0.6 mm (nearly identical to that of

field-caught females, 26.69/1.1 mm, Boggs 1987). Newly

emerged females were hand-mated and placed in cylind-

rical cages made from lantern globes and lined with wax

paper (15 cm diameter�/20 cm height). Females were

provided fresh violet leaves for oviposition, and were fed

twice daily. Of 15 healthy experimental females, two were

removed mid-way through because excessive wing da-

mage made them difficult to hold for feeding. Thus, N�/

13 females for fecundity and lifespan estimates, 5 of

which were hand-fed quantitatively.

Heliconius charitonia

H. charitonia (Nymphalidae: Heliconiinae: Heliconini:

Heliconiiti) is a sub-tropical butterfly, which cannot

diapause. Adults feed on both nectar and pollen. A free-

flying greenhouse colony of H. charitonia is maintained

at Stanford University, founded from individuals col-

lected in Costa Rica and Yucatan, Mexico. Adults in the

colony oviposit on potted Passiflora caerula and feed

from potted Lantana camara and Psiguria umbrosa .

Honey water feeders supplement these natural sources of

nectar and pollen. Experimental females were collected

as pupae from the colony and mated upon emergence.

Average female forewing length was 38.69/0.6 mm.

H. charitonia requires more space than the other

butterflies to fly and oviposit normally; therefore, mated

females were placed individually into 1.8�/1.0�/1.0

meter cages and each was given a potted Passiflora

caerula plant for oviposition. Females were provided

experimental sugar solutions ad lib in feeders colored

to resemble Psiguria umbrosa flowers, and sugar solu-

tions were changed daily. Although H. charitonia

normally supplement their diet with pollen, females in

this experiment were not provided with pollen. Eggs

were collected daily. Nine females initiated oviposition

and were used in isotope measurements; of those, two

died prematurely (N�/7 for lifespan and fecundity

estimation).

Colias eurytheme

Colias eurytheme (Pieridae) is a temperate zone butterfly,

which does not diapause. Colias eurytheme eggs were

provided by Ward Watt from his colony at Stanford

University, which derived from females collected near

Tracy, California, USA. Larvae were reared on hydro-

ponically grown Vicia villosa (Watt, unpubl.), and pupae

were housed at 208C until emergence. Newly emerged

adult females were hand-mated the same day. Average

female forewing length was 28.79/0.05 mm. Mated

females were housed in 20�/20�/30 cm mesh cages

and provided potted vetch plants for oviposition.

Females were hand-fed twice daily. Of 18 females that

started the experiment, 4 laid fewer than 500 eggs, lived

fewer than 10 days, or skipped multiple days of

oviposition in the first week. These butterflies were

considered non-representative and were removed. Of 14

healthy ovipositing females used for isotope analyses,

two were prematurely removed due to injury or escape

and were not included in our estimates of lifespan and

fecundity (N�/12).

Experimental methods

Experiments were carried out between February and

May, 2000 (except where noted), at the Herrin Green-

houses at Stanford University. Greenhouses were main-

tained on a 16:8 L:D photoperiod, with daytime and

nighttime temperatures of 278C and 15.58C, respectively.

All butterflies were fed 30% sucrose solution (wt/wt) as

adults. Approximately half of the individuals received

beet sugar solution (C3) and half received cane sugar

solution (C4). A single batch was mixed for each sugar

solution, and stored as frozen aliquots until fed to the

butterflies. A subset of the experimental females were fed

quantitatively from 25 or 50 ml Hamilton graduated

syringes, as described elsewhere (Boggs and Ross 1993),

to evaluate their intake quantitatively under the experi-

mental conditions. Eggs were collected daily into labeled

0.5 ml Eppendorf tubes, which were placed open into a

drying oven at 508C. After 3 days, tubes were removed

from the oven, sealed, and stored at room temperature

until isotope analysis.

OIKOS 105:2 (2004) 281

d13C analysis

Isotope ratios are given a d13C�/(Rsample/Rstandard

�/1)�/1000, in which R�/13C/12C and the standard is

PDB limestone. Dried egg samples weighing 0.30 to 0.35

mg were analyzed for d13C using a CHNS analyzer (CE

Instruments, model NC2500) interfaced to a Finnigan

Delta Plus XL isotope ratio mass spectrometer via the

Finnigan Conflo II interface. The CHNS analyzer also

measured %C and %N for each sample. Acetanilide

standards (0.2�/0.25 mg.) were run with each batch of

samples (�/1 standard per 5 samples), and were used to

correct for consistent variation in d13C among batches

run at different times. The standard deviation of

acetanilide d13C within a batch was typically B/0.15�.

Turnover model

We parameterize the pattern of dietary carbon allocation

with the model of O’Brien et al. (O’Brien et al. 2000).

Briefly, the model assumes two classes of egg nutrients:

one that shifts from larval to adult sources as the

butterfly feeds, and one that retains an unchanging

larval isotopic signature. We label these the ‘‘mixing’ and

‘non-mixing’’ carbon sources, respectively. The propor-

tion of egg carbon in these two nutrient classes is given

by pmax, which represents the maximum contribution of

adult dietary carbon to eggs:

egg d13C�(d13Cmixing)(pmax)�(d13Cnon-mixing)(1�pmax)

(1)

Because d13Cnon-mixing will be constant, whereas d13

Cmixing will change over time, the isotopic composition

of the two nutrient pools is modeled as:

d13Cnon-mixing�d13Clarval C (2)

d13Cmixing�d13Clarval C�((d13Cdiet�fa)�d13Clarval C)

� ( 1�e�rt) (3)

Thus, egg

d13C�Cd13Clarval C�((d13Cdiet�fa)�d13Clarval C)

� ( 1�e�rt)) (pmax) �Cd13Clarval C)(1�pmax) (4)

where

pmax�/the maximum % of egg carbon to derive from the

adult diet

r�/the fractional turnover rate of carbon from larval to

adult sources

fa�/the isotopic fractionation associated with incorpor-

ating adult dietary carbon into eggs

d13Clarval C�/the isotopic composition of egg carbon

deriving from the larval diet.

d13Cdiet�/the isotopic composition of the adult diet

(known)

t�/days since adult emergence (known)

By applying this model to our data, we can estimate

the values of the parameters in bold. In particular, we

are interested in pmax, which describes the maximum

contribution of adult dietary carbon to eggs, and r,

which indicates the rate at which butterflies transition

from larval to adult sources of egg carbon.

Calculating % contribution of the adult diet

The magnitude of the difference in egg d13C between C3

vs C4 �/ fed females on a given day reflects how much of

the carbon in those eggs comes from the adult diet. We

term the proportional contribution of the adult diet to

eggs ‘‘p’’, and quantify it as follows (O’Brien et al. 2002):

p�(d13CC4 egg�d13CC3 egg)=(d13CC4 diet�d13CC3 diet)

(5)

We use the lines of best fit through the data from C3

and C4 fed females to calculate an average daily ‘‘p’’ for

each species.

Amino acid composition

Egg amino acid compositions were analyzed by post-

column ninhydrin-based amino acid analyses at the

Molecular Structure Facility at the University of Cali-

fornia, Davis (Smith 1997). This technique provides data

on 16 of the 20 amino acids found in proteins: cysteine

and tryptophan are partially or completely destroyed by

the analysis, and glutamine and asparagine become

indistinguishable from glutamic acid and aspartic acid,

respectively, after acid hydrolysis (Smith 1997).

Oocyte provisioning at adult emergence

Asking whether adult-derived nutrients are used in egg

manufacture implies that at least some oocytes are not

mature at the time of adult emergence. In order to

evaluate the extent of oocyte provisioning during

metamorphosis, we dissected the ovaries of newly

emerged females and calculated the volume of the

most mature and least mature oocytes assuming a

cylindrical egg. We used the same method to measure

the volume of newly laid eggs for all species, except

Euphydyras chalcedona , which has chorionated eggs in

the ovaries at emergence, which therefore cannot change

in volume. Oocyte volume as a % of the volume of newly

laid eggs was then calculated for both the most and least

mature eggs in the ovaries. Because females emerge with

a full complement of oocytes, these measurements

represent the first and last eggs potentially laid, and

hence the endpoints of eggs used in our isotopic analysis.

Note that Heliconius charitonia exhibits oogenesis as an

adult only when fed pollen (Dunlap-Pianka et al. 1977);

282 OIKOS 105:2 (2004)

hence its ovarian dynamics here resemble the other

species in that new oocytes are not produced.

Statistical analyses

All statistical analyses were performed in JMP version

5.0 (SAS Institute, Inc.) Means are given9/SD unless

otherwise noted. Species differences in lifespan, fecund-

ity, egg weight, and egg C/N were evaluated with

Analysis of Variance (ANOVA). Variation among spe-

cies in meal size over time was tested with Analysis of

Covariance (ANCOVA). Within each species, changes in

egg mass and C/N over time were evaluated using

ANCOVA to control for individual differences. With

each application of ANOVA or ANCOVA, means were

compared using Tukey’s HSD contrasts and residuals

were tested for normality using the Shapiro-Wilks test.

Non-linear fitting was iteratively performed using least

squares minimization until estimates converged. Briefly,

non-linear fitting applies a model with unknown para-

meters to a set of known data and modifies the

parameters from pre-set values to achieve the best fit

(via least-squares minimization). We tested whether

nutrient allocation differed between species pairs by

evaluating whether the data were best described by a

single carbon model or separate models, as proposed by

Ratkowsky and outlined in Motulsky and Ransas

(Motulsky and Ransnas 1987). In these comparisons,

we used p�/0.008 (Bonferroni correction) as the sig-

nificance threshold to account for multiple tests.

Results

Lifespan and fecundity

Average adult lifespan in this study ranged from 15 days

for Euphydryas chalcedona to 41 days for Heliconius

charitonia , and average lifetime fecundity ranged from

138 eggs in H. charitonia to 1065 eggs in E. chalcedona

(Fig. 1, Table 1, species were different at PB/0.0001).

Age-specific fecundity (mean9/SE) is presented for each

species in Fig. 2. E. chalcedona began to deposit eggs in

large clusters immediately after mating, typically laying

�/500 eggs within the first 5 days of adult life. In

contrast, H. charitonia began to oviposit more than a

week after emergence and mating, laid fewer eggs daily,

and lived for weeks longer than the other three butterfly

species. H. charitonia is a species that normally supple-

ments its nectar diet with pollen, (Dunlap-Pianka et al.

1977, Boggs 1979), but still lays relatively few eggs per

day (�/10�/15, Dunlap-Pianka et al. 1977, Boggs 1997a).

Speyeria mormonia fell between H. charitonia and E.

chalcedona in the timing of its reproductive effort, its

lifespan, and its fecundity. Colias eurytheme, from the

family Pieridae, was also intermediate; it achieved

greater fecundity than S. mormonia, but did so with

smaller eggs (see below). The data are consistent with

previously published life history studies on the three of

these species for which records are available (Stern and

Smith 1960, Dunlap-Pianka et al. 1977, Boggs 1986,

1990).

Egg weight and composition

Egg weight and % carbon and nitrogen are presented for

each species in Table 1. Egg weight declined slightly

(between 1�/5 mg dry weight/day) but significantly with

age in C. eurytheme, E. chalcedona and S. mormonia

(ANCOVA, Table 2), consistently with many other

butterfly species (Boggs 1986, Karlsson 1987, Wickman

and Karlsson 1987). Speyeria mormonia eggs had a

markedly lower protein content compared to the other

species, as indicated by their high egg C/N (Table 1). Egg

C/N increased slightly over time in H. charitonia

(ANCOVA, Day effect P�/0.0152, slope�/0.014/day),

but did so in no other species.

Oocyte provisioning at adult emergence

Euphydryas chalcedona , with an average of 489/6 (n�/3

newly emerged females) oocytes mature and chorionated

at emergence, had its most mature egg equal to 100% of

the volume of a laid egg. The other species showed lower

percentages: 7% (n�/2 newly emerged females) for S.

mormonia and 4% for both C. eurytheme (n�/2) and H.

charitonia (n�/1). The volume of least mature oocytes

was invariant within species and similar among species,

at 0.0008 mm3 for S. mormonia and C. eurytheme and

0.0005 mm3 for E. chalcedona and H. charitonia . These

volumes are 0.1% of that of a laid egg for E. chalcedona

and H. charitonia , 0.2% for S. mormonia and 0.4% for

C. eurytheme.

Fig. 1. Species differences in lifespan and fecundity for thisexperiment. Shown are means9/SD, sample sizes are given inparentheses next to the species names.

OIKOS 105:2 (2004) 283

Nectar intake and carbon budget

Euphydryas chalcedona had the largest average meal size

(36.19/1.9 ml; mean9/SD), followed by S. mormonia

(28.09/1.4 ml) and C. eurytheme (17.49/1.4 ml). In all

species, meal size declined an average of 0.97 ml/day.

Both the differences among species and the decline in

meal size were significant (Fig. 3, ANCOVA with Tukey’s

HSD; species PB/0.0001, day PB/0.0001, age�/species

interaction not sig.). Heliconius charitonia was not fed

quantitatively.

We converted meal sizes (ml) to mg carbon consumed

using 0.143 mg C/ml sucrose solution (determined

empirically). We calculated the mg carbon contained in

Table 1. Species differences in oviposition, egg characteristics and total carbon budget. All values are means9/SD.

Character E. chalcedona S. mormonia C. eurytheme H. charitonia

Fecundity 10659/282 5049/124 8049/166 1379/24Dry egg weight (mg)1 0.0619/0.009 (6, 17 ) 0.0819/0.010 (11, 38 ) 0.0279/0.003 (5, 27 ) 0.0939/0.010 (7, 26 )Total egg output (mg) 65.09/26.8 40.89/15.1 21.79/7.0 12.79/3.6Egg % C (wt) 50.909/0.94 (8, 29 ) 57.839/2.83 (11, 24 ) 49.739/0.09 (13, 37 ) 43.719/1.25 (7, 32 )Egg % N (wt) 8.459/0.54 (8, 29 ) 5.749/0.21 (11, 24 ) 9.479/0.51 (13, 37 ) 7.619/0.19 (7, 32 )Egg C/N (mol) 7.069/0.49 (8, 29 ) 11.779/0.67 (11, 24 ) 6.159/0.33 (13, 37 ) 6.719/0.19 (7, 32 )

Total C in (mg) 95.39/36.8 84.99/15.7 56.29/14.4 �/

Egg C out (mg) 35.39/9.4 23.69/5.8 10.79/2.2 5.69/1.0C in/Egg C out 2.79/1.8 3.69/1.5 5.39/2.4 �/

1Multiple samples were evaluated per individual, which were averaged before calculating species means and SD. Numbers inparentheses give the No. of individuals (N) and the total number of samples measured (in italics).

Fig. 2. (A�/D) Mean numberof eggs laid daily for eachspecies. Data are given asmeans9/SE. Sample sizes foreach mean (when differentfrom the preceding mean) aregiven in italics below each datapoint. (E) Comparison ofoviposition among species,with uniform scaling. Linesconnect mean egg numbers foreach day.

284 OIKOS 105:2 (2004)

eggs from mean egg weights and % C for each species

(Table 1). Total carbon gain and loss were calculated for

each butterfly, and the mean ratio for each species is

presented in Table 1. For each species, the total carbon

consumed in nectar exceeds the total carbon lost in eggs.

Egg amino acid composition

Egg amino acid composition (in mol%) is given in Table

3. Data from the hawkmoth Amphion floridensis are also

presented for comparison (O’Brien et al. 2002). For each

species, amino acids are ranked from most to least

prevalent. Amino acid composition was similar (but not

identical) in all four species. Glutamic acid was the most

prevalent amino acid in all four species, followed by

alanine, glycine, and aspartic acid in varying order

(totaling 42�/46% of egg amino acids). Serine, leucine,

lysine, valine and proline, in varying order, were the next

most abundant amino acids (30�/34%). Threonine,

arginine, isoleucine, and tyrosine were the next four

most abundant (16�/19% of egg amino acids). Finally,

phenylalanine, histidine, and methionine were the least

abundant amino acids (6�/8% of egg amino acids). These

amino acid compositions, together with the measured

%C and %N for each species, allowed us to calculate the

% egg carbon deriving from different classes of amino

acids (Table 4).

d13C Dietary constituents

The d13C of the larval hostplant (in parentheses) for each

butterfly species were as follows: E. chalcedona (Scro-

phularia californicus ), �/28.259/1.61� (mean9/SD;

N�/3), S. mormonia (Viola soraria ) �/33.049/1.94�(N�/10), H. charitonia (Passiflora caerulea ) �/31.869/

0.92� (N�/3) and C. eurytheme (Vicia villosa ),

�/25.859/0.56� (N�/4). Although E. chalcedona lar-

vae were field collected on both S. californicus and

Mimulus aurantiacus, the majority of their growth was

on S. californicus in the greenhouse. The d13C of the

adult diets were �/11.149/0.10� (C4 cane sugar) and

�/24.849/0.13� (C3 beet sugar). All butterflies were fed

from frozen aliquots from single batches of sugar

solution; thus, the standard deviation around the value

for adult diet represents measurement error.

d13C Butterfly eggs

The change over time in isotopic composition of egg

carbon (d13C) for each species is presented in Fig. 4. In

Table 2. The effect of female age (in days of adult lifespan) on egg weight (mg.). The relationship was evaluated for each speciesseparately with ANCOVA, to remove significant inter-individual differences in egg mass. Where age significantly affected mass, themagnitude of the effect is given by the slope (in mg/day).

Species Effect F ratio P value N Slope (mg/day)

C. eurytheme Individual (5)1 13.93 B/0.0001 27 �/

Age 57.08 B/0.0001 �/0.0005E. chalcedona Individual (6) 5.0844 0.0141 17 �/

Age 18.0215 0.0017 �/0.0011S. mormonia Individual (11) 0.72 0.7029 38 �/

Age 11.35 B/0.0024 �/0.0018H. charitonia Individual (7) 15.90 B/0.0001 26 �/

Age 1.83 0.1932 N/A

1Individual N are given in parenthesis.

Fig. 3. Daily meal size (in ml) for E. chalcedona , C. eurytheme and S. mormonia . Data are given as means9/SD. Sample sizes foreach mean (when different from the preceding mean) are given in italics below each data point. Note that the scaling of axes isconstant among species. Mean meal size differs significantly among species, however, the decline in meal size over time does not (seeresults).

OIKOS 105:2 (2004) 285

all four species, egg d13C reflected the d13C of the C3 or

C4 adult diet (triangles vs circles, Fig. 4). Egg d13C

increased asymptotically over time (Fig. 4), as found

elsewhere (O’Brien et al. 2000). The extent of the

increase depended on the species, and on the lag between

emergence and oviposition. For example, larval to adult

carbon turnover in H. charitonia stabilized prior to the

onset of oviposition on day 8, and thus egg d13C changed

little if at all (Fig. 4). In E. chalcedona, S. mormonia and

C. eurytheme, eggs laid within several days of emergence

were predominantly larval in carbon composition, and

rapidly shifted to a more adult-derived isotopic signal.

The data were well described by the turnover model

outlined in the Methods (lines of best fit in Fig. 4,

parameters given in Table 5).

Use of adult diet in egg provisioning

The daily proportion of egg carbon deriving from the

adult diet is summarized in Fig. 5 for each species.

Species varied widely in the maximum percentage of egg

carbon that derives from the adult diet (from 44% in E.

chalcedona to nearly 80% in S. mormonia , Fig. 5, Table

5). In contrast, species were all relatively similar in their

turnover rate (r), the inverse of the time required to

reach half turnover (Fig. 5). The turnover rate indicates

how rapidly the shift from larval to adult sources occurs.

These varied from 0.18 day�1 in S. mormonia (most

rapid turnover) to 0.32 day�1 in C. eurytheme (least

rapid, Table 5).

Comparing allocation patterns among species

In order to evaluate whether species differed significantly

in their patterns of adult and larval resource use, we

tested whether the parameters fit to each species’ data by

the turnover model could also adequately describe that

of the other species. In order to do this we used a test

that compares whether the goodness of fit of a non-

linear model significantly worsens when two sets of data

Table 3. Amino acid composition of butterfly eggs. Each column gives averaged data from 2 individuals, with inter-individualdifferences less than 0.1% on average. Data are ranked from highest to lowest prevalence for each species. Glx�/glutamic acid�/

glutamine, Asx�/aspartic acid�/asparagine. Horizontal lines separate groups of amino acids that share similar prevalence (but invarying order).

E. chalcedona S. mormonia H. charitonia C. eurytheme A. floridensis1

aa mol% aa mol% aa mol% aa mol% aa mol%

glx 13.0 glx 12.5 glx 13.0 glx 12.5 gly 15.6asx 10.5 gly 11.4 gly 11.2 asx 10.1 ala 11.6ala 9.7 asx 9.4 asx 11.0 gly 9.8 glx 11.1gly 8.7 ala 8.7 ala 10.3 ala 9.6 asx 8.2

leu 7.2 pro 7.6 ser 7.8 ser 7.6 leu 7.0ser 7.1 val 7.2 leu 6.6 leu 7.1 val 6.0val 6.1 leu 6.9 lys 5.7 lys 6.1 pro 5.9lys 6.1 ser 6.3 pro 5.5 val 5.8 ser 5.7pro 5.7 lys 5.4 val 5.0 pro 5.8 tyr 5.0

thr 4.7 ileu 4.9 tyr 4.7 thr 4.7 lys 4.4arg 4.7 tyr 4.5 thr 4.3 arg 4.6 thr 4.3ileu 4.6 thr 4.3 arg 4.1 ileu 4.4 arg 3.8tyr 4.3 arg 3.7 ileu 3.5 tyr 4.3 ileu 3.8

phe 3.3 phe 3.1 phe 2.9 phe 3.3 phe 2.8his 2.3 his 2.0 his 2.0 his 2.3 his 2.4met 1.9 met 1.6 met 2.0 met 1.8 met 1.7

1Data originally published in (O’Brien et al. 2002).

Table 4. Calculations of egg protein and EAA content, based on egg amino acid composition, %C and %N.

E. chalcedona S. mormonia H. charitonia C. eurytheme

g N g�1 egg1 0.085 0.057 0.076 0.095g protein g�1 N2 5.713 5.817 5.716 5.697g protein g�1 egg3 0.486 0.331 0.434 0.541

g C g�1 egg1 0.51 0.58 0.44 0.497g EAA C g�1 protein2 0.292 0.301 0.283 0.302g EAA C g�1 C4 0.278 0.172 0.279 0.329

1Data are from the Elemental Analyzer (Table 1).2Calculated from the amino acid composition data (Table 3).3Calculated from the above two lines.4Calculated from the above three lines.

286 OIKOS 105:2 (2004)

are grouped for analysis, vs analyzed separately (see

Methods, Motulsky and Ransnas 1987). We compared

all two-way species combinations using this test and

found that each species differed significantly from the

others in diet use dynamics (Table 6).

Lifetime dietary resource use

Although a butterfly may come to rely heavily on the

adult diet to manufacture eggs later in life, the bulk of

her egg-laying effort may occur much earlier. Thus, her

total larval vs adult resource use will reflect both her

oviposition strategy (Fig. 2) and the shifting sources of

egg nutrients (Fig. 5). Figure 6 presents the mg. of larval

and adult carbon invested in eggs per day by each

species, and the summation of these daily investments to

give a lifetime total. The ratio of these lifetime totals

(adult /larval) for each species was 0.46 in E. chalcedona ,

Fig. 4. Egg carbon isotopiccomposition (d13C) vs femaleage in days since emergencefrom pupation. Trianglesdenote eggs laid by females fedcane sugar solution as adults(C4: �/11.15�), circles denoteeggs laid by females fed beetsugar solution as adults (C3:�/24.85�). Lines through thedata were generated by themodel given in Eq. 4, usingnon-linear fitting to estimateparameters. (A) E. chalcedona ,N�/33 samples from 8 females.(B) C. eurytheme ; N�/47samples from 14 females. (C) S.mormonia , N�/56 samplesfrom 13 females. (D) H.charitonia ; N�/32 samplesfrom 9 females.

Table 5. Parameters estimated by the larval to adult turnover model for each species, given with estimated standard errors.

Parameter estimated E. chalcedona S. mormonia H. charitonia C. eurytheme

Max. % adult carbon (p) 0.449/0.05 0.799/0.05 0.669/0.02 0.559/0.02Turnover rate (r) 0.249/0.06 0.189/0.03 0.289/0.05 0.329/0.04Adult diet fractionation (fa) 3.79/1.9 �/1.89/0.9 �/1.899/0.34 0.49/1.0d13C larval C �/29.09/0.8 �/33.89/1.4 see below1 �/27.29/1.0

d13C (measured) of late-instar larvae (N) �/29.09/0.07 (2) �/34.49/0.2 (11) �/329/0.9 (3) �/26.59/0.1 (6)

1Model was unable to estimate d13C larval C for H. charitonia . Because the estimated values of ‘‘d13C larval C’’ were close to measuredd13C values of larvae (within 1�) in the other species, H. charitonia d13C larval was fixed at the measured larval value in order to fitthe other parameters.

Fig. 5. The proportion of egg carbon deriving from the adultdiet, vs female age, for each species. Proportions for each daywere calculated according to Eq. 5, using the model (Eq. 4) toestimate the mean value of d13C C3 egg and d13C C4 egg for eachday (see Methods). We also present the equivalent curve for thehawkmoth A. floridensis, published elsewhere (O’Brien et al.2000).

OIKOS 105:2 (2004) 287

1.04 in C. eurytheme, 1.79 in S. mormonia and 1.88 in H.

charitonia . Euphydryas chalcedona incorporated a lower

percentage of adult diet into eggs than the other species,

and also laid the majority of its eggs before the bulk of

larval to adult carbon turnover has occurred.

Discussion

All four butterflies used nectar carbon in egg manufac-

ture, to varying extents. Interestingly, the pattern of

resource use was similar in all four species, despite their

different life histories. Each species exhibited a shift from

larval to adult sources of egg carbon that was well-

described by a turnover function, with eggs coming to

reflect a stable ratio of larval and adult resources. The

pattern is similar to that found in a hawkmoth (family

Sphingidae, O’Brien et al. 2000), a species that is only

distantly related to the butterflies examined here. It is

also characteristic of adult resource allocation to repro-

duction in parasitioid wasps (Rivero et al. 2001). Tissue-

specific turnover has been well documented in animals

that undergo diet shifts (Tieszen et al. 1983, Hobson

1995, Webb et al. 1998), and is relatively straightforward

to model and understand physiologically (Penry and

Jumars 1987). It is directly related to nutrient pool sizes

and flow rates; thus, these results suggest that resource

allocation should be determined by factors such as

dietary intake rate, total nutrient pool size, and nutrient

output in metabolism and egg manufacture.

The turnover of egg carbon from larval to adult

dietary sources illuminates the underlying physiology of

nutrient use in egg manufacture. This pattern can be

characterized with two biologically-relevant parameters:

the fractional turnover rate, which describes the rate of

the shift from larval to adult egg carbon sources, and the

maximal contribution of the adult diet (the value at

which egg composition stabilizes). The fractional turn-

Table 6. Comparison of model fit between pairs of species: do species differ significantly in their turnover from larval to adultderived egg carbon?

Comparison F ratio P value Significantly different?(PB/0.008)1

C. eurytheme vs S. mormonia 69.19 PB/0.0001 yesC. eurytheme vs H. charitonia 84.81 PB/0.0001 yesS. mormonia vs E. chalcedona 23.20 PB/0.0001 yesH. charitonia vs E. chalcedona 26.12 PB/0.0001 yesC. eurytheme vs E. chalcedona 13.94 PB/0.0001 yesH. charitonia vs S. mormonia 4.39 P�/0.006 yes

Fig. 6. The mg of carbon fromthe larval and adult diets useddaily in egg manufacture, foreach species. Lifetime totals arepresented on each legend. Thelarval diet is indicated by blackbars and adult diet by graybars. Note that the axes aredifferently scaled amongspecies.

288 OIKOS 105:2 (2004)

over rate was quite consistent across all species studied

to date; from 3�/5 days to achieve 50% turnover.

Another factor contributing to dietary resource use is

how much sugar-derived dietary carbon a species can or

will use in their eggs. This parameter, termed ‘‘pmax’’,

varied widely among the different species in this study.

At one extreme, E. chalcedona eggs never contained

more than 44% adult carbon, whereas S. mormonia eggs

came to comprise nearly 80% adult carbon. All of the

butterflies appeared to consume sufficient carbon to

make up for losses to eggs, although a caveat is that

carbon lost in respiration and excretion was not

quantified in this study. Egg composition is likely to

play a role in determining the extent to which adult

dietary carbon is used. Eggs are biochemically complex,

containing primarily protein and lipid, but also glycogen

granules, hydrocarbons, and nucleic acids (Nation 2002).

Approximately half of the carbon in egg protein derives

from essential amino acids (Table 4). These amino acids

have been shown to derive exclusively from the larval

diet in Amphion floridensis (O’Brien et al. 2002).

Essential fatty acids (and the fatty acids that derive

from them) also occur in lepidopteran eggs, estimated

for one species as 33% of all egg fatty acids (Forte et al.

2002). Thus, requirements for essential nutrients not

abundant in nectar diets limit the extent to which a

species can use adult dietary nutrients in egg manufac-

ture. For example, pollen-feeding H. charitonia have

access to essential amino acids (O’Brien et al. 2003), and

would be expected to rely more heavily on the adult diet

than the exclusively sucrose-fed H. charitonia in this

study.

For the species studied here, the percentage of egg

carbon deriving from essential amino acids is presented

in Table 4. This percentage approximates the minimum

possible carbon contribution from the larval diet (if

essential amino acids were the only carbon compound

not synthesized from sugar). We plotted these values for

each species against the % egg carbon that is exclusively

larval in origin (1�/pmax) (Fig. 7), including data

previously published from the hawkmoth A. floridensis

(O’Brien et al. 2000). This relationship provides some

insight into the differences among species in carbon

utilization. S. mormonia, H. charitonia, A. floridensis

and (to a lesser degree) C. eurytheme all fall fairly close

to the line y�/x, indicating that they are incorporating

nearly the maximum amount of adult dietary carbon

possible, given their individual egg composition. E.

chalcedona is notably different: it seems to incorporate

much less adult dietary carbon than it theoretically

‘‘could’’, based on egg composition.

The difference in E. chalcedona may lie in the extent of

egg provisioning that takes place prior to adult emer-

gence. Based on ovarian dissections, E. chalcedona

provisions its eggs at a much higher level prior to adult

emergence than do the other three species. These data

suggest two diet use ‘‘strategies’’ here: one in which a

butterfly makes as much of its eggs from adult diet as is

biochemically possible (S. mormonia, H. charitonia,C.

eurytheme and A. floridensis ), and the other in which the

butterfly foregoes the potential carbon influx from the

adult diet in favor of egg provisioning during pupal

development, and more rapid reproduction (E. chalce-

dona ). The smallest oocytes (corresponding to the last

eggs to be laid) were similarly sized among all four

species at emergence, suggesting at least the possibility

that E. chalcedona could use more adult resources to

provision the eggs laid latest in life. However, E.

chalcedona concentrates oviposition in the first week of

adult life; therefore, it is likely that even the least mature

oocytes are provisioned in early adulthood, before much

turnover to adult nutrients has occurred.

S. mormonia has a higher proportion of non-protein

egg nutrient than the other butterflies (high C/N), and

exhibits the highest % adult dietary carbon in its eggs. A

possible explanation for the high egg C/N is glycogen.

Glycogen stores are converted into the colligative anti-

freezes glycerol and sorbitol in freeze-avoiding Lepidop-

tera (Chino 1957, Storey 1990, Pullin et al. 1991).

Because S. mormonia overwinters as unfed, 1st instar

hatchlings at high altitude in the Rocky Mountains,

antifreeze production is likely to be a factor in over-

winter survivorship. Thus, we predict that S. mormonia

eggs are especially glycogen rich, causing nectar feeding

to be especially important to egg provisioning in this

species.

By combining the data on egg carbon turnover with

age-specific fecundity, we can calculate the total con-

tribution of the larval and adult diets to egg manufac-

ture, summed across a female’s lifetime (Fig. 6). The

larval diet supplied the majority of carbon for egg

Fig. 7. The proportion of egg carbon for each species that isexclusively larval in origin, plotted against the proportion of eggcarbon that derives from essential amino acids. The lineindicates y�/x. Species that fall close to y�/x are deriving amaximum of egg raw materials from the adult diet; those thatfall far away rely to a greater degree on larval resources thanbiochemical composition requires.

OIKOS 105:2 (2004) 289

manufacture in E. chalcedona (69%), the butterfly that

oviposits immediately after mating. In C. eurytheme,

input from the larval and the adult diet is fairly even (49

vs 51%), whereas in both S. mormonia and H. charitonia

the adult diet supplies the majority of egg carbon (65%).

One might expect S. mormonia to resemble C. eurytheme

in carbon allocation because their timing of oviposition

is similar; however, S. mormonia uses more adult dietary

carbon on a per egg basis. This difference has a

significant impact on lifetime resource use. Thus, the

extent to which dietary resources are important in egg

manufacture in butterflies depends on development (egg

provisioning in teneral adults), behavior (timing of

oviposition) and nutritional physiology (nutrient synth-

esis and turnover). Further understanding of this inter-

play is critical if we are to interpret variation in nutrient

use in light of reproductive strategies.

Acknowledgements �/ We would like to thank Jesse Boyett, ZoeChafe, Becky Chaplin, Liz Forwand, Sharon Komarow, JanetLarsen, Margie Mayfield, Sarah Oehm, Joanne White andGwen Yoshimura for their help in butterfly rearing and care,and Ward Watt for providing Colias eurytheme. We also thankKlaus Fischer for his helpful comments on this manuscript.This research was supported by NSF Grant IBN 98�/83044.

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