OIKOS 105: 279Á/291, 2004

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 role of life history and dietary carbon turnover in reproductive resource allocation. Á/ Oikos 105: 279Á/291.

The diets of many and moths change dramatically with development: from herbivory in the larvae to nectarivory in the adults. These diets are nutritionally distinct, and thus are likely to contribute differentially to egg manufacture. We examine the use of dietary resources in egg manufacture by four butterfly species with different patterns of oviposition and lifespan; three in the (Euphydryas chalcedona, mormonia and Heliconius charitonia), and one in the Pieridae (Colias eurytheme). Each species was fed two isotopically distinct adult diets based on sucrose, both of which differed from the larval hostplant in 13C content. Egg isotopic composition was analyzed to quantify the contribution of carbon from the larval and adult diets to egg manufacture. In all four species, egg 13C content increased to an asymptotic maximum with time, indicating that adult diet is an increasingly important source of egg carbon . The 13C increase closely resembled that of a nectar-feeding hawkmoth, and was well-described by a model of carbon flow proposed for that species. This similarity suggests that the turnover from larval to adult dietary support of egg manufacture is conserved among nectar-feeding . Species varied widely 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 the extent of oocyte provisioning prior to adult emergence, and to egg composition. A species’ lifetime use of larval vs adult resources in egg manufacture reflected both the carbon turnover of the eggs and the timing of oviposition. 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 synthesis and turnover).

D. M. O’Brien and C. Boggs, Center for , Dept of Biological Sciences, Stanford Univ., Stanford, CA 94305. Present address for DMO: Dept of Biological Sciences, Wellesley College, 106 Central St., Wellesley, MA 02481, USA ([email protected]). Á/ M. Fogel, Geophysical Laboratory, Carnegie Institution of Washington, 5251 Broad Branch Rd. NW, Washington DC 20015, USA.

Assumptions about how organisms use nutritional as a currency for reproduction and survival, other resources are fundamental to predictions about life nutrients, such as nitrogen (Slansky and Feeny 1977, history and evolutionary ecology (Reznick 1985, Stearns Brodbeck et al. 1993), phosphorous (Elser et al. 1996, 1992, Rose and Bradley 1998). Nutrition is a key Markow et al. 1999), or essential carbon compounds determinant of reproduction (Wheeler 1996); therefore, (Douglas 1998, O’Brien et al. 2002) may pose greater reproductive strategies need to be considered in light of constraints upon reproduction and population growth. nutrient availability (Boggs 1992, de Jong and van The complexities of nutrient requirements, synthesis, Noordwijk 1992). Although energy is commonly used storage, conversion and metabolism are poorly under-

Accepted 25 September 2003 Copyright # OIKOS 2004 ISSN 0030-1299

OIKOS 105:2 (2004) 279 stood, especially for free-living with mixed or enabled them to trace carbon use in eggs with high complex diets. There is a growing appreciation for the accuracy, and they proposed a descriptive carbon turn- physiological complexity of resource allocation (Rose over model to describe how egg nutrients were incorpo- and Bradley 1998, Rivero and Casas 1999, Zera and rated into eggs (O’Brien et al. 2000). Harshman 2001, Zhao and Zera 2002), in particular, for In this study, we apply a similar approach to hormonal controls of nutrient use (Sinervo and Svensson investigate the dietary sources of reproductive nutrients 1998, Zera et al. 1998). A similar focus on nutritional in four nectar-feeding butterfly species, three from the physiology will further help to strengthen the connec- family Nymphalidae and one from the family Pieridae. tions between diet and life history. The species selected exhibit a range of variation in Resource allocation in holometabolous is lifespan and age-specific fecundity, and differ in degree particularly interesting because diets and energetic needs of shared phylogenetic history. We investigate whether change between life stages, causing nutrient intake and the turnover pattern modeled in hawkmoth eggs is nutrient demand to be temporally separated (Boggs exhibited in other nectar-feeding Lepidoptera. If so, we 1981, 1986, Rivero et al. 2001). In these insects, evaluate whether the extent of adult diet use or the mechanisms for appropriately timed nutrient storage rapidity of larval to adult diet turnover vary according to and release are extremely important (Delobel et al. 1993, differences in life history. We predict that the reliance on Wheeler and Buck 1995, Snigirevskaya et al. 1997, adult dietary carbon in egg manufacture will be higher in Djawdan et al. 1998, Wheeler et al. 2000, Pan and Telfer those species whose reproductive effort takes place later 2001). In most butterflies and many moths, diets change in the adult lifespan. dramatically from herbivory in the larvae to nectarivory This study uses sucrose solution as an experimental in the adults. These diets are nutritionally distinct, and proxy for the adult nectar diet, as it elicts a strong thus are likely to contribute differently to physiological feeding response from butterflies (Romeis and Wa¨ckers demands. Nectar is rich in sugars but relatively depau- 2000) and is easy to acquire in different isotopic forms. perate in amino acids, fatty acids, and other nutritionally This proxy is a nutritional oversimplication, especially as important compounds (Baker and Baker 1983). The some butterflies have been shown to detect and slightly larval diet is the primary supply of protein, which is prefer nectars containing amino acids (Alm et al. 1990, stored for use during metamorphosis and oogenesis Rusterholz and Erhardt 1998, Mevi-Schutz and Erhardt (Wheeler et al. 2000, Pan and Telfer 2001). Butterfly 2002). We do not suggest that other dilute components researchers have long appreciated that both larval and of nectar could not play some nutritional role in adult diets are important to reproduction and are both oviposition. This study might slightly underestimate used in egg manufacture (Norris 1934, Dunlap-Pianka et the importance of the adult diet in species that capitalize on nectar amino acids. However, as the vast majority of al. 1977, Hill 1989, Hill and Pierce 1989, Alm et al. 1990, carbon provided by the adult diet is in the form of sugar Boggs and Ross 1993, Boggs 1997a). Because their life (Baker and Baker 1983), sugar is a reasonable proxy histories vary, the Lepidoptera have also provided a given our carbon isotope mass-balance approach. 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 Methods deriving from the larval and adult diets in butterflies with nectivorous adults, but differing lifespans and age- Study organisms specific fecundities. 13C content differs between plants Euphydryas chalcedona with C3 and C4 photosynthesis, but also can vary among Euphydryas chalcedona (Nymphalidae: Nymphalinae; plants sharing a photosynthetic mode (O’Leary 1981, Melitaeini) is a univoltine butterfly distributed through- 1988). Thus, it can be a powerful tracer of carbon flow out western North America. Individuals were field from multiple plant sources. Natural abundances of light collected as 5th and 6th instar larvae from Scrophularia stable isotopes are a well-established tracer of nutrient californicus and Mimulus aurantiacus at the Silver Creek flow, within both organisms (Metges et al. 1990, Hobson Hills Butterfly Reserve near San Jose, California, USA. et al. 1997, Schmidt et al. 1999) and ecosystems Larvae were placed in mesh bags on potted S. californi- (Rounick and Winterbourn 1986, Peterson and Fry cus for the remainder of their larval development, and 1987, Ostrom et al. 1997, Wolf and Martı´nez del Rio bags were moved as needed to ensure a constant supply 2000). In a previous study, O’Brien and colleagues (2000) of fresh leaves. Pupae were stored at 208C until adults used carbon isotopes to trace the use of larval and adult emerged. Average female forewing length was 29.89/0.4 diets in reproduction by a hawkmoth. Females were fed mm. Females mated either the day of emergence or the one of two sucrose-based adult diets made from beet day following. Once mated, females were placed in wire (C3) or cane (C4) sugar, both of which differed from the mesh cages (20/20/30 cm) and provided fresh S. natural larval hostplant in 13C content. This approach californicus leaves for oviposition. Females oviposited

280 OIKOS 105:2 (2004) daily or every other day in clusters of typically 100Á/200 to resemble Psiguria umbrosa flowers, and sugar solu- eggs (all other species laid eggs singly). Females were tions were changed daily. Although H. charitonia handfed twice daily. Of the 10 females to successfully normally supplement their diet with pollen, females in mate and start the experiment, 2 were removed because this experiment were not provided with pollen. Eggs they either waited unusually long to oviposit, or didn’t were collected daily. Nine females initiated oviposition eat consistently. The remaining 8 butterflies were used in and were used in isotope measurements; of those, two both isotope analyses and feeding and fecundity mea- died prematurely (N/7 for lifespan and fecundity surements. estimation).

Speyeria mormonia Colias eurytheme Speyeria mormonia (Nymphalidae: Heliconiinae: Heli- coniini: Argynniti) is a temperate zone univoltine Colias eurytheme (Pieridae) is a temperate zone butterfly, butterfly that occurs in montane and subalpine habitat which does not diapause. Colias eurytheme eggs were in western North America. Adult females were captured provided by Ward Watt from his colony at Stanford during August 1999 near Gothic, Gunnison County, University, which derived from females collected near Colorado, USA, and oviposited in captivity. Speyeria Tracy, California, USA. Larvae were reared on hydro- mormonia overwinters as unfed first instar larvae; ponically grown Vicia villosa (Watt, unpubl.), and pupae therefore, their hatchlings were placed in humid vials were housed at 208C until emergence. Newly emerged and stored at 48C. The following February (2000), larvae adult females were hand-mated the same day. Average were warmed to room temperature and placed in mesh female forewing length was 28.79/0.05 mm. Mated bags tied over leaves of potted violets ( soraria). females were housed in 20/20/30 cm mesh cages Bags were moved as needed to provide larvae with a and provided potted vetch plants for oviposition. constant supply of fresh leaves. Pupae were stored at Females were hand-fed twice daily. Of 18 females that 208C until adults emerged. Average female forewing started the experiment, 4 laid fewer than 500 eggs, lived length was 26.99/0.6 mm (nearly identical to that of fewer than 10 days, or skipped multiple days of field-caught females, 26.69/1.1 mm, Boggs 1987). Newly oviposition in the first week. These butterflies were emerged females were hand-mated and placed in cylind- considered non-representative and were removed. Of 14 rical cages made from lantern globes and lined with wax healthy ovipositing females used for isotope analyses, paper (15 cm diameter/20 cm height). Females were two were prematurely removed due to injury or escape provided fresh violet leaves for oviposition, and were fed and were not included in our estimates of lifespan and twice daily. Of 15 healthy experimental females, two were fecundity (N/12). 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. Experimental methods Heliconius charitonia Experiments were carried out between February and May, 2000 (except where noted), at the Herrin Green- H. charitonia (Nymphalidae: Heliconiinae: Heliconini: houses at Stanford University. Greenhouses were main- Heliconiiti) is a sub-tropical butterfly, which cannot tained on a 16:8 L:D photoperiod, with daytime and diapause. Adults feed on both nectar and pollen. A free- 8 8 flying greenhouse colony of H. charitonia is maintained nighttime temperatures of 27 C and 15.5 C, respectively. at Stanford University, founded from individuals col- All butterflies were fed 30% sucrose solution (wt/wt) as lected in Costa Rica and Yucatan, Mexico. Adults in the adults. Approximately half of the individuals received colony oviposit on potted Passiflora caerula and feed beet sugar solution (C3) and half received cane sugar from potted Lantana camara and Psiguria umbrosa. solution (C4). A single batch was mixed for each sugar Honey water feeders supplement these natural sources of solution, and stored as frozen aliquots until fed to the nectar and pollen. Experimental females were collected butterflies. A subset of the experimental females were fed as pupae from the colony and mated upon emergence. quantitatively from 25 or 50 ml Hamilton graduated

Average female forewing length was 38.69/0.6 mm. syringes, as described elsewhere (Boggs and Ross 1993), H. charitonia requires more space than the other to evaluate their intake quantitatively under the experi- butterflies to fly and oviposit normally; therefore, mated mental conditions. Eggs were collected daily into labeled females were placed individually into 1.8/1.0/1.0 0.5 ml Eppendorf tubes, which were placed open into a meter cages and each was given a potted Passiflora drying oven at 508C. After 3 days, tubes were removed caerula plant for oviposition. Females were provided from the oven, sealed, and stored at room temperature experimental sugar solutions ad lib in feeders colored until isotope analysis.

OIKOS 105:2 (2004) 281 d13C analysis By applying this model to our data, we can estimate

13 the values of the parameters in bold. In particular, we Isotope ratios are given a d C/(Rsample/Rstandard 13 12 are interested in pmax, which describes the maximum /1)/1000, in which R/ C/ C and the standard is contribution of adult dietary carbon to eggs, and r, PDB limestone. Dried egg samples weighing 0.30 to 0.35 13 which indicates the rate at which butterflies transition mg were analyzed for d C using a CHNS analyzer (CE from larval to adult sources of egg carbon. 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 Calculating % contribution of the adult diet 13 standards (0.2/0.25 mg.) were run with each batch of The magnitude of the difference in egg d C between C3 samples (Â/1 standard per 5 samples), and were used to vs C4 Á/ fed females on a given day reflects how much of 13 correct for consistent variation in d C among batches the carbon in those eggs comes from the adult diet. We run at different times. The standard deviation of term the proportional contribution of the adult diet to 13 acetanilide d C within a batch was typically B/0.15˜. eggs ‘‘p’’, and quantify it as follows (O’Brien et al. 2002):

13 13 13 p(d CC4 egg d CC3 egg)=(d13CC4 diet d CC3 diet) (5) Turnover model We use the lines of best fit through the data from C3 We parameterize the pattern of dietary carbon allocation and C4 fed females to calculate an average daily ‘‘p’’ for with the model of O’Brien et al. (O’Brien et al. 2000). each species. 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 Amino acid composition larval isotopic signature. We label these the ‘‘mixing’ and ‘non-mixing’’ carbon sources, respectively. The propor- Egg amino acid compositions were analyzed by post- tion of egg carbon in these two nutrient classes is given column ninhydrin-based amino acid analyses at the by pmax, which represents the maximum contribution of Molecular Structure Facility at the University of Cali- adult dietary carbon to eggs: fornia, Davis (Smith 1997). This technique provides data on 16 of the 20 amino acids found in proteins: cysteine d13 (d13 )( ) (d13 )( ) egg C Cmixing pmax  Cnon-mixing 1pmax and tryptophan are partially or completely destroyed by (1) the analysis, and glutamine and asparagine become 13 13 indistinguishable from glutamic acid and aspartic acid, Because d Cnon-mixing will be constant, whereas d respectively, after acid hydrolysis (Smith 1997). Cmixing will change over time, the isotopic composition of the two nutrient pools is modeled as: d13C d13C (2) non-mixing  larval C Oocyte provisioning at adult emergence 13 13 13 13 d Cmixing d Clarval C ((d Cdiet fa)d Clarval C) Asking whether adult-derived nutrients are used in egg  ( 1ert)(3) manufacture implies that at least some oocytes are not mature at the time of adult emergence. In order to Thus, egg evaluate the extent of oocyte provisioning during 13 13 13 d CCd Clarval C ((d Cdiet fa)d13Clarval C) metamorphosis, we dissected the ovaries of newly emerged females and calculated the volume of the  ( 1ert)) (p ) Cd13C )(1p )(4) max larval C max most mature and least mature oocytes assuming a where cylindrical egg. We used the same method to measure pmax /the maximum % of egg carbon to derive from the the volume of newly laid eggs for all species, except adult diet Euphydyras chalcedona, which has chorionated eggs in r/the fractional turnover rate of carbon from larval to the ovaries at emergence, which therefore cannot change adult sources in volume. Oocyte volume as a % of the volume of newly fa /the isotopic fractionation associated with incorpor- laid eggs was then calculated for both the most and least ating adult dietary carbon into eggs mature eggs in the ovaries. Because females emerge with 13 d Clarval C /the isotopic composition of egg carbon a full complement of oocytes, these measurements deriving from the larval diet. represent the first and last eggs potentially laid, and 13 d Cdiet /the isotopic composition of the adult diet hence the endpoints of eggs used in our isotopic analysis. (known) Note that Heliconius charitonia exhibits oogenesis as an t/days since adult emergence (known) 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 Fig. 1. Species differences in lifespan and fecundity for this each application of ANOVA or ANCOVA, means were experiment. Shown are means9/SD, sample sizes are given in compared using Tukey’s HSD contrasts and residuals parentheses next to the species names. were tested for normality using the Shapiro-Wilks test. Non-linear fitting was iteratively performed using least greater fecundity than S. mormonia, but did so with squares minimization until estimates converged. Briefly, smaller eggs (see below). The data are consistent with non-linear fitting applies a model with unknown para- previously published life history studies on the three of meters to a set of known data and modifies the these species for which records are available (Stern and parameters from pre-set values to achieve the best fit Smith 1960, Dunlap-Pianka et al. 1977, Boggs 1986, (via least-squares minimization). We tested whether 1990). 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 Egg weight and composition Ratkowsky and outlined in Motulsky and Ransas (Motulsky and Ransnas 1987). In these comparisons, Egg weight and % carbon and nitrogen are presented for each species in Table 1. Egg weight declined slightly we used p/0.008 (Bonferroni correction) as the sig- nificance threshold to account for multiple tests. (between 1Á/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 Results and Karlsson 1987). Speyeria mormonia eggs had a markedly lower protein content compared to the other Lifespan and fecundity species, as indicated by their high egg C/N (Table 1). Egg Average adult lifespan in this study ranged from 15 days C/N increased slightly over time in H. charitonia for Euphydryas chalcedona to 41 days for Heliconius (ANCOVA, Day effect P/0.0152, slope/0.014/day), charitonia, and average lifetime fecundity ranged from but did so in no other species. 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 Oocyte provisioning at adult emergence species in Fig. 2. E. chalcedona began to deposit eggs in large clusters immediately after mating, typically laying Euphydryas chalcedona, with an average of 489/6(n/3 /500 eggs within the first 5 days of adult life. In newly emerged females) oocytes mature and chorionated contrast, H. charitonia began to oviposit more than a at emergence, had its most mature egg equal to 100% of week after emergence and mating, laid fewer eggs daily, the volume of a laid egg. The other species showed lower and lived for weeks longer than the other three butterfly percentages: 7% (n/2 newly emerged females) for S. species. H. charitonia is a species that normally supple- mormonia and 4% for both C. eurytheme (n/2) and H. ments its nectar diet with pollen, (Dunlap-Pianka et al. charitonia (n/1). The volume of least mature oocytes 1977, Boggs 1979), but still lays relatively few eggs per was invariant within species and similar among species, 3 day (Â/10Á/15, Dunlap-Pianka et al. 1977, Boggs 1997a). at 0.0008 mm for S. mormonia and C. eurytheme and Speyeria mormonia fell between H. charitonia and E. 0.0005 mm3 for E. chalcedona and H. charitonia. These chalcedona in the timing of its reproductive effort, its volumes are 0.1% of that of a laid egg for E. chalcedona lifespan, and its fecundity. Colias eurytheme, from the and H. charitonia, 0.2% for S. mormonia and 0.4% for family Pieridae, was also intermediate; it achieved C. eurytheme.

OIKOS 105:2 (2004) 283 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 1379/24 1 Dry egg weight (mg) 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 12.79/3.6 Egg % 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 Á/ Egg C out (mg) 35.39/9.4 23.69/5.8 10.79/2.2 5.69/1.0 C in/Egg C out 2.79/1.8 3.69/1.5 5.39/2.4 Á/

1Multiple samples were evaluated per individual, which were averaged before calculating species means and SD. Numbers in parentheses give the No. of individuals (N) and the total number of samples measured (in italics). Nectar intake and carbon budget meal size were significant (Fig. 3, ANCOVAwith Tukey’s HSD; species PB/0.0001, day PB/0.0001, age/species Euphydryas chalcedona had the largest average meal size interaction not sig.). Heliconius charitonia was not fed (36.19/1.9 ml; mean9/SD), followed by S. mormonia quantitatively. (28.09/1.4 ml) and C. eurytheme (17.49/1.4 ml). In all We converted meal sizes (ml) to mg carbon consumed species, meal size declined an average of 0.97 ml/day. using 0.143 mg C/ml sucrose solution (determined Both the differences among species and the decline in empirically). We calculated the mg carbon contained in

Fig. 2. (AÁ/D) Mean number of eggs laid daily for each species. Data are given as means9/SE. Sample sizes for each mean (when different from the preceding mean) are given in italics below each data point. (E) Comparison of oviposition among species, with uniform scaling. Lines connect mean egg numbers for each day.

284 OIKOS 105:2 (2004) Table 2. The effect of female age (in days of adult lifespan) on egg weight (mg.). The relationship was evaluated for each species separately with ANCOVA, to remove significant inter-individual differences in egg mass. Where age significantly affected mass, the magnitude of the effect is given by the slope (in mg/day).

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

1 C. eurytheme Individual (5) 13.93 B/0.0001 27 Á/ Age 57.08 B/0.0001 /0.0005 E. chalcedona Individual (6) 5.0844 0.0141 17 Á/ Age 18.0215 0.0017 /0.0011 S. mormonia Individual (11) 0.72 0.7029 38 Á/ Age 11.35 B/0.0024 /0.0018 H. charitonia Individual (7) 15.90 B/0.0001 26 Á/ 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 for each mean (when different from the preceding mean) are given in italics below each data point. Note that the scaling of axes is constant among species. Mean meal size differs significantly among species, however, the decline in meal size over time does not (see results). eggs from mean egg weights and % C for each species % egg carbon deriving from different classes of amino (Table 1). Total carbon gain and loss were calculated for acids (Table 4). 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. d13C Dietary constituents The d13C of the larval hostplant (in parentheses) for each butterfly species were as follows: E. chalcedona (Scro- Egg amino acid composition phularia californicus), /28.259/1.61˜ (mean9/SD; N/3), S. mormonia (Viola soraria) /33.049/1.94˜ Egg amino acid composition (in mol%) is given in Table (N/10), H. charitonia (Passiflora caerulea) /31.869/

3. Data from the hawkmoth Amphion floridensis are also 0.92˜ (N/3) and C. eurytheme (Vicia villosa), presented for comparison (O’Brien et al. 2002). For each /25.859/0.56˜ (N/4). Although E. chalcedona lar- species, amino acids are ranked from most to least vae were field collected on both S. californicus and prevalent. Amino acid composition was similar (but not Mimulus aurantiacus, the majority of their growth was identical) in all four species. Glutamic acid was the most on S. californicus in the greenhouse. The d13C of the prevalent amino acid in all four species, followed by adult diets were /11.149/0.10˜ (C4 cane sugar) and alanine, glycine, and aspartic acid in varying order /24.849/0.13˜ (C3 beet sugar). All butterflies were fed (totaling 42Á/46% of egg amino acids). Serine, leucine, from frozen aliquots from single batches of sugar lysine, valine and proline, in varying order, were the next solution; thus, the standard deviation around the value most abundant amino acids (30Á/34%). Threonine, for adult diet represents measurement error. arginine, isoleucine, and tyrosine were the next four most abundant (16Á/19% of egg amino acids). Finally, phenylalanine, histidine, and methionine were the least d13C Butterfly eggs abundant amino acids (6Á/8% of egg amino acids). These amino acid compositions, together with the measured The change over time in isotopic composition of egg %C and %N for each species, allowed us to calculate the carbon (d13C) for each species is presented in Fig. 4. In

OIKOS 105:2 (2004) 285 Table 3. Amino acid composition of butterfly eggs. Each column gives averaged data from 2 individuals, with inter-individual differences 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 in varying 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 15.6 asx 10.5 gly 11.4 gly 11.2 asx 10.1 ala 11.6 ala 9.7 asx 9.4 asx 11.0 gly 9.8 glx 11.1 gly 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 7.0 ser 7.1 val 7.2 leu 6.6 leu 7.1 val 6.0 val 6.1 leu 6.9 lys 5.7 lys 6.1 pro 5.9 lys 6.1 ser 6.3 pro 5.5 val 5.8 ser 5.7 pro 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 4.4 arg 4.7 tyr 4.5 thr 4.3 arg 4.6 thr 4.3 ileu 4.6 thr 4.3 arg 4.1 ileu 4.4 arg 3.8 tyr 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 2.8 his 2.3 his 2.0 his 2.0 his 2.3 his 2.4 met 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 gNg1 egg1 0.085 0.057 0.076 0.095 g protein g1 N2 5.713 5.817 5.716 5.697 g protein g1 egg3 0.486 0.331 0.434 0.541 gCg1 egg1 0.51 0.58 0.44 0.497 g EAA C g1 protein2 0.292 0.301 0.283 0.302 g EAA C g1 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.

13 13 all four species, egg d C reflected the d C of the C3 or Species varied widely in the maximum percentage of egg 13 C4 adult diet (triangles vs circles, Fig. 4). Egg d C carbon that derives from the adult diet (from 44% in E. increased asymptotically over time (Fig. 4), as found chalcedona to nearly 80% in S. mormonia, Fig. 5, Table elsewhere (O’Brien et al. 2000). The extent of the 5). In contrast, species were all relatively similar in their increase depended on the species, and on the lag between turnover rate (r), the inverse of the time required to emergence and oviposition. For example, larval to adult reach half turnover (Fig. 5). The turnover rate indicates carbon turnover in H. charitonia stabilized prior to the how rapidly the shift from larval to adult sources occurs. onset of oviposition on day 8, and thus egg d13C changed These varied from 0.18 day1 in S. mormonia (most little if at all (Fig. 4). In E. chalcedona, S. mormonia and rapid turnover) to 0.32 day1 in C. eurytheme (least C. eurytheme, eggs laid within several days of emergence rapid, Table 5). 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, Comparing allocation patterns among species parameters given in Table 5). 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 Use of adult diet in egg provisioning the turnover model could also adequately describe that of the other species. In order to do this we used a test The daily proportion of egg carbon deriving from the that compares whether the goodness of fit of a non- adult diet is summarized in Fig. 5 for each species. linear model significantly worsens when two sets of data

286 OIKOS 105:2 (2004) Fig. 4. Egg carbon isotopic composition (d13C) vs female age in days since emergence from pupation. Triangles denote eggs laid by females fed cane sugar solution as adults (C4: /11.15˜), circles denote eggs laid by females fed beet sugar solution as adults (C3: /24.85˜). Lines through the data were generated by the model given in Eq. 4, using non-linear fitting to estimate parameters. (A) E. chalcedona, N/33 samples from 8 females. (B) C. eurytheme;N/47 samples from 14 females. (C) S. mormonia,N/56 samples from 13 females. (D) H. charitonia;N/32 samples from 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 0.559/0.02 Turnover rate (r) 0.249/0.06 0.189/0.03 0.289/0.05 0.329/0.04 Adult diet fractionation (fa) 3.79/1.9 /1.89/0.9 /1.899/0.34 0.49/1.0 13 1 d C larval C /29.09/0.8 /33.89/1.4 see below /27.29/1.0 13 d C (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)

1 13 13 Model was unable to estimate d C larval C for H. charitonia. Because the estimated values of ‘‘d C larval C’’ were close to measured 13 13 d C values of larvae (within 1˜) in the other species, H. charitonia d C larval was fixed at the measured larval value in order to fit the other parameters.

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 Fig. 5. The proportion of egg carbon deriving from the adult oviposition strategy (Fig. 2) and the shifting sources of diet, vs female age, for each species. Proportions for each day egg nutrients (Fig. 5). Figure 6 presents the mg. of larval were calculated according to Eq. 5, using the model (Eq. 4) to and adult carbon invested in eggs per day by each 13 13 estimate the mean value of d C C3 egg and d C C4 egg for each species, and the summation of these daily investments to day (see Methods). We also present the equivalent curve for the hawkmoth A. floridensis, published elsewhere (O’Brien et al. give a lifetime total. The ratio of these lifetime totals 2000). (adult /larval) for each species was 0.46 in E. chalcedona,

OIKOS 105:2 (2004) 287 Table 6. Comparison of model fit between pairs of species: do species differ significantly in their turnover from larval to adult derived egg carbon?

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

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

1.04 in C. eurytheme, 1.79 in S. mormonia and 1.88 in H. also characteristic of adult resource allocation to repro- charitonia. Euphydryas chalcedona incorporated a lower duction in parasitioid wasps (Rivero et al. 2001). Tissue- percentage of adult diet into eggs than the other species, specific turnover has been well documented in animals and also laid the majority of its eggs before the bulk of that undergo diet shifts (Tieszen et al. 1983, Hobson larval to adult carbon turnover has occurred. 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 Discussion allocation should be determined by factors such as All four butterflies used nectar carbon in egg manufac- dietary intake rate, total nutrient pool size, and nutrient ture, to varying extents. Interestingly, the pattern of output in metabolism and egg manufacture. resource use was similar in all four species, despite their The turnover of egg carbon from larval to adult different life histories. Each species exhibited a shift from dietary sources illuminates the underlying physiology of larval to adult sources of egg carbon that was well- nutrient use in egg manufacture. This pattern can be described by a turnover function, with eggs coming to characterized with two biologically-relevant parameters: reflect a stable ratio of larval and adult resources. The the fractional turnover rate, which describes the rate of pattern is similar to that found in a hawkmoth (family the shift from larval to adult egg carbon sources, and the Sphingidae, O’Brien et al. 2000), a species that is only maximal contribution of the adult diet (the value at distantly related to the butterflies examined here. It is which egg composition stabilizes). The fractional turn-

Fig. 6. The mg of carbon from the larval and adult diets used daily in egg manufacture, for each species. Lifetime totals are presented on each legend. The larval diet is indicated by black bars and adult diet by gray bars. Note that the axes are differently scaled among species.

288 OIKOS 105:2 (2004) over rate was quite consistent across all species studied to date; from 3Á/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 Fig. 7. The proportion of egg carbon for each species that is exclusively larval in origin, plotted against the proportion of egg granules, hydrocarbons, and nucleic acids (Nation 2002). carbon that derives from essential amino acids. The line Approximately half of the carbon in egg protein derives indicates y/x. Species that fall close to y/x are deriving a from essential amino acids (Table 4). These amino acids maximum of egg raw materials from the adult diet; those that fall far away rely to a greater degree on larval resources than have been shown to derive exclusively from the larval biochemical composition requires. diet in Amphion floridensis (O’Brien et al. 2002). Essential fatty acids (and the fatty acids that derive suggest two diet use ‘‘strategies’’ here: one in which a from them) also occur in lepidopteran eggs, estimated butterfly makes as much of its eggs from adult diet as is for one species as 33% of all egg fatty acids (Forte et al. biochemically possible (S. mormonia, H. charitonia,C. 2002). Thus, requirements for essential nutrients not eurytheme and A. floridensis), and the other in which the abundant in nectar diets limit the extent to which a butterfly foregoes the potential carbon influx from the species can use adult dietary nutrients in egg manufac- adult diet in favor of egg provisioning during pupal ture. For example, pollen-feeding H. charitonia have development, and more rapid reproduction (E. chalce- access to essential amino acids (O’Brien et al. 2003), and dona). The smallest oocytes (corresponding to the last would be expected to rely more heavily on the adult diet eggs to be laid) were similarly sized among all four than the exclusively sucrose-fed H. charitonia in this species at emergence, suggesting at least the possibility study. that E. chalcedona could use more adult resources to For the species studied here, the percentage of egg provision the eggs laid latest in life. However, E. carbon deriving from essential amino acids is presented chalcedona concentrates oviposition in the first week of in Table 4. This percentage approximates the minimum adult life; therefore, it is likely that even the least mature possible carbon contribution from the larval diet (if oocytes are provisioned in early adulthood, before much essential amino acids were the only carbon compound turnover to adult nutrients has occurred. not synthesized from sugar). We plotted these values for S. mormonia has a higher proportion of non-protein each species against the % egg carbon that is exclusively egg nutrient than the other butterflies (high C/N), and larval in origin (1/pmax) (Fig. 7), including data exhibits the highest % adult dietary carbon in its eggs. A previously published from the hawkmoth A. floridensis possible explanation for the high egg C/N is glycogen. (O’Brien et al. 2000). This relationship provides some Glycogen stores are converted into the colligative anti- insight into the differences among species in carbon freezes glycerol and sorbitol in freeze-avoiding Lepidop- utilization. S. mormonia, H. charitonia, A. floridensis tera (Chino 1957, Storey 1990, Pullin et al. 1991). and (to a lesser degree) C. eurytheme all fall fairly close Because S. mormonia overwinters as unfed, 1st instar to the line y/x, indicating that they are incorporating hatchlings at high altitude in the Rocky Mountains, nearly the maximum amount of adult dietary carbon antifreeze production is likely to be a factor in over- possible, given their individual egg composition. E. winter survivorship. Thus, we predict that S. mormonia chalcedona is notably different: it seems to incorporate eggs are especially glycogen rich, causing nectar feeding much less adult dietary carbon than it theoretically to be especially important to egg provisioning in this ‘‘could’’, based on egg composition. species. The difference in E. chalcedona may lie in the extent of By combining the data on egg carbon turnover with egg provisioning that takes place prior to adult emer- age-specific fecundity, we can calculate the total con- gence. Based on ovarian dissections, E. chalcedona tribution of the larval and adult diets to egg manufac- provisions its eggs at a much higher level prior to adult ture, summed across a female’s lifetime (Fig. 6). The emergence than do the other three species. These data larval diet supplied the majority of carbon for egg

OIKOS 105:2 (2004) 289 manufacture in E. chalcedona (69%), the butterfly that Brodbeck, B. V., Mizell III, R. F. and Andersen, P. C. 1993. oviposits immediately after mating. In C. eurytheme, Physiological and behavioral adaptations of three species of leafhoppers in response to the dilute nutrient content of input from the larval and the adult diet is fairly even (49 xylem fluid. Á/ J. Physiol. 39: 73Á/81. vs 51%), whereas in both S. mormonia and H. charitonia Chino, H. 1957. Conversion of glycogen to sorbitol and glycerol the adult diet supplies the majority of egg carbon (65%). in the diapause egg of the Bombyx silkworm. Á/ Nature 180: 606Á/607. One might expect S. mormonia to resemble C. eurytheme de Jong, G. and van Noordwijk, A. J. 1992. Aquisition and in carbon allocation because their timing of oviposition allocation of resources: genetic (co)variances, selection, and is similar; however, S. mormonia uses more adult dietary life histories. Á/ Am. 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